MICROSCOPIC AND MOLECULAR METHODS FOR

Intergovernmental

Oceanographic

Commission

Manuals and Guides

MICROSCOPIC AND MOLECULAR

METHODS FOR QUANTITATIVE

PHYTOPLANKTON ANALYSIS

2010 UNESCO

IOC Manuals and Guides

No. Title

1 rev. 2 Guide to IGOSS Data Archives and Exchange (BATHY and TESAC). 1993. 27 pp. (English,

French, Spanish, Russian)

2 International Catalogue of Ocean Data Station. 1976. (Out of stock)

3 rev. 3 Guide to Operational Procedures for the Collection and Exchange of JCOMM Oceanographic

Data. Third Revised Edition, 1999. 38 pp. (English, French, Spanish, Russian)

4 Guide to Oceanographic and Marine Meteorological Instruments and Observing Practices.

1975. 54 pp. (English)

5 rev. 2 Guide for Establishing a National Oceanographic Data Centre. Second Revised Edition, 2008.

27 pp. (English) (Electronic only)

6 rev. Wave Reporting Procedures for Tide Observers in the Tsunami Warning System. 1968. 30 pp.

(English)

7 Guide to Operational Procedures for the IGOSS Pilot Project on Marine Pollution (Petroleum)

Monitoring. 1976. 50 pp. (French, Spanish)

8 (Superseded by IOC Manuals and Guides No. 16)

9 rev. Manual on International Oceanographic Data Exchange. (Fifth Edition). 1991. 82 pp. (French,

Spanish, Russian)

9 Annex I (Superseded by IOC Manuals and Guides No. 17)

9 Annex II Guide for Responsible National Oceanographic Data Centres. 1982. 29 pp. (English, French,

Spanish, Russian)

10 (Superseded by IOC Manuals and Guides No. 16)

11 The Determination of Petroleum Hydrocarbons in Sediments. 1982. 38 pp. (French, Spanish,

Russian)

12 Chemical Methods for Use in Marine Environment Monitoring. 1983. 53 pp. (English)

13 Manual for Monitoring Oil and Dissolved/Dispersed Petroleum Hydrocarbons in Marine Wa-

ters and on Beaches. 1984. 35 pp. (English, French, Spanish, Russian)

14 Manual on Sea-Level Measurements and Interpretation. (English, French, Spanish, Russian)

Vol. I: Basic Procedure. 1985. 83 pp. (English)

Vol. II: Emerging Technologies. 1994. 72 pp. (English)

Vol. III: Reappraisals and Recommendations as of the year 2000. 2002. 55 pp. (English)

Vol. IV: An Update to 2006. 2006. 78 pp. (English)

15 Operational Procedures for Sampling the Sea-Surface Microlayer. 1985. 15 pp. (English)

16 Marine Environmental Data Information Referral Catalogue. Third Edition. 1993. 157 pp.

(Composite English/French/Spanish/Russian)

17 GF3: A General Formatting System for Geo-referenced Data

Vol. 1: Introductory Guide to the GF3 Formatting System. 1993. 35 pp. (English, French, Spa-

nish, Russian)

Vol. 2: Technical Description of the GF3 Format and Code Tables. 1987. 111 pp. (English,

French, Spanish, Russian).

17 Vol. 3: Standard Subsets of GF3. 1996. 67 pp. (English)

Vol. 4: User Guide to the GF3-Proc Software. 1989. 23 pp. (English, French, Spanish, Rus-

sian)

Vol. 5: Reference Manual for the GF3-Proc Software. 1992. 67 pp. (English, French, Spanish,

Russian)

Vol. 6: Quick Reference Sheets for GF3 and GF3-Proc. 1989. 22 pp. (English, French, Spa-

nish, Russian)

18 User Guide for the Exchange of Measured Wave Data. 1987. 81 pp. (English, French, Spa-

nish, Russian)

To be continued on page 113

MICROSCOPIC AND MOLECULAR METHODS FOR

QUANTITATIVE PHYTOPLANKTON ANALYSIS

editors

Bengt Karlson, Caroline Cusack and Eileen Bresnan

Intergovernmental

Oceanographic

Commission

Manuals and Guides

2010 UNESCO

The designations employed and the presentations of the

material in this publication do not imply the expression of

any opinion whatsoever on the part of the Secretariats of

UNESCO and IOC concerning legal status of any country

or territory, or its authorities, or concerning the delimita-

tions of the frontiers of any country or territory.

For bibliographic purposes, this document should be

cited as follows:

Intergovernmental Oceanographic Commission of

©UNESCO. 2010. Karlson, B., Cusack, C. and Bresnan, E.

(editors). Microscopic and molecular methods for quantita-

tive phytoplankton analysis. Paris, UNESCO. (IOC Manuals

and Guides, no. 55.) (IOC/2010/MG/55)

110 pages.

(English only)

Published in 2010

by the United Nations Educational, Scientiﬁc and Cultural

Organization

7, Place de Fontenoy, 75352, Paris 07 SP

UNESCO 2010

Printed in Spain

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Contents

Preamble 2

Foreword 3

1 Introduction to methods for quantitative phytoplankton analysis 5

2 The Utermöhl method for quantitative phytoplankton analysis 13

3 Settlement bottle method for quantitative phytoplankton analysis 21

4 Counting chamber methods for quantitative phytoplankton analysis - 25

haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

5 Filtering – calcoﬂuor staining – quantitative epiﬂuorescence microscopy for phytoplankton analysis 31

6 Filtering – semitransparent ﬁlters for quantitative phytoplankton analysis 37

7 The ﬁlter - transfer - freeze method for quantitative phytoplankton analysis 41

8 Imaging ﬂow cytometry for quantitative phytoplankton analysis — FlowCAM 47

9 Detecting intact algal cells with whole cell hybridisation assays 55

10 Electrochemical detection of toxic algae with a biosensor 67

11 Hybridisation and microarray ﬂuorescent detection of phytoplankton 77

12 Toxic algal detection using rRNA-targeted probes in a semi-automated sandwich hybridization format 87

13 Quantitative PCR for detection and enumeration of phytoplankton 95

14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation 103

Appendix: Acronyms and Notation 109

IOC Manuals & Guides no 55

Preamble

Henrik Enevoldsen*

IOC Science and Communication Centre on Harmful Algae

University of Copenhagen,Øster Farimagsgade 2D, DK-1353 Copenhagen K, Denmark

*e-mail address: h.enev[email protected]

The Intergovernmental Oceanographic Commission of UNESCO has since 1992 given attention to activities aimed at devel-

oping capacity in research and management of harmful microalgae. With this IOC Manual & Guide we wish to ﬁll a gap for

information and guidance, in an easy accessible and low cost format, to comparison between traditional and modern methods

for enumeration of phytoplankton. Enumeration of harmful phytoplankton species is a key element in many monitoring pro-

grammes to protect public health, seafood safety, markets, tourism, etc. However, phytoplankton enumeration has self evidently

much broader application that just monitoring of harmful microalgae species.

One important task of the IOC and UNESCO is to synthesize the available ﬁeld and laboratory research techniques for ap-

plications to help solve problems of society as well as facilitate further research and especially systematic observations and data

gathering. The results include the publications in the ‘IOC Manuals and Guides’ series, and the UNESCO series ‘Monographs

in Oceanographic Methodology’. The easy access to manuals and guides of this type is essential to facilitate knowledge exchange

and transfer, the related capacity building, and for the establishment of ocean and coastal observations in the framework of the

Global Ocean Observing System.

The IOC is highly appreciative of the efforts of the ICES-IOC Working Group on Harmful Algal Bloom Dynamics in organ-

izing the Joint ICES-IOC Intercomparison Workshop on New and Classic Techniques for Estimation of Phytoplankton Abun-

dance at the Kristineberg Marine Research Station in Sweden 2005, and not the least the efforts of the scientists who prepared

the manuscripts for this IOC Manual & Guide. The IOC wishes to express its particular thanks to Dr. Bengt Karlson, SMHI

Sweden, Editor-in-Chief, for his determination to produce this volume.

The scientiﬁc opinions expressed in this work are those of the authors and are not necessarily those of UNESCO and its IOC.

Equipment and materials have been cited as examples of those most currently used by the authors, and their inclusion does not

imply that they should be considered as preferable to others available at that time or developed since.

The publication of this IOC Manual & Guide has been made possible through support from the United States National Oceanic

and Atmospheric Administration and the Department of Biology, University of Copenhagen, Denmark.

Henrik Enevoldsen

IOC Harmful Algal Bloom Programme

http://ioc.unesco.org/hab

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Foreword

Phytoplankton occupy the base of the food web of the sea. It plays a vital role in the global carbon cycle and is also of importance

since some phytoplankton may cause harmful algal blooms, a problem e.g. for aquaculture. Man induced changes in the envi-

ronment, e.g. eutrophication, can be manifested in changes in the phytoplankton community and there is now some evidence

that climate change may also be having an effect. Phytoplankton analysis is an essential part in the process of understanding and

predicting changes in our environment. Recent introduction of new methods, several based on molecular biology, has led to a

perceived need for a manual on quantitative phytoplankton analysis.

The aim of this publication is to provide a guide for phytoplankton analysis methods. A number of different methods are de-

scribed and information about applicability, cost, training, equipment etc. is included to facilitate information on choosing the

right method for a certain purpose. The costs of equipment, consumables, etc. are based on 2009 prices. Although the methods

described are for marine plankton they are also applicable for freshwater plankton. The method descriptions are more detailed

than what is usually found in scientiﬁc articles to make the descriptions useful when setting up monitoring or research pro-

grammes that include inexperienced researchers. Some of the methods described are relatively old and well tested while a few

must be considered to be emerging technology. We hope that this publication will supplement existing literature and that the

distribution of the book freely using the Internet will make it useful in environmental monitoring and for students, researchers

and regulators. A book like this can never be complete. Some methods are missing and newer techniques are under development.

The production of this book was initiated during an international workshop at Kristineberg Marine Research Station in Sweden

2005. Participants in the Joint ICES/IOC Intercomparison Workshop on New and Classic Techniques for Estimation of Phytoplankton

Abundance (WKNCT) agreed to write chapters of the book. A scientiﬁc paper describing the results of this workshop can be

found in Godhe et al. (2007). Co-authors have joined some of the workshop participants. The Harmful Algal Bloom programme

of the Intergovernmental Oceanographic Commission, of UNESCO, has aided in the production and also ﬁnanced the print-

ing of the book. We would like to express our gratitude to everyone who has been involved in the production of this book. In

particular the editors would like to acknowledge the time and effort contributed to the ﬁnal edits and proof reading by Jacob

Larsen and Pia Haecky.

Bengt Karlson, Caroline Cusack and Eileen Bresnan

Reference

Godhe A, Cusack C, Pedersen J, Andersen P, Anderson DM, Bresnan E, Cembella A, Dahl E, Diercks S, Elbrächter M, Edler L, Galuzzi L,

Gescher C, Gladstone M, Karlson B, Kulis D, LeGresley M, Lindahl O, Marin R, McDermott G, Medlin MK, Naustvoll L-J, Penna A, Töbe

K (2007) Intercalibration of classical and molecular techniques for identiﬁcation of Alexandrium fundyense (Dinophyceae) and estimation of

cell densities. Harmful Algae 6: 56-72

IOC Manuals & Guides no 55

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

Background

Phytoplankton is a critical component of the marine ecosys-

tem as they are responsible for approximately half of the glo-

bal (terrestrial and marine) net primary production (Field et

al. 1998). Today approximately 4000 marine phytoplankton

species have been described (Simon et al. 2009). They have

the potential to serve as indicators of hydro-climatic change

resulting from global warming as well as other environmen-

tal impacts, such as ocean acidiﬁcation due to combustion of

fossil fuels and eutrophication. Under certain environmental

conditions phytoplankton can experience elevated growth

rates and attain high cell densities. This is known as an al-

gal bloom. There are different types of algal blooms. Some

are natural events such as the spring diatom bloom where, at

temperate latitudes, there is a burst of diatom growth during

spring time as a response to increasing light availability, tem-

perature and water column stabilisation. This is part of the

annual phytoplankton cycle in these regions. Some blooms

can have a negative impact on the marine system and aqua-

culture industry and are termed ‘Harmful Algal Blooms’

(HABs). Some HAB species such as the dinoﬂagellate, Kare-

nia mikimotoi, form high density blooms with millions of

cells per Litre discolouring the water and causing anoxia as

the bloom dies off. This can result in benthic mortalities such

as starﬁsh, lugworms and ﬁsh. In contrast, low cell densities

of species of the dinoﬂagellate genus Alexandrium (2,000 cells

-1

) have been associated with closures of shellﬁsh harvest-

ing areas owing to elevated levels of the toxins responsible for

paralytic shellﬁsh poisoning. These are also called HABs even

though they are present at low cell densities.

Many regions of the world implement phytoplankton moni-

toring programmes to protect their aquaculture industry.

These programmes provide advice about the potential for

toxic events and improve local knowledge of the dynamics of

toxic phytoplankton in the area. The European Union (EU)

member states are legally obliged to monitor their shellﬁsh

production areas for the presence of toxin producing phy-

toplankton. Marine environmental policy has increased in

importance and a number of directives has been developed

to monitor water quality. The Water Framework Directive

(WFD) uses phytoplankton as one of the ecosystem compo-

nents required to monitor the quality status of marine and

freshwater bodies. Phytoplankton is also a required biological

component of the EU Marine Strategy Framework Directive,

devised to protect and conserve the marine environment. The

1 Introduction to methods for quantitative

phytoplankton analysis

Bengt Karlson

, Anna Godhe

, Caroline Cusack

and Eileen Bresnan

Swedish Meteorological and Hydrological Institute, Research & development, Oceanography, Sven Källfelts gata 15, SE-426 71 Västra

Frölunda, Sweden

Department of Marine Ecology, Göteborg university, Carl Skottbergs Gata 22, SE-413 19 Göteborg, Sweden

Marine Institute, Rinville, Oranmore, Co. Galway, Ireland

Marine Scotland Marine Laboratory, 375 Victoria Road, Aberdeen AB11 9DB, UK

*Author for correspondence e-mail: bengt.kar[email protected]

International Maritime Organization (IMO) adopted the Bal-

last Water Convention in 2004 although it has not yet been

ratiﬁed. This convention includes a ballast water discharge

standard whereby ships will be required to treat or manage

ballast water to ensure that no more than 10 organisms per

mL in the size category >10 µm - < 50 µm and no more than

10 organisms per m

>50 µm are discharged.

Thus, there is a requirement to be able to describe and

monitor the abundance, composition and diversity of the

phytoplankton community. A variety of different methods

have been developed to identify and enumerate phytoplank-

ton. Descriptions of many of these can be found in two

UNESCO-produced volumes: The Phytoplankton manual,

edited by Sournia, was published in 1978. This volume pro-

vides a comprehensive description of many traditional light

microscopy methods used to enumerate phytoplankton. It

is currently out of print and many laboratories have found

it difﬁcult to obtain a copy. The Manual on Harmful Ma-

rine Microalgae edited by Hallegraeff et al. was ﬁrst published

in 1995 with a revised second edition published in 2004. It

provides information on the taxonomy and methodology in-

volved in operating phytoplankton and biotoxin monitoring

programmes.

The present manual aims to provide detailed step by step

guides on how to use microscope based and molecular meth-

ods for phytoplankton analysis. Most of the molecular meth-

ods are aimed only at selected target species while some of

the microscope based methods can be used for a large part

of the phytoplankton community. Methods for analyzing

autotrophic picoplankton are not included in this manual.

Common methods for this important group include ﬂuores-

cence microscopy (Platt and Li 1986 and references therein)

and ﬂow cytometry (e.g. Simon et al 1994) as well as molecu-

lar methods. The decision on which method to use will ulti-

mately depend on the purpose of the monitoring programme

and the facilities and resource available. Information about

sampling strategies are found in Franks and Keafer (2004).

Although the sampling methods are outside the scope of this

manual an overview of the steps from sampling to presenta-

tion of results to end users is presented in Fig. 1. Examples

of sampling devices are found in Figs. 2-7. In addition to

these automated sampling systems on Ships of Opportunity

(SOOP, e.g. FerryBox systems), buoys, Autonomous Under-

water Vehicles (AUV’s) etc. are used (Babin et al. 2008).

IOC Manuals & Guides no 55

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

Quantitative

sampling

Preservation

Concentration

Storage

Printed report

Filtering Centrifugation Sedimentation

Quality control

Often made by analyst when entering data into

electronic database . Double checked by someone else

Homogenisation and DNA

extraction for some

molecular techniques

End users

Interpretation of results

Web site and

other media

Identification

of organisms

and estimation of

cell concentrations

and biomass

Microscopy Flow cytometers

Molecular biological

techniques

Lugol ’ s iodine

acid

neutral

alkaline

Aldehydes

Saline ethanol

Water bottles

( discrete depths )

Keep in dark and refrigerate . Analyse as quickly as possible

Tube

( integrating )

Sonication

Scientific

publication

Comparison with existing data, statistical analysis ,

inclusion of other data, e.g . oceanographic data

and data on algal toxins in shellfish

( None )

Results

Number of organisms or biomass per litre

and species composition ( biodiversity )

Freezing of

raw sample

Ring tests with other laboratories , test for repeatability ,

estimation of variability due to method or persons

performing analysis , documentation of methods

> accredited analysis and laboratory

Automated

sampling devices

Figure 1. Schematic drawing of the steps from sampling to results.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

Microscopy based techniques

The historical development of microscope based

phytoplankton analysis techniques

Many historic reports exist of phytoplankton blooms. Some

believe the description of the Nile water changing to blood in

the bible and resultant ﬁsh mortalities (Exodus 7:14-25) is an

account of the occurrence of a HAB. The invention of the mi-

croscope by Anton van Leeuwenhoek (1632-1723) in the 17

century allowed more detailed observations of phytoplankton

to be made with Christian Gottfried Ehrenberg (1795-1876)

and Ernst Heinrich Philipp August Haeckel (1834-1919) be-

coming pioneers in observations of microalgae. Over the last

150 years a number of techniques for analysis of phytoplank-

ton have been developed and adopted in analytical laborato-

ries throughout the world. The Swedish chemist, Per Teodor

Cleve (1840-1905), was one of the ﬁrst researchers to under-

take more quantitative surveys of the phytoplankton commu-

nity. He used silk plankton nets to investigate the distribution

of phytoplankton in the North Sea Skagerrak-Kattegat area

(1897). Hans Lohmann (1863-1934) ﬁrst used a centrifuge

to concentrate plankton and discovered the nanoplankton

(phytoplankton 2 – 20 µm in size) (Lohmann 1911). The

classic sedimentation chamber technique still used in many

laboratories today was developed by Utermöhl (1931, 1958).

In the 1970s the ﬂuorescence microscope was ﬁrst used for

quantitative analysis of bacteria in seawater (e.g. Hobbie et al.

1977). A similar technique was used to reveal the ubiquitous

distribution of autotrophic picoplankton (size 0.2 – 2 µm) in

the sea (Johnson and Sieburth 1979, Waterbury et al. 1979).

In the 1980s auto- and heterotrophic nanoplankton were in-

vestigated using various stains and ﬁltration techniques (e.g.

Caron 1983).

Training and literature for identiﬁcation of phytoplankton

using microscopes

Microscope based methods involve the identiﬁcation of phy-

toplankton species based on morphological and other visible

criteria. Phytoplankton taxonomists should have a high de-

gree of skill and experience in the identiﬁcation of the spe-

cies present in their waters and appropriate training should

be incorporated into their work programme. Access to key

literature for phytoplankton identiﬁcation, such as Horner

(2002), Tomas (1997) and Throndsen et al. (2003, 2007) is

essential. Access to older scientiﬁc literature is often necessary

for detailed species descriptions, however, these may be dif-

ﬁcult to access. Attendance at phytoplankton identiﬁcation

training courses when possible is the most successful way to

allow analysts to continue to learn and develop their skills.

This is especially important since the systematics and nomen-

clature of phytoplankton is constantly under revision. Species

lists and images of phytoplankton are presented in a variety

of web sites, see examples listed in Table 1. While a wealth of

information is available on the internet, they cannot replace

teaching and guidance from an experienced taxonomist.

Microscopes for phytoplankton identiﬁcation and

enumeration

A high quality microscope is essential for the enumeration

and identiﬁcation of phytoplankton species. Although the

initial cost will be high, a microscope, if serviced on a regular

basis, can remain in use for many years. Two types of mi-

croscopes are commonly used: (1) the standard compound

(upright) microscope and (2) the inverted microscope (Figs.

8 - 9). With the inverted microscope, the objectives are posi-

tioned underneath the stage holding the sample. This is nec-

essary for examination of samples in sedimentation chambers

and ﬂasks where the phytoplankton cells have settled onto the

bottom. Oculars should be ﬁtted with a graticule and a stage

micrometer is used to determine and calibrate the length of

the scale bars of the eyepiece graticule under each objective

magniﬁcation. In Fig. 10 examples of how Alexandrium fun-

dyense is viewed in the microscope using different micrsocope

and staining techniques are presented. The digital photo-

graphs were taken during a workshop comparing micrsocopic

a and molecular biological techniques for quantiative phyto-

plankton analysis. Results from the workshop are found in

Godhe et al. (2007).

Because many phytoplankton species are partially transpar-

ent when viewed under a light microscope, different tech-

Species information URL

AlgaeBase www.algaebase.org

World Register of Marine Species, WoRMS www.marinespecies.org

IOC-UNESCO Taxonomic Reference List of Harmful

Micro Algae

www.marinespecies.org/hab/index.php

European Register of Marine Species, ERMS www.marbef.org

Integrated Taxonomic Information System, ITIS www.itis.gov

Micro*scope starcentral.mbl.edu/microscope/

Plankton*net www.planktonnet.eu

Encyclopedia of Life www.eol.org

Gene sequences etc.

Genbank www.ncbi.nlm.nih.gov/Genbank/

European Molecular Biological Laboratory www.embl.org

National Center for Biotechnology Information www.ncbi.nlm.nih.gov

Table 1. Examples of web sites that provide useful information for phytoplankton analysts.

IOC Manuals & Guides no 55

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

Figure 2. Reversing water sampler of the modiﬁed Nansen type.

Figure 4. CTD with rosette and Niskin-type water bottles. An in situ

chlorophyll a ﬂuorometer is also mounted.

Figure 5. Phytoplankton net. This is not used for quantitative sampling

but for collecting rare, non fragile species.

Figure 6. Tube for integrated water sampling.

Figure 7. The Continuous Plankton Recorder. This device is mainly

aimed for sampling zooplankton but may be useful for collecting

larger, non fragile phytoplankton species. Photo courtesy of the Sir

Alister Hardy Foundation for Ocean Science, SAHFOS

http://www.sahfos.ac.uk/.

Figure 3. Water sampler of the Ruttner type.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

niques to improve contrast are used. Differential Interference

Contrast (DIC, also called Normarski) and Phase Contrast

are popular. DIC is considered by many to be the optimal

method for general phytoplankton analysis. Most plastic con-

tainers, however, cannot be used with this method as many

plastics depolarize the required polarized light. It is also more

expensive than Phase Contrast and requires a different set of

objectives, polarizing ﬁlters etc. to function properly.

Natural ﬂuorescence

Fluorescence generated from photosynthetic and other pig-

ments in phytoplankton can be used as an aid for the identi-

ﬁcation and enumeration of species. This works best with live

samples and samples preserved with formaldehyde or glutar-

aldehyde. If Lugols iodine is used for preservation, the natural

ﬂuorescence is not visble. Fluorescence can also be used to dif-

ferentiate between heterotrophic and autotrophic organisms.

The microscope must be equipped with objectives suitable for

ﬂuorescence, a lamp housing for ﬂuorescence (e.g. mercury

lamp 50 or 100 W), the required ﬁlter sets. A useful ﬁlter set

to observe ﬂuorescence from both chlorophyll a and phyco-

erythrin consists of a ﬁlter for excitation at 450-490 nm and a

long pass ﬁlter for emission at 515 nm.

Staining of cells

Different stains are used to aid the identiﬁcation of phyto-

plankton species. In this volume only ﬂuorescent stains (ﬂuor-

ochromes) are discussed. The stain used in chapters 2 and 5,

calcoﬂuor, binds to the cellulose theca in armoured dinoﬂag-

ellates and allows a detailed examination of the plate structure

to be performed. This stain is very useful when morphologi-

cally similar species, e.g. Alexandrium spp., are present. Fluor-

ochromes are also often used in connection with antibodies

or RNA targeted probes to identify phytoplankton. Some of

these are covered in chapter 9. It should be noted that some

microscope objective lenses do not transmit ultraviolet light

and are unsuitable for work with ﬂuorochromes that require

UV-light excitation, e.g. calcoﬂuor.

Image analysis

Manual phytoplankton analysis with microscopy may be time

consuming and analysts must possess the necessary skills to

allow the identiﬁcation of cells using morphological features.

This has led to interest in the use of automated image analysis

of phytoplankton samples. Basic image analysis methods do

not generally discriminate between phytoplankton and other

material such as detritus and sediment in samples thereby

presenting a problem in the application to routine ﬁeld sam-

ples. This technique may be more useful for the analysis of

cultures and monospeciﬁc high density blooms. Researchers

have tried more advanced methods such as artiﬁcial neural

networks (ANN) to identify species automatically by pattern

recognition. Some ANN software includes functions which

train the ANN to identify certain species. One such instru-

ment under development is the HAB Buoy, which uses the

Dinoﬂagellate Categorisation by Artiﬁcial Neural Network

(DICANN) recognition system software (Culverhouse et al.

2006). Other examples of software currently under evaluation

for automated phytoplankton identiﬁcation are used in Flow

Cytometers (see next paragraph), e.g. the FlowCAM (chapter

8) and the method described by Sosik and Olson (2007). To

date, these methods require a highly trained phytoplankton

identiﬁcation specialist to train the software to recognise the

images and carry out a quality control on the results of the

automated image analysis.

Flow cytometry

A ﬂow cytometer is a type of particle counter initially devel-

oped for use in medical science. Today instruments have been

developed for use speciﬁcally in aquatic sciences. Autoﬂuores-

cence and scattering properties are used to discriminate dif-

ferent types of phytoplankton. The different phytoplankton

groups are in general not well distinguished taxonomically

when a standard instrument is used. A standard ﬂow cytom-

eter is very useful to estimate abundance of e.g. autotrophic

picoplankton. A more advanced type of ﬂow cytometer has a

camera that produces images of each particle/organism. Auto-

mated image analysis makes it possible to identify organisms.

Manual inspection of images by an experienced phytoplank-

ton identiﬁcation specialist is required for quality control and

for training the automated image analysis system. A desk top

system is described in chapter 8. An example of an in situ

system is described by Sosik and Olsen (2007) and Olsen and

Sosik (2007).

Molecular techniques

Signiﬁcance of molecular based phytoplankton analysis

techniques

Owing to some of the difﬁculties and limitations of mor-

phological identiﬁcation techniques, microalgal studies are

increasingly exploring the use of molecular methods. Most

molecular techniques have their origin in the medical science,

and during the last three decades these various techniques

have been tested, modiﬁed, and reﬁned for the use in algal

identiﬁcation, detection and quantiﬁcation.

The development of molecular tools for the identiﬁcation and

detection of microalgae has inﬂuenced and improved other

ﬁelds of phycological research. Molecular data are gaining in-

ﬂuence when the systematic position of an organism is estab-

lished. Today, the description of new species, erection of new

genera, or rearrangement of a species to a different genus is

usually supported by molecular data in addition to morpho-

logical structures, ultrastructure, and information on biogeo-

graphic distribution (e.g. Fraga et al. 2008). Thus, the un-

derstanding of evolutionary relationships among microalgal

taxa has been immensely improved (Saldarriaga et al. 2001).

Spatially separated populations of microalgal species might

display different properties, such as toxin production. By

studying minor differences within the genome, populations

can be conﬁned to certain locations, and human assisted and/

or natural migration of populations can be investigated (e.g.

Persich et al. 2006, Nagai et al. 2007). Also, the increasing

information on the structure of genes and new tools for inves-

tigating their expressions, have enhanced our understanding

of algal physiological processes (Maheswari et al. 2009).

Laboratory requirements for molecular techniques

Different types of molecular techniques have very different

requirements for laboratory facilities and instruments. The

range is from very well equipped laboratories to ﬁeld instru-

ments. In chapters 9-14 examples of laboratory methods are

IOC Manuals & Guides no 55

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

found. In situ systems are under development (e.g. Paul et al.

2007 and Scholin et al. 2009).

Identiﬁcation and quantiﬁcation of phytoplankton species

by molecular methods

Molecular methodologies aim to move away from species

identiﬁcation and classiﬁcation using morphological charac-

teristics that often require highly specialist equipment such as

electron microscopes, or very skilled techniques such as single

cell dissections. Instead molecular techniques exploit differ-

ences between species at a genetic level. Molecular analysis

requires the use of specialised equipment and personnel and

most importantly requires a previous knowledge of the genet-

ic diversity of the phytoplankton in a speciﬁc region. To date,

molecular methods have been used to support HAB monitor-

ing programmes in New Zealand and the USA (Rhodes et al.

1998, Scholin et al. 2000, Bowers et al. 2006).

In this present manual, methods based on ribosomal RNA

(rRNA) and DNA (rDNA) targeted oligonucleotides and

polymerase chain reaction (PCR) are described. Oligonucle-

otides and PCR primers are short strains of synthetic RNA or

DNA that is complementary to the target RNA/DNA. Mo-

lecular sequencing of phytoplankton cells has generated DNA

sequence information from many species around the world.

This has allowed the design of oligonucleotide probes and

PCR primers for speciﬁc microalgal species. Some oligonu-

cleotide probes, which hybridize with complementary target

rRNA or rDNA, have a ﬂuorescent tag attached and can act

as a direct detection method using ﬂuorescence microscopy.

PCR primers enable the ampliﬁcation of target genes through

PCR. The primers serve as start and end points for in vitro

DNA synthesis, which is catalysed by a DNA polymerase.

The PCR consists of repetitive cycles, where in the ﬁrst step,

DNA is heated in order to separate the two strands in the

DNA helix. In the second step during cooling, the primers,

which are present in large excess, are allowed to hybridize with

the complementary DNA. In a third step, the DNA polymer-

ase and the four deoxyribonucleoside triphosphates (dNTPs)

complete extension of a complementary DNA strand down-

stream from the primer site. For effective DNA ampliﬁca-

tion, the three steps are repeated in 20-35 cycles (Alberts et

al. 1989). A useful volume covering the basics of molecular

methods and general applications is Molecular Systematics

edited by Hillis et al. (1996).

Most of the molecular methods described here, with the ex-

ception of the whole cell assay (chapter 9 and 14), do not

require the cells to remain intact. In these methods the rRNA

molecules in the cell’s cytoplasm or the nuclear DNA are re-

leased during nucleic acid extraction and are targeted by the

probes or PCR primers. During the whole cell assay, the target

rRNA/rDNA within intact cells is labelled with ﬂuorescently

tagged probes. It is therefore vital that the laboratory protocol

used ensures that the probes can penetrate the cell wall in

order to access target genetic region and label them. Tyramide

Signal Ampliﬁcation has been used with FISH (TSA-FISH)

to further enhance ﬂuorescence signals (see chapter 14). The

ﬂuorescent tag can then be read using a ﬂuorescent micro-

scope as with the whole cell assays (FISH chapter 9) or addi-

tional technology is employed to allow these ﬂuorescent tags

to be read automatically e.g. using a sandwich hybridization

technique (chapter 12) and PCR (chapter 13).

The hand held device and DNA-biosensor with disposable

sensorchip (sandwich hybridisation, electrochemical detec-

tion) and DNA microarray technology (ﬂuorescent detec-

tion) methods discussed in this manual are still at the ﬁnal

development stages (see chapters 10 and 11). Within the

next decade these methods may be ready to be incorporated

into monitoring programmes. The authors suggest that fu-

ture advances in this ﬁeld will include microarray/DNA chip

(sometimes called “phylochips”) technologies with probes for

multiple species applied in situ to an environmental sample

simultaneously.

Alternative molecular based methods such as lectin (protein

and sugar) binding and antibody based assays (e.g. immuno-

ﬂuorescence assays) are not included in this manual. Infor-

mation on these molecular diagnostic tools may be found

in chapter 5 of The Manual on Harmful Marine Microalgae

(Hallegraeff et al. 2004).

Molecular method validation

rDNA and rRNA have become the most popular target re-

gions for microalgal species identiﬁcation. These regions are

attractive for primer and probe design because they contain

both conserved and variable regions and are ubiquitous in

Figure 8 Compound microscope

Figure 9. Inverted microscope

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 1 Introduction to methods for quantitative phytoplankton analysis

all organisms. In addition, a large number of sequences are

available in molecular web based databases, e.g. GENBANK,

for sequence comparative analyses (Table 1) and design of

oligonucleotide probes and PCR primers. Despite extensive

sequence analysis of cultured phytoplankton species, cross

reactivity with other organisms in the wild may occur, it is

therefore crucial to test the developed probes/primers with

the target species and several non-target species. Method

development, although time consuming, is essential if these

methods are to be implemented. It is the responsibility of the

end user to ensure that speciﬁcity to the target organism is

evaluated appropriately.

Quality control

As with all scientiﬁc research, it is necessary to investigate the

variability of the methods used before employment into any

monitoring programme. The variability of the result can be

affected by cell abundance which can dictate the method of

choice. Further information on this can be found in chapter

2 and of Venrick (1978 a,b,c) and Andersen and Throndsen

(2004). Many laboratories have achieved national accredita-

tion for techniques described in this manual. This involves

developing protocols with levels of traceability and reproduc-

ibility in line with deﬁned criteria. Participation in interna-

tionally recognised inter-laboratory comparisons are strongly

recommended.

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tion of marine phytoplankton, C. R. Biologies 332: 159-170

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Verh. int. Ver. theor. angew. Limnol. 5: 567-596.

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Phytoplankton manual. Unesco, Paris, pp. 7-16.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 2 The Utermöhl method

2 The Utermöhl method for quantitative phytoplankton analysis

Lars Edler*

and Malte Elbrächter

WEAQ, Doktorsgatan 9 D., SE-262 52 Ängelholm, Sweden

Deutsches Zentrum für Marine Diversitätsfor-schung Forschungsinstitut Senckenberg, Wattenmeerstation Sylt, Hafenstr. 43, D-25992

List/Sylt, Germany

*Author for correspondence: e-mail [email protected]

Introduction

The Utermöhl method (Utermöhl 1931, 1958) has an ad-

vantage over other methods of phytoplankton analysis in that

algal cells can be both identiﬁed and enumerated. Using this

method, it is also possible to determine individual cell size,

form, biovolume and resting stage.

The Utermöhl method is based on the assumption that cells

are poisson distributed in the counting chamber. The method

is based on the sedimentation of an aliquot of a water sample

in a chamber. Gravity causes the phytoplankton cells to settle

on the bottom of the chamber. The settled phytoplankton

cells can then be identiﬁed and enumerated using an inverted

microscope. To quantify the result as cells per Litre a conver-

sion factor must be determined.

Materials

Equipment

Sample Bottles

If samples are analysed immediately or within a few days

plastic vials may be used. Note that the preservatives may be

absorbed by the plastic. For long term storage, glass sample

bottles should be used to minimise any chemical reaction

with the preservative. Clear glass bottles allow the state of

Lugol’s iodine preservation to be easily monitored (Fig. 1).

These samples must be stored in the dark to prevent the de-

gradation of Lugol’s iodine in light. It is important that the

bottle cap is securely tightened to avoid spillage of the sample

and evaporation of the preservative. Utermöhl (1958) recom-

mended that the bottle is ﬁlled to 75-80% of its volume. This

facilitates the homogenisation of the sample before dispensing

into the sedimentation chamber.

Preservation agents

Preservation agents must be chosen depending on the objec-

tive of the study. The most commonly used is potassium iodi-

ne; Lugol’s iodine solution – acidic, neutral or alkaline (Table

1; Andersen and Throndsen 2004). If samples are stored for

long periods they may be preserved with neutral formalde-

hyde (Table 2).

Sedimentation chambers

The sedimentation chamber consists of two parts, an upper

cylinder (chimney) and a bottom plate with a thin glass (Fig.

2). They are usually made of perspex in volumes of 2, 5, 10,

25 or 50 mL. The thickness of the glass base plate should not

exceed 0.2 mm, as this will affect the resolution achievable

by the microscope. Counting chambers should be calibrated.

This is achieved by ﬁrst weighing the chamber while empty

and then ﬁlled with water to conﬁrm the volume.

The inverted microscope

For quantitative analysis using sedimentation chambers, an

inverted microscope is required (Fig. 3). The optical quality of

the microscope is crucial for facilitating phytoplankton iden-

tiﬁcation. Phase- and/or differential interference-contrast is

helpful for the identiﬁcation of most phytoplankton, whereas

bright-eld may be advantageous for coccolithophorids (He-

imdahl 1978).

Epiﬂuorescence equipment is a great advantage for counting

and identiﬁcation of organisms with cellulose cell walls, e.g.,

thecate dinoﬂagellates, chlorophytes and “fungi”. A stain is

applied to the sample which causes cellulose to ﬂuoresce.

One eyepiece should be equipped with a calibrated ocular

micrometer. The other eyepiece should be equipped with

two parallel threads forming a transect. A third thread per-

pendicular to the other two facilitates the counting procedure

(Fig. 4 a). It is also possible to have the eyepiece equipped

with other graticules such as a square ﬁeld or grids (Fig. 4

b). The eyepiece micrometer and counting graticule must be

calibrated for each magniﬁcation using a stage micrometer.

Acidic Alkaline Neutral

20 g potassium iodide (KI) 20 g potassium iodide (KI) 20 g potassium iodide (KI)

10 g iodine (I

) 10 g iodine (I

)

20 g conc. acetic acid 50 g sodium acetate 200 mL distilled water

200 mL distilled water 200 mL distilled water

Table 1. Recipes for Lugol’s iodine solution (acidic, alkaline and neutral).

(from: Utermöhl 1958, Willén 1962, Andersen and Throndsen, 2004).

Table 2. Recipe for neutral formaldehyde. (from: Throndsen 1978,

Edler 1979, Andersen and Throndsen 2004). Filter after one week

to remove any precipitates.

Neutral formaldehyde

500 mL 40% formaldehyde

500 mL distilled water

100 g hexamethylentetramid

pH 7.3 – 7.9

IOC Manuals & Guides no 55

Chapter 2 The Utermöhl method

Scope

Qualitative and quantitative analysis of phytoplankton.

Detection range

Detection range is dependent on the volume of sample settled.

Counting all of the cells in a 50 mL chamber will give a detec-

tion limit of 20 cells per Litre.

Advantages

Qualitative as well as quantitative analysis. Identiﬁcation and

quantiﬁcation of muliple or single species. Detection of harm-

ful species.

Drawbacks

This is a time consuming analysis that requires skilled person-

nel. Sedimentation time prevents the immediate analysis of

samples. Autotrophic picoplankton is not analysed using the

Utermöhl method.

Type of training needed

Analysis requires continuous training over years with in-depth

knowledge of taxonomic literature.

Essential Equipment

Inverted microscope, sedimentation chambers, microscope

camera, identiﬁcation literature, (epiﬂuorescense equipment,

counting programme).

Equipment cost*

Inverted microscope: 7,500 – 50,000 € (11,000 – 70,000 US $).

Sedimentation chamber: 150 € (200 US$).

Microscope camera: 3,000 – 8,000 € (4,300 – 11,000 US $).

Identiﬁcation literature: 1,000 – 3,000 € (1,400 – 4,300 US $).

Epiﬂuorescense equipment: 10,000 € (14,000 US $).

Counting programme: 500 – 5,000 € (700 – 7,000 US $).

Consumables, cost per sample**

Less than 5 €/4 US $.

Processing time per sample before analysis

App. 10 minutres for ﬁlling and assembling sedimentation

chamber.

3-24 hours sedimtation time depending on volume and analysis

type.

Analysis time per sample

2-10 hours or more depending on type of sample and analysis.

Sample throughput per person per day

1-4 depending on type of sample and analysis.

No. of samples processed in parallel

One per analyst.

Health and Safety issues

Analysis sitting at the microscope is tiresome for eyes, neck and

shoulder. Frequent breaks are needed. If formalin is used as pre-

servation agent appropriate health and safety guidelines must

be followed.

*service contracts not included

**salaries not included

The fundamentals of

The Utermöhl method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 2 The Utermöhl method

The microscope should have objectives of 4-6X, 10X, 20X

and 40-60X. For detailed examination a 100X oil immersion

objective may also be used. If epiﬂuorescence microscopy is

to be used, the microscope must be equipped with the appro-

priate objective lenses. In order to survey the entire bottom

plate the microscope must be equipped with a movable me-

chanical stage.

Cell counters

A cell counter with 12 or more keys is a useful device. Medical

blood cell counters (Fig. 5) are commonly used. If these are

not availabe single tally counters can be used as appropriate. It

is also common to have a computerised counting programme

(Fig. 6) beside the microscope, so that the observed species are

registered directly into a database.

Laboratory facilities

Laboratory facilities necessary for the quantitative analysis

of phytoplankton require amenities for storing, handling

(mixing and pouring samples) and washing of sedimentation

chambers. Preserved samples should be stored in cool and

dark conditions. During sedimentation the chambers should

be placed on a level, horizontal and solid surface. This will

prevent any non random accumulation of phytoplankton cells.

Methods

Preparation of sample

Preservation

Once the sample has been collected from the ﬁeld and poured

into the sample bottle it should be immediately preserved

using either:

Lugol’s iodine solution;

0.2 – 0.5 mL per 100 mL water sample.

Neutralised formaldehyde;

2 mL per 100 mL water sample.

The advantage of Lugol’s iodine solution is that it has an in-

stant effect and increases the weight of the organisms redu-

cing sedimentation time. Lugol’s iodine solution will cause

discolouration of some phytoplankton making identiﬁcation

difﬁcult. To reduce this effect, the sample can be bleached us-To reduce this effect, the sample can be bleached us-

ing sodium thiosulfate prior to analysis.

The advantage of formaldehyde is that preserved samples re-

main viable for a long time. Formaldehyde is not suitable for

ﬁxation of naked algal cells, as the cell shape is distorted and

ﬂagella are lost. Some naked algal forms may also disintegra-

te when formaldehyde is used (CEN 2005). Formaldehyde

should be used with care because of its toxicity to humans

(Andersen and Throndsen 2004).

Figure 1. Sample bottles: glass and plastic. Bottle of Lugol’s iodine

solution to the right.

Figure 2. Sedimentation chambers. From left to right: bottom plate

with cover glass, 10 mL chamber, 25 mL chamber and 50 mL

chamber.

Figure 3. Inverted microscope.

Figure 4. Counting aids mounted in the eyepiece. a) parallel

threads, with a transverse thread. b) grids.

A B

IOC Manuals & Guides no 55

Chapter 2 The Utermöhl method

Storage of samples

Preserved phytoplankton samples should be stored in cool

and dark conditions. When using Lugol’s iodine solution, the

colour of the sample should be checked regularly and if neces-

sary, more preservative added. Preserved samples should be

analysed without delay. Samples stored more than a year are

of little use (Helcom Combine 2006).

Temperature adaptation

The ﬁrst step in the analysis procedure is to adapt the

phytoplankton sample and the sedimentation chamber to

room temperature. This prevents convection currents and air

bubbles forming in the sedimentation chamber. If this is not

carried out non-random settling of the phytoplankton cells

may occur.

Chamber preparation

Sediment chambers must be clean and dust free to avoid con-

tamination from previous samples. Many laboratories use a

new base plate after every sample. Sometimes it is necessary

to grease the chimney bottom with a small amount of vaseline

to ensure the chamber parts are tightly sealed (Andersen and

Throndsen 2004).

In studies where the succession of the phytoplankton is exa-

mined over a period of time it is important to use the same

chamber volume for the analysis (Hasle 1978a). At times, the

“standard” chamber size may be either too small (extreme

winter situations) or too large (phytoplankton blooms) and

another chamber size must be used.

Sample homogenisation

Before the sample is poured into the sedimentation chamber,

the bottle should be shaken ﬁrmly, but gently, in irregular

jerks to homogenise the contents. Violent shaking will pro-

duce bubbles, which can be difﬁcult to eliminate. A rule of

thumb is to shake the bottle at least 50 times. It is recom-It is recom-

mended to check the homogenous distribution a couple of

times per year by counting 3 subsamples from the same stock-

sample.

Concentration/dilution of samples

Although it is possible to concentrate and dilute samples that

are either too sparse or too dense it is not recommended as

all additional handling steps may interfere with the sample

contents. Instead it is recommended that a sediment chamber

of an appropriate size be used to allow accurate identiﬁcation

and enumeration of cells.

Filling the sedimentation chamber

After homogenisation, the sedimentation chamber is placed

on a horizontal surface and gently ﬁlled from the sample

bottle (Fig. 7a and 7b). The chamber is then sealed with a

cover glass. It is important that no air bubbles are left in the

chamber. It may be necessary to grease the cover glass with a

little vaseline to maintain a tight seal.

Sedimentation

The sedimentation should take place at room temperature

and out of direct sunlight. In order to minimise evaporation

the sedimentation chamber may be covered with a plastic box

and a Petri dish containing water should be placed beside the

chamber (Fig. 8). Settling time is dependent on the height

of the chamber and the preservative used (Lund et al. 1958,

Nauwerck 1963). Recommended settling times for Lugol’s

preserved samples are shown in Table 3. According to Hasle

(1978a) formaldehyde preserved samples need a settling time

of up to 40 hours independent of chamber size.

After sedimentation the chimney of the sedimentation cham-

ber is gently slid off from the bottom plate and replaced by a

cover glass. Care should be taken not to introduce airbubbles

at this stage (Fig. 9). The transfer of the bottom plate to the

microscope will not affect the distribution of the settled phy-

toplankton cells if there are no air bubbles present. The bot-

tom plate is placed on the inverted microscope (Fig. 10) and

the phytoplankton cells are identiﬁed and counted.

Figure 5. Laboratory cell counter.

Figure 6. Computerised counting programme.

Table 3. Recommended settling times for Lugol’s iodine preserved

samples (from Edler 1979).

Chamber volume

(mL)

Chamber height

approx. (cm)

Settling time (hr)

2 1 3

10 2 8

25 5 16

50 10 24

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 2 The Utermöhl method

Counting procedure

The quantitative analysis should start with a scan of the entire

chamber bottom at a low magniﬁcation. This will help to give

an overview of the density and distribution of phytoplankton.

If the distribution is considered uneven the sample must be

discarded. During this scan it is also convenient to make a

preliminary species list, which may help to select the counting

strategy.

Organisms should be identiﬁed to the lowest taxonomic le-

vel that time and skill permits (Hasle 1978b). Ultimately the

objective of the study will decide the level of identiﬁcation

accuracy.

Counting begins at the lowest magniﬁcation, followed by ana-

lysis at successively higher magniﬁcation. For adequate com-

parison between samples, regions and seasons it is important

to always count the speciﬁc species at the same magniﬁcation.

In special situations, such as bloom conditions, however, this

may not be possible. Large species which are easy to identify

(e.g. Ceratium spp.) and also usually relatively sparse can be

counted at the lowest magniﬁcation over the entire chamber

bottom. Smaller species are counted at higher magniﬁcations,

and if needed, only on a part of the chamber bottom. In Table

4, the recommended magniﬁcations for different phytoplank-

ton sizes are listed.

Counting the whole chamber bottom is done by traversing

back and forth across the chamber bottom. The parallel ey-

epiece threads delimit the transect where the phytoplankton

are counted (Fig. 11).

Counting part of the chamber bottom can be done in diffe-

rent ways. If half the chamber bottom is to be analysed every

second transect of the whole chamber is counted. If a smaller

part is to be analysed one, two, three or more diameter tran-

sects are counted. After each transect is counted the chamber

is rotated 25-45

(Fig. 12).

When counting sections of the chamber using transects it

is important to be consistent as to which cells lying on the

border lines are to be counted. The easiest way is to decide

that cells lying on the upper or right line should be counted,

whereas cells on the lower or left line should be omitted.

In order to obtain a statistically robust result from the quanti-

tative analysis it is necessary to count a certain number of

counting units (cells, colonies or ﬁlaments). The precision

Table 4. Recommended magniﬁcation for counting of different size

classes of phytoplankton (Edler, 1979, Andersen and Throndsen

2004).

Size class Magniﬁcation

0.2 – 2.0 µm (picoplankton)* 1000 x

2.0 – 20.0 µm (nanoplankton) 100 – 400 x

>20.0 µm (microplankton) 100 x

* picoplankton are normally not analysed using

the Utermöhl method.

Figure 8. Sedimentation, with a Petri dish ﬁlled with water. A

plastic box covers the sedimentation chamber and the Petri dish to

maintain the humidity.

Figure 9. Replacing the sedimentation chimney with a cover glass.

Figure 10. Chamber bottom placed in microscope ready for

analysis.

Figure 7A and 7B. Filling of sedimentation chamber.

IOC Manuals & Guides no 55

Chapter 2 The Utermöhl method

desired decides how many units to count. The precision is

usually expressed as the 95% conﬁdence limit as a propor-

tion of the mean. Table 5 and Figure 13 show the relationship

between number of units counted and the accuracy. In many

studies it has been decided that counting of 50 units of the

dominant species, giving a 95% conﬁdence limit of 28% is

sufﬁcient. Increasing the precision to e.g. 20% or 10% would

need a dramatic increase in counted units, 100 and 400 re-

spectively (Venrick 1978, Edler 1979). The precision is given

by the following equation:

It is clear that it will not be possible to count 50 units of all

species present in a sample. Some species may not be sufﬁ-

cently abundant which will decrease the overall precision. To

maintain an acceptable precision for the entire sample a total

of at least 500 units should be counted (Edler 1979).

The counting unit of most phytoplankton species is the cell.

In some cases this is not practical. For ﬁlamentous cyano-

bacteria, for instance, the practical counting unit is a certain

length of the ﬁlament, usually 100 µm (Helcom Combine

2006). In some colony forming species and coenobia it may

be difﬁcult to count the individual cells. In such cases the co-

lony/coenobium should be the counting unit. If desired, the

calculation of cells per colony/coenobium can be approxima-

ted by a thorough counting and mean calculation of a certain

number of colonies/coenobia.

The transformation of the microscopic counts to the concen-

tration or density of phytoplankton of a desired water volume

(usually Litre or millilitre) can be achieved using this equa-

tion:

V: volume of counting chamber (mL)

: total area of the counting chamber (mm

)

: counted area of the counting chamber (mm

)

N: number of units (cells) of speciﬁc species counted

C: concentration (density) of the speciﬁc species

Table 5. Relationship between number of cells counted and

conﬁdence limit at 95% signiﬁcance level (Edler 1979, Andersen

and Throndsen 2004).

No of counted

cells

Conﬁdence limit

+/- (%)

Absolute limit if cell

density is estimated at

500 cells L

-1

1 200 500 ± 1000

2 141 500 ± 705

3 116 500 ± 580

4 100 500 ± 500

5 89 500 ± 445

6 82 500 ± 410

7 76 500 ± 380

8 71 500 ± 355

9 67 500 ± 335

10 63 500 ± 315

15 52 500 ± 260

20 45 500 ± 225

25 40 500 ± 200

50 28 500 ± 140

100 20 500 ± 100

200 14 500 ± 70

400 10 500 ± 50

500 9 500 ± 45

1000 6 500 ± 30

Figure 12. Counting of diameter transects.

Figure 11. Counting of the whole chamber bottom with the parallel

eyepiece threads indicating the counted area.

Figure 13. Relationship between number of cells counted and

conﬁdence limit at the 95% signiﬁcance level.

100

125

150

175

200

0 50 100 150 200 250 300 350 400 450 500

no of counted cells

Confidence limit (%)

countedcellsofnumber

100*2

%Precision =

V A

N mLCells

* *















−

V A

N LCells

1000

* *













−

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 2 The Utermöhl method

Cleaning of sedimentation chambers

The cleaning of sedimentation chambers is a critical part of

the Utermöhl method. The chambers should be cleaned im-

mediately after analysis to prevent salt precipitate formation.

A soft brush and general purpose detergent should be used

(Edler 1979, Tikkanen and Willén 1992). To clean the cham-

ber margin properly a tooth pick can be used. Usually it is

sufﬁcient to clean the chamber bottom without dissembling

the bottom glass. Sometimes, however, it is necessary to sepa-

rate the bottom glass from the chamber, either to clean it or

to replace it. This is easily done by loosening the ring holding

the bottom glass with the key. Care should be taken as the

bottom glasses are very delicate. Counting chambers should

be checked regularly to ensure that no organisms stick to the

bottom glass. This can be achieved by ﬁlling the chambers

with distilled water.

Quality assurance

To ensure high quality results all steps of the method must be

validated. Ideally this is performed on natural samples, but

in some instances it may be helpful to spike the sample with

cultured algae. Steps in the Utermöhl method to validate are

• homogenisation of sample

• sedimentation/sinking

• distribution on chamber bottom

• repeatability and reproducibility

Ultimatley the quality of the result from this method is de-

pendent on the skill of the analyst. The variation of paral-

lel samples counted by the same analyst and the variation in

parallel samples counted by different analysts are two of the

most important considerations in quality assurance (Willén

1976). When possible laboratories should take part in interla-

boratory comparisons.

Epiﬂuorescence microscopy

Epiﬂuorescence microscopy is an effective method to enhance

detection and identiﬁcation of certain organisms (Fritz and

Triemer 1985, Elbrächter 1994). In formalin ﬁxed samples,

autoﬂuorescence of the chlorophyll can easily be detected by

epiﬂuorescence. This will be specially important among di-

noﬂagellates and euglenids, in which both phototrophic and

obligate heterotrophic genera/species are present. Phycobilins

of cyanobacteria, rhodophytes and cryptophytes have a spe-

cial autoﬂuorescence, thus this method is particularly suited

to detect and count cryptophytes and small coccoid cyano-

bacteria. In addition, staining of organisms can help to en-

hance counting effort and identiﬁcation of certain organisms.

Applying this method, the inverted microscope should have

an epiﬂuorescence equipment. The lenses should be suitable

for ﬂuorescence microscopy. For the respective excitation ﬁl-

ter and barrier ﬁlter to be used to detect the different epiﬂuo-

rescence emissions, the supplier of the respective microscope

should be contacted. Some information on ﬁlter combina-

tions is provided by Elbrächter (1994). A common method

is to induce epiﬂuorescence in organisms with cellulose cell

walls (e.g. thecate dinoﬂagellates, chlorophytes, “fungi” and

others) by Fluorescent Brightener (Fritz and Triemer 1985).

Protocol for staining and use of epiﬂuorescence

• Prepare a 0.1% stock solution of Fluorescent Brightener.

• The ﬂuorescent brightener solution should be added to

the sedimentation chamber before ﬁlling it with the sam-

ple. The ﬁnal concentration should be 0.02 %.

• Switch on the mercury lamp for about 10 min. before

starting to analyse the sample.

• Use Exitation Filter BP 390-490 and Barrier Filter LP

515 or ﬁlters recommended by the microscope brand.

This will give dinoﬂagellate thecae a clear intensive blue epi-

ﬂuorescence including the sutures of the plates (Fig. 14). Oth-

er cellulose items like chlorophyte cell walls, cell walls of fungi

parasitising in diatoms etc. will also ﬂuoresce.

Note that the intensity of epiﬂuorescence is pH dependent, in

acidic samples epiﬂuorescence is absent or poor.

Discussion

The Utermöhl method for the examination of phytoplankton

communities is probably the most widely used method for

the quantitative analysis of phytoplankton. Through the years

both microscopes and sedimentation chambers have develo-

ped considerably, yet it is the taxonomic skill of the analyst

that sets the standard of the results.

The Utermöhl method determines both the quantity and di-

versity of phytoplankton in water samples. Moreover, with

only a little extra effort, the biovolume of the different species

can also be elucidated. The method allows very detailed anal-

ysis and with high quality lenses the resolution of phytoplank-

ton morphology can be very good. The Utermöhl method has

some disadvantages. It is very time consuming and thus also

very costly. In order to achieve reliable results the analyst has

to be skilled, with a good knowledge of the taxonomic litera-

ture. It is commonly agreed that analysts take some years to

train and must then keep up to date with the literature.

Figure 14. Alexandrium ostenfeldii, epiﬂuorescence light micros-

copy, stained with Fluorescent Brightener. Note the clear indication

of the sutures and the large ventral pore, characteristic for this

species.

IOC Manuals & Guides no 55

Chapter 2 The Utermöhl method

References

Andersen P, Throndsen, J (2004) Estimating cell numbers. In Hal-

legraeff, GM, Anderson DM, Cembella AD (eds) Manual on

Harmful Marine Microalgae. Monographs on Oceanographic

Methodology no. 11. p. 99-130. UNESCO Publishing

CEN/TC 230 (2005) Water quality — Guidance on quantitative

and qualitative sampling of marine phytoplankton. pp. 26.

Edler L (1979) Recommendations on methods for Marine Biologi-

cal Studies in the Baltic Sea. Phytoplankton and Chlorophyll. Bal-

tic Marine Biologists Publication No. 5, p 38

Elbrächter, M, 1994. Green autoﬂuorescence – a new taxonomic

feature for living dinoﬂagellate cysts and vegetative cells. Rev. Pal-

aeobot. Palynol. 84: 101-105.

Fritz, L., Triemer, RE 1985. A rapid simple technique using cal-

coﬂuor White M2R for the study of dinoﬂagellate thecal plates. J.

Phycol. 21: 662-664.

Hallegraeff GM, Anderson, DM, Cembella AD (2004) Manual on

Harmful Marine Microalgae. Unesco Publishing, Paris. ISBN 92-

3-103871-0

Hasle GR (1978a) The inverted-microscope method. In: Sournia,

A. (ed.) Phytoplankton manual. UNESCO Monogr. Oceanogr.

Method. 6: 88-96

Hasle GR (1978b) Identiﬁcation problems. General recommen-

dations. In: Sournia A (ed.):Phytoplankton manual. UNESCO

Monogr. Oceanogr. Method. 6: 125-128

Heimdal BR (1978) Coccolithophorids. In: Sournia A (ed.):

Phytoplankton manual. UNESCO Monogr. Oceanogr. Method.

6: 148-150

HELCOM Combine Manual, 2006. http://www.helcom.ﬁ/Monas/

CombineManual

Lund JWG., Kipling C, Le Cren ED. (1958) The inverted micro-

scope method of estimating algal numbers and the statistical basis

of estimations by counting. Hydrobiologia 11:2, pp. 143-170

Nauwerck A (1963) Die Beziehungen zwischen Zooplankton und

Phytoplankton im See Erken. Symb. Bot. Ups. 17(5): 1-163

Throndsen J (1978) Chapter 4. Preservation and storage. In: Sour-

nia A (ed.): Phytoplankton manual. UNESCO Monogr. Ocea-

nogr. Method. UNESCO. 6: 69-74

Tikkanen T, Willén T (1992) Växtplanktonﬂora. Naturvårdsverket.

ISBN 91-620-1115-4. pp. 280

Utermöhl H (1931) Neue Wege in der quantitativen Erfassung des

Planktons. (Mit besonderer Berücksichtigun des Ultraplanktons).

Verh. Int. Ver. Theor. angew. Limnol. vol. 5, no. 2. p. 567-596

Utermöhl H (1958) Zur Vervollkommnung der quantitativen

Phytoplankton-Methodik. Mitt int. Verein. theor. angew. Lim-

nol. 9: 1-38

Venrick EL (1978) How many cells to count? In: Sournia A (ed.):

Phytoplankton manual. UNESCO Monogr. Oceanogr. Method.

6: 167-180

Willén E (1976) A simpliﬁed method of phytoplankton counting.

British Phycology Journal. 11:265-278

Willén T (1962) Studies on the phytoplankton of some lakes con-

nected with or recently isolated from the Baltic. Oikos. 13: 169-199

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 3 Settlement bottle method

3 Settlement bottle method for quantitative

phytoplankton analysis

Georgina McDermott*

and Robin Raine

Environmental Protection Agency, (Castlebar), Co. Mayo, Ireland

Martin Ryan Institute, National University of Ireland, Galway, Ireland

*Author for correspondence: G.McDer[email protected]

Introduction

The settlement bottle technique is a modiﬁed Utermöhl tech-

nique (Hasle 1978) for quantifying phytoplankton. It relies

on the observation and enumeration of phytoplankton cells

after sedimentation using an inverted microscope. It differs

from other similar methodologies as once a sample has been

taken and is preserved, there is no further requirement for

any more sub-sampling or manipulation. Once a water sam-

ple has been taken, it is transferred directly into a plastic tis-

sue culture ﬂask, a preservative is added and the sample is

stored until analysis. For counting, the contents of the ﬂask

are gently shaken, allowed to settle onto one of the ﬂat sides

of the ﬂask which is then placed directly onto an inverted

microscope. As with other Utermöhl methods, it requires the

skills of an analyst experienced in the identiﬁcation of phyto-

plankton cells.

Materials

Laboratory facilities

The settlement bottle method is simple in that all it requires

are tissue culture ﬂasks (acting as sample jars and counting

chambers), some preservative and an inverted microscope.

Thus all that is required is a room, preferably without direct

sunlight, with enough space and a power supply to use an

inverted microscope.

Equipment

Tissue culture bottles of 50-60 mL capacity are used. Larger

volume bottles are difﬁcult to place on a microscope and suf-

fer from movement of water inside them during examination.

Smaller sized bottles will probably not contain enough sam-

ple. The bottles should be rectangular in shape, as opposed to

having a triangular form, as this makes the calculation at the

end much simpler.

The base of the tissue culture bottles are examined using an

inverted microscope. This should be equipped with 10X, 20X

and 40X objective lenses. The 20X and 40X lenses should

have a long focal length. The technique can be applied using a

choice of either brightﬁeld or phase contract microscopy. Dif-

ferential interference contrast (DIC) and epiﬂuorescence mi-

croscopy are unsuitable. A specialised plate for secure mount-

ing of the settlement bottle onto the stage of the inverted

microscope may need to be manufactured. See Appendix for

examples of equipment.

Chemicals and consumables

The only chemicals that are required are solutions of preserva-

tive. Lugol’s iodine, neutral formalin or glutaraldehyde are all

suitable.

Methods

Preparation of sample

Before taking a sample, a tissue culture bottle should be la-

belled with appropriate information (Date, location, station

number, depth) with a permanent marker pen. Labels should

be written on the edge or narrow side of the bottle, not on the

broad side (see Fig. 1) so that it does not interfere with the

identiﬁcation and enumeration of cells.

The tissue culture bottle should be ﬁlled to the top with the

water sample, leaving just enough air space to add preserva-

tive. This prevents the introduction of large air bubbles which

can degrade the optical path when examining through or near

the edge of the bubble.

Before the sample is allowed to settle it should be wiped clean

and acclimated to room temperature for 24 hours and then

gently shaken to disrupt any aggregation of phytoplankton

cells. This can be achieved by a combination of horizontally

rolling and vertically turning the sample bottle upside down

as gently as possible to prevent the break-up of colonies and

the accumulation of air bubbles. The contents of the tissue

culture ﬂask should be allowed to settle for a period of at least

six hours.

Analysis of sample

The phytoplankton cells can then be counted using an in-

verted microscope. The entire base of the bottle or a number

of strips, going across the length or width of the bottle, can be

examined and the cells of each species or type are scored (see

Fig. 2 for details).

Preservation and storage

As with all samples of phytoplankton, deterioration is ex-

tremely fast in direct sunlight. All samples should therefore

be stored in the dark, stacked, preferably horizontally, to

minimise storage space. Samples should also be kept cool, al-

though refrigeration is not absolutely necessary. Storage over

long periods of time (months) is not as effective using Lugol’s

iodine, as opposed to formalin or glutaraldehyde, as leaching

of iodine into the plastic will occur which will deteriorate the

quality of microscopic observations.

IOC Manuals & Guides no 55

Chapter 3 Settlement bottle method

Scope

Qualitative and quantitative analysis of phytoplankton.

Detection range

Detection range is dependent on the volume of sample settled

and the number of strips analysed. Counting over the base of

a 50 mL settlement bottle will give a detection limit of ca. 20

cells per Litre.

Advantages

Avoidance of errors arising from sub sampling. Qualitative as

well as quantitative analysis. Identiﬁcation and quantiﬁcation

of multiple or single species. Detection of harmful species. Se-

diment bottles are available at low cost.

Drawbacks

Optical resolution is reduced due to the thickness of the wall of

the settlement bottles. This is a time consuming analysis that

requires skilled personnel. Sedimentation time prevents the im-

mediate analysis of samples. DIC and epiﬂuorescence micros-

copy are not suitable with this method. Special long distance

objectives must be used at higher magniﬁcations.

Type of training needed

Analysis requires continuous training over years with in-depth

knowledge of taxonomic literature.

Essential Equipment

Inverted microscope, settlement bottles, identiﬁcation litera-

ture.

Equipment cost*

Inverted microscope: 7,500 – 50,000 € (11,000 – 70,000 US $).

Identiﬁcation literature: 1,000 – 3,000 € (1,400 – 4,300 US $).

Consumables, cost per sample**

Less than 5 €/4 US $.

Processing time per sample before analysis

A minimum of 6 hours sedimentation time for a 50 mL sett-

lement bottle.

Analysis time per sample

2-10 hours or more depending on type of sample and analysis.

Sample throughput per person per day

1-4 depending on type of sample and analysis.

No. of samples processed in parallel

One per analyst.

Health and Safety issues

Analysis sitting at the microscope is tiresome for eyes, neck and

shoulder. Frequent breaks are needed. If formalin is used as pre-

servation agent appropriate health and safety guidelines must

be followed.

*service contracts not included

**salaries not included

The fundamentals of

The settlement bottle method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 3 Settlement bottle method

Calculation of results

Once a count of a particular species or type has been com-

pleted, this must be multiplied by a conversion factor (in

this case F) to calculate the cell density per unit volume. This

type of conversion applies to all sedimentation techniques for

enumerating phytoplankton cells. The general formula for

achieving a value of F to derive the cell density in cells per

Litre is:

The value of F will vary when counting in strips along the

bottle. This will depend on the magniﬁcation of the objective

lens, the number of strips across the bottle which were exam-

ined, and whether the strips were oriented along or across the

bottle. The width of the ﬁeld of view (i.e. the width of each

strip) must be predetermined using a calibrated graticule at

each magniﬁcation. If one is scanning the long side of the bot-

tle, the length of the short side across the bottle is required,

and vice versa.

A worked example

A tissue culture bottle of 50 mL capacity was ﬁlled with a sea

water sample and preserved with Lugol’s iodine. The content

of the bottle was gently shaken and allowed to settle onto the

ﬂat side of the bottle; dimensions 60 x 30 mm. Five strips

across the long length of this ﬂat side were examined using a

20X objective lens. The width of the ﬁeld of view was previ-

ously estimated as 0.95 mm at this magniﬁcation. A total of

135 cells of Karenia mikimotoi were counted.

In this example ﬁve strips across the long side were originally

examined (as shown in Fig. 2b) the value for F then becomes:

Where 30 equals the width of the bottle (mm)

5 equals the number of strips counted

0.95 equals the width of the transect counted (mm)

The density of K. mikimotoi cells in the sample then be-

comes:

135 * 126.3 = 17,000 cells per Litre.

The standard error (SE) using this technique, as a percentage,

is typically the square root of the number of cells counted,

expressed as a proportion of the cells counted:

This gives an overall result of a cell density for K. mikimotoi of

17,000 cells per Litre ± 1,500. Note the number of signiﬁcant

ﬁgures in this result (1,500 has been rounded from 1,467).

Discussion

A common problem in plankton identiﬁcation and enu-

meration is archiving a particular sample for future reference,

veriﬁcation of the identity of a species or even accurate inter-

calibration studies. A particular advantage of the settlement

bottle technique is the minimisation of sample handling

which can, at times, introduce serious errors. A sample is di-

Figure 2. Methods of counting strips (shaded areas) across a cell

culture bottle. If, for example, the width of the ﬁeld of view, and

hence of each strip is 0.95 mm, then the F factor may be calcula-

ted. In a) where the strips are widthways the ratio of total area to

the area counted is 60/(5 * 0.95). In b) with the strips lengthways

this ratio is 30/(5 * 0.5). With a sample volume inside the bottle of

50 mL, the F factor for a) then becomes (60/(5 * 0.95)) * (1000/50)

= 12.6 * 20 = 252. In b) the F factor is (30/(5*0.95))*(1000/50)

= 6.3 * 20 = 126. F factors can become very high using the cell

culture bottle technique unless a suitable number of strips are

counted.

Strips scanned across the

width of the bottle. The width

of the strip is the width of the

field counted. In the example

this is 0.5 mm.

Settlement bottle length 60 mm

Settlement bottle

width 30 mm

Figure 1. A comparison of a) the Utermöhl settlement chamber

and b) the settlement bottle method for counting phytoplankton

cells using an inverted microscope. In the example above a 10

mL settlement chamber shows a similar height of water from

which samples are sedimented as the 50 mL cell culture bottle

used in b), allowing the same density of cells on the base of each.

However, a much bigger surface area can be examined when

using the settlement bottle, allowing more accurate estimations

of, in particular, the larger species since more cells are counted.

This can be done quite rapidly at lower power magniﬁcations, for

example using a X10 objective lens. Note the sample label of sta-

tion number and depth is written on the side of the bottle (date and

location are on the opposite side). Note also that special mounts

on the movable stage are required for both types of settlement

container.

A B

Sample volume

1000 (mL)

Settling area

Area analysed

F =

95.0*5

1000

= 126.3

8.6%

135

11.6

135

IOC Manuals & Guides no 55

Chapter 3 Settlement bottle method

rectly put into a tissue culture bottle, preserved and capped in

the ﬁeld, after which there is no further need for any future

sub-sampling. Samples ﬁxed with formalin can be stored in-

tact for an extended period of time. If noxious chemicals such

as formalin or glutaraldehyde are used as preservatives, then

there is no danger of contamination or fumes resulting from

them as the sample is in an air- and water-tight container.

Additionally, many samples can be prepared for analysis at

any given time as there is no need for an extended range of

relatively expensive settlement chambers.

No technique is without imperfections. In the case of the set-

tlement bottle method the most irritating of these is probably

the leaching of iodine into the sides of the tissue culture bottle

if either acidic or neutral Lugol’s iodine is used to preserve the

samples. This occurs after a few weeks, and reduces the qual-

ity of the observations. As the sample is enclosed, sedimented

specimens of phytoplankton cannot be manipulated which

can on occasion make identiﬁcation very difﬁcult. Observa-

tions of cells at the extreme edges sides of the bottle can be

difﬁcult. However, the method is ideal for long-term storage

especially if the samples are stored with formalin. The method

is also very low cost relative to other techniques.

Acknowledgements

One of the authors (GMcD) wishes to acknowledge the ﬁ-

nancial assistance of Bord Iascaigh Mhara for attendance at

the WKNCT workshop in Kristineberg, Sweden.

References

Hasle GR (1978) Counting phytoplankton cells In Sournia A (ed.)

Phytoplankton Manual. UNESCO, Paris, p337.

Equipment Supplier and model reference € US $

Inverted Microscope e.g. Nikon TS100F, Olympus CKX41 5,000-6000

†

6,500-8,500

Tissue Culture Bottles Any medical laboratory supplier 0.5-1 each Ca. 1 each

Table 1. Equipment and suppliers.

Appendix

†

The price can vary considerably depending on the quality of the objective lenses used. The price here

is for a reasonable quality set of 10X, 20X and 40X lenses.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

General introduction

The counting chamber methods are established methods

to count phytoplankton. The three most common types of

counting chambers for phytoplankton enumeration are the

Sedgewick-Rafter counting slide, the Palmer-Maloney coun-

ting slide and the haemocytometer counting slide. All three

methods are easy to learn and use, requiring preserved sample,

a good quality compound microscope and counting slides.

The set up cost is low and it is beneﬁcial to have a selec-

tion of all three on hand in the laboratory. These methods are

particularly suitable for samples containing a high concentra-

tion of cells, as in bloom situations or in phytoplankton cul-

tures with the haemocytometer being reserved for extremely

high cell densities of small organisms. The capability of the

Sedgewick-Rafter and the Palmer-Maloney to view the whole

phytoplankton community including the presence of harmful

species is a deﬁnite asset. Nevertheless correct identiﬁcation

of the phytoplankton community still requires highly trained

analysts for its implementation.

Sedgewick-Rafter counting slide

Introduction

The Sedgewick-Rafter counting slide is a traditional counting

method using a compound microscope and a highly trained

taxonomist. This is a rapid method for quantifying samples

with high cell numbers. The slide is comprised of a transpa-

rent base, which has a centrally mounted chamber (50 mm

x 20 mm x 1 mm deep) and can hold 1 mL of sample. The

base of this chamber has a ruled 1 mm grid, so that the 1 mL

sample is subdivided into single microlitres. This chamber is

covered over by a cover glass, which protects the sample from

drying out and disturbances by air currents. The sample is

then counted using a compound microscope.

Materials

Equipment

• A standard, compound microscope with 10X and 20X

objectives and brightﬁeld and phase contrast. A 40X ob-

jective may not be able to be used for this analysis, this

depends on the working distance of the objective lenses.

• A Sedgewick-Rafter slide: This can be made from plastic

or glass.

• A tally counter is useful when counting high cell num-

bers.

4 Counting chamber methods for quantitative phytoplankton

analysis - haemocytometer, Palmer-Maloney cell and

Sedgewick-Rafter cell

Murielle LeGresley*

and Georgina McDermott

Fisheries & Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, NB E5B 2L9, Canada

Environmental Protection Agency, John Moore Road, Castlebar, Co. Mayo, Ireland

*Author for correspondence e-mail: Murielle.LeGresley@dfo-mpo.gc.ca

Chemicals and consumables

• A pipette is needed to dispense the sample into the cell.

• Sample bottles for storing the samples.

Solutions for preservation

• Lugol’s iodine or formalin.

Methods

1 Prior to analysis the sample should ﬁrst be homogenised.

This is achieved using a combination of horizontally rol-

ling and vertically turning the sample bottle as gently as

possible to prevent the breakup of colonies and the ac-

cumulation of bubbles;

2 From a well mixed sample, 1 mL is removed using a pi-

pette. The pipette should have a wide opening that does

not restrict the movement of larger phytoplankton spe-

cies (such as Noctiluca scintillans, Ceratium species). This

is especially important when using a 1 mL pipette with

removable tips, the end of the tip should be cut off to

widen the opening;

3 The cover glass should be placed carefully onto the coun-

ting slide, perpendicular to the long axis of the slide, so

one corner is left open for ﬁlling and another for the es-

cape of air;

4 The sample aliquot is then dispensed into the counting

cell (Fig. 1a – f):

5 Slowly swing the cover glass so that it completely covers

the sample. Careful alignment of the cover glass will pre-

vent air bubbles from being introduced into the sample

and will ensure that the sample holds its complete vo-

lume. If a bubble develops, reﬁll the counting cell.

6 Preserved samples should be left to settle for 15 minutes

before enumeration;

7 The sample is then examined using a compound micros-

cope. The slide should ﬁrst be scanned under low mag-

niﬁcation to estimate the concentration of cells. Using

this information a counting strategy is decided upon as

to whether the whole slide or a noted fraction is to be

counted;

8 If the concentration of phytoplankton in the Sedgewick-

Rafter slide is too dense and the cells are overlapping thus

hindering identiﬁcation, a dilution step should be perfor-

med using ﬁltered seawater for marine samples;

9 After analysis is completed the slide is washed and clea-

ned between samples to prevent cross-contamination. A

pure detergent like soap is recommended (Hallegraeff et

al. 2004);

IOC Manuals & Guides no 55

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

The fundamentals of

The counting chamber methods

Counting chamber Sedgewick-Rafter Palmer-Maloney Haemocytometer

Scope

Cultures and high cell numbers Cultures and high cell densities as in bloom

conditions

Cultures and extremely high cell concentration of

small organisms

Detection range

1,000 cells L

-1

Limit of Detection (LOD) 10,000 cells L

-1

(LOD) 10,000,000 cells L

-1

(LOD)

Advantages

A rapid estimate of cell concentrations A rapid estimate of high cell concentrations A rapid estimate of extremely high cell

concentrations

Drawbacks

Accurate results only when sample contains high

phytoplankton cell densities

Accurate results only when sample contains very

high cell densities

Accurate results only when sample contains

extremely high cell densities

Type of training needed

Method-easy to learn and use.

Highly trained taxonomist needed for verication of

species identication

Method-easy to learn and use.

Highly trained taxonomist needed for verication

of species identication.

Method-easy to learn and use.

Highly trained taxonomist needed for verication of

species identication

Essential Equipment

Compound Microscope

Cover slips

Pipettes

Compound Microscope

Cover slips

Pipettes

Compound Microscope

Pipettes

Sedgewick-Rafter slides Palmer Maloney slides Haemocytometer slide with cover glass

Equipment cost

Compound Microscope: € 2500 / 3250 US $ Compound Microscope: € 2500 / 3250 US $ Compound Microscope: € 2500 / 3250 US $

Sedgewick-Rafter slides:

Perspex :€50/ 65 US $

Glass: €166/ 213 US $

Palmer-Maloney slides:

Ceramic- €60/ 80 US $

Stainless Steel-€170/$230 US $

Haemocytometer slide:

€200/ 230 US $

Consumables, cost per sample

€ 1/$1.3 US $ € 1/$1.3 US $ € 1/$1.3 US $

Processing time/sample:

20 minutes 5 minutes 5 minutes

Analysis time /sample:

This depends on the sample density 10-30 min/ sample depending on the sample

density

< 20 min / sample depending on the sample density

Sample/throughput/person/ day

This depends on the sample density 14-20 dependent on target species and density of

samples

< 30 dependent on target species

Samples processed in parallel

Only one sample at a time Only one sample at a time Only one sample at a time

Health and Safety issues

Dependent on preservative used Dependent on preservative used Dependent on preservative used

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

Preservation and storage

Samples should be stored in the dark (closed boxes) to avoid

direct light (which affects the Lugol’s iodine preservative) and

at room temperature. Samples preserved with Lugol’s iodine

can deteriorate over time resulting in a paler solution which

could impare preservation. If the samples are stored for ex-

tended periods, preservation should be checked regularly and

further Lugol’s iodine added as appropriate.

Formulas for calculating results

The Sedgewick-Rafter slide has a volume of 1 mL with the

base of the cell being divided into 1,000 squares (50 rows

by 20 rows), each representing 1/1,000 of the volume of the

slide.

To obtain a ﬁnal result expressed as cells L

-1

, the following

equation is used to calculate the multiplication factor (F). F

is dependent on the number of squares of the base of the cell

counted during the analysis.

Examples of F for the Sedgewick-Rafter slide:

4 rows (200 squares) are counted.

50 rows (1000 squares) or the entire slide is counted.

Discussion

The Sedgewick-Rafter slide is best used when analysing cul-

tures or high biomass blooms. As this method does not re-

quire an overnight settling period, it is rapid and can provide

a quick assessment of a water sample. It has been proven to

provide accurate results between 10,000 (ICES 2006) and

100,000 cells L

-1

(McAlice 1971). The set up cost is low due

to the use of a compound microscope.

The Sedgewick-Rafter slide tends to perform better with

samples containing larger phytoplankton cells. Another coun-

ting method may have to be used for samples with low cell

densities. It may be possible to pre-concentrate cells using a

ﬁltering/settling step when the target organism is present in

low concentrations.

Plastic Sedgewick-Rafter slides tend to scratch easily and care

must be taken when cleaning the cell. Scratches may hinder

the accurate identiﬁcation of cells. Due to the design of the

Sedgewick-Rafter slide it may be difﬁcult to use the 40X mag-

niﬁcation. This could prove a problem in the identiﬁcation of

smaller (10-15 µm) phytoplankton cells. In addition exteme

care must be taken to load the Sedgewick-Rafter slide cor-

rectly and avoid the introduction of air bubbles to ensure the

even distribution of phytoplankton in the slide.

000,1*

000,1

countedSquaresofNumber

F =

000,5000,1*5000,1*

200

000,1

===F

000,1000,1*1000,1*

000,1

===F

Figure 1. A – F: Loading Lugol’s iodine preserved sample into

Sedgewick-Rafter cell.

IOC Manuals & Guides no 55

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

Palmer-Maloney counting slide

Introduction

The Palmer-Maloney counting slide method is a rapid and

straightforward technique that was ﬁrst employed to enume-

rate nanoplankton. The counting chamber is round, measures

17.9 mm in diameter, 400 µm depth and holds a volume 0.1

mL of sample. Two loading channels are located on either

side of the counting slide (Fig. 2 a-d). The Palmer-Maloney

slide does not have any rulings or grid. This counting slide is

useful with cultures, samples with high cell densities or pre-

concentrated samples. The detection level is 10,000 cells L

-1

or 10 cells mL

-1

(Guillard 1978).

Equipment

A standard compound microscope or an inverted microscope

with 10X and 20X objectives. Phase contrast and epiﬂuores-

cence capability are valuable asset to the identiﬁcation of phy-

toplankton.

• Palmer-Maloney counting slide

• Cover glass (22mm X 22mm or 50mm X 22 mm)

• Pipettes (Pasteur or disposable) needed to dispense the

sample

• A tally counter is useful when counting high cell num-

bers

Chemicals and consumables

• Preservative (Lugol’s iodine, Formalin) (see chapter 2 for

recipes)

• Alcohol for cleaning slides

Method

1 The cover glass should initially be placed over the coun-

ting chamber;

2 The preserved sample should be inverted gently about

10-20 times to ensure homogenisation. A pipette is ﬁlled

with the well-mixed sample;

3 The chamber of the counting slide is then ﬁlled by gradu-

ally dispensing an aliquot of sample from the pipette into

one of the loading channels (Fig. 2 a-d). It is important

that no air bubbles are present in the chamber. Trapped

air may be removed by slowly sliding the cover slip back

then replacing it in its original position however it may

be nessecary to repeat steps 1-3;

4 The slide should be left to settle for 5 minutes;

5 Counting organisms should begin at the top or bottom

edge and continue until all area of the chamber is exa-

mined excluding loading channels. The centre of the lo-

ading channels are effective reference points to count half

of the slide;

6 Once the count has been completed the slide and cover

glass should be rinsed thoroughly with water then with

alcohol and wiped clean with lint-free wipes.

Preservation and storage

Any preserved samples can be used with this method.

Note: samples preserved in Lugol’s iodine- should be kept in

the dark and checked periodically for light tea colour, adding

more preservative if needed.

Formulas for calculating results

Since the volume of a Palmer-Maloney slide is 0.1 mL, mul-

tiply the total count by 10,000 to obtain the number of

cells L

-1

For example:

Cells per Litre= total cell count * 10,000

Final count in the Palmer-Maloney slide is 200 cells; 200 *

10,000= 2,000,000 cells L

-1

Discussion

The Palmer-Maloney counting slide method is excellent to

enumerate dense blooms, net tows, cultures or pre-concentra-

ted samples. It is an inexpensive and rapid counting method

in which the entire phytoplankton community may be obser-

ved including harmful algae species.

This counting slide is appropriate for enumerating most spe-

cies but is not as useful for counting large organisms (>150

µm) or long chain-forming diatoms as these may not be dist-

ributed evenly in the sample (Guillard and Sieracki 2005).

Because of the thickness of the Palmer-Maloney slide the hig-

hest magniﬁcation objective lenses possible to use with some

compound microscopes is 10X or 20X. Thus this slide is not

a good choice when the proper identiﬁcation or enumeration

of an organism requires a higher magniﬁcation. This can be

addressed by using an inverted microscope with a higher ob-

jective. The Palmer-Maloney slide may also be used with cal-

coﬂuor stain and an epiﬂuorescence microscope for thecate

dinoﬂagellate identiﬁcation.

When using the small cover slips (22 mm x 22 mm) the sam-

ple may have a tendency to evaporate. This problem can be

solved by using longer cover slips (22 mm x 55 mm) or by

applying paraﬁlm to cover the loading channels. This method

may be used to monitor target species that are deemed to be

harmful only at very high cell concentrations.

Figure 2 A –D: Loading sample into the Palmer Maloney cell

A B

C D

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

Haemocytometer counting method

Introduction

The haemocytometer (also used for counting blood cells) is a

counting slide method specially practical for cultures and ex-

tremely high concentrations of cells of small sized organisms

(< 30 µm). The middle of the slide has the appearance of an

“H” which separates the 2 thin silver-coloured chambers both

engraved with a nine-square grid. Haemocytometers may be

purchased in either 0.1 mm or 0.2 mm depth and may possess

different grid subdivisions. The most common slide of this

type is 0.1 mm deep with Improved Neubauer ruling (Fig.

3); each chamber holding nine 1-mm large squares separated

by double or triple rulings. The volume in nine large squares

is 0.0009 mL, with the 2 chambers having 18 squares with a

total volume of 0.0018 mL.

Equipment

• A standard compound microscope or an inverted micros-

cope with 10X and 20X objectives, phase contrast

• Haemocytometer counting slide (Improved Neubauer

rulings)

• Cover glass supplied with haemacytometer

• Pipettes (Pasteur)

Chemicals and consumables

• Preservative (Lugol’s iodine, Formalin) ( see chapter 2 for

recipes)

• Alcohol for cleaning slides

Methods

1 A cover glass is placed over both chambers of the haemo-

cytometer;

2 With a soft undulating motion, the preserved sample is

gently inverted appoximately 10-20 times to ensure the

sample is mixed thoroughly;

3 A Pasteur pipette is ﬁlled with the well-mixed sample;

4 Each chamber of the haemocytometer is loaded by hol-

ding pipette at a 30 to 45 degree angle with the open

dispensing tip in the V-shaped slash, allowing the pipette

tip to touch the slot then slowly expelling a drop of the

liquid. The capillary action will ﬁll the chamber with the

sample. It is important to check that the liquid spreads

over the silver- coloured chamber without overﬂowing

into the moats. (Fig 4);

5 Step 2-4 is repeated to ﬁll the other side of the chamber.

Allow 2-3 minutes for cells to settle;

6 The slide should be scanned initially in the microscope to

determine the counting strategy. The whole slide or a se-

lected number of large squares should be counted to ob-

tain a statistically signiﬁcant number of cells (Andersen

and Throndsen 2004). Each side of the haemocytometer

slide has a grid with nine large 1 mm

squares which are

further subdivided depending on the type of haemocyto-

meter;

Figure 3. Drawing of haemocytometer with improved Neubauer ruling.

Figure 4 A-C: Loading sample into haemocytometer.

1 mm

IOC Manuals & Guides no 55

Chapter 4 Counting chamber methods - Haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell

7 The number of organisms and the number of squares

counted should both be noted. To avoid counting cells

twice, it must be determined beforehand to include cells

that touch 2 of the 4 sides of each square (i.e. the top and

left side of each large square while ignoring the cells that

touch the bottom and right side);

8 After the count has been completed the haemocytometer

slide must be cleaned thoroughly by rinsing the slide and

cover glass with running water then with alcohol, wiping

clean with lint-free wipes.

Preservation and storage

Any preserved samples can be used.

Note: samples preserved in Lugol’s iodine- should be kept in

the dark and checked periodically for light tea colour, adding

more preservative if needed.

Formulas for calculating results

Total the cells and divide by the number of large 1 mm squa-

res counted to obtain the average number of cells per square.

Multiply this average cell number by 10,000 to obtain num-

ber of cells per mL. (Alternately to obtain the number of

cells per Litre, multiply the average number of cells per large

square by 10,000,000).

The average number of cells per mL = average count per large

square X 10,000

For example: In total 200 cells in 4 large squares are counted:

Discussion

The haemocytometer counting method is excellent for coun-

ting cultures or an extremely high concentration of small cells.

The slide can be used with 10X objective with the compound

microscope or with 10X or 20X objectives with an inverted

microscope. This method is not suitable for routine water

monitoring because of the high cell biomass needed to get

statistically signiﬁcant numbers. It will not give an overview

of the whole phytoplankton community especially organisms

with a low cell density. It is not compatible with large orga-

nisms because of the shallow 0.1 mm depth of the slide. It is

better to use the slide for extremely high cell estimates.

Pre-concentration of sample

Concentration of sample may be necessary when cell density

is low. This can be achieved using a settlement method where

a sample is poured into a graduated cylinder (of volume (A))

and allowed sufﬁcient time for cells to settle (one hour for

each cm height of cylinder or overnight). After settling, the

water from the upper portion of the sample is gently removed

and the ﬁnal volume (B) noted. Another method involves ﬁl-

tering the sample through a 10 or 20 µm mesh (i.e. plankton

net). The concentration factor (CF) is calculated by dividing

the initial volume (A) by the ﬁnal volume (B). The remain-

ing volume should be mixed well and the instructions of the

counting method followed, remembering to divide the total

cell count by the CF.

Example:

The original volume (A) = 100 mL

Final volume (B) = 10 mL

Acknowledgements

One of the authors (GMcD) wishes to acknowledge the as-

sistance of Bord Iascaigh Mhara (The Irish Fisheries Board).

for assistance in attending the WKNCT workshop in Kris-

tineberg, Sweden.

References

Andersen P, Throndsen J (2004) Estimating cell numbers. In: Halle-

graeff GM, Anderson DM, Cembella AD (eds) Manual on Harm-

ful Marine Microalgae 99-129

Guillard RRL (1978) Counting Slides. In: A. Sournia (ed) Phyto-

plankton Manual. UNESCO Publishing 182-189

Guillard RRL, Sieracki MS (2005) Counting cells in cultures with

the light microscope. In: Andersen RA (ed) Algal Culturing

Techniques Elsevier Academic Press 239-252

Hallegraeff GM, Anderson DM, Cembella AD (2004) Manual on

Harmful Marine Microalgae. UNESCO Publishing 793pp

ICES (2006) Report on the ICES/IOC workshop on new and clas-

sic techniques for the determination of numerical abundance and

biovolume of HAB species – evaluation of the cost, time-efﬁ-

ciency and intercalibration methods (WKNCT), 22-27 August

2005, Krinstineberg, Sweden. ICES CM 2005/C:10

McAlice BJ (1971) Phytoplankton sampling with the Sedgewick-

Rafter cell. Limnol Oceanogr 16(1)19-28.

100

===

200

==countedcellsofnumberAverage

000,500000,10*50

−

 mLcellsmL perCells

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

Materials

Laboratory facilities

The method can be used in a basic laboratory. No special fa-

cilities are required.

Equipment

A ﬁltration unit and a vacuum pump as well as an epiﬂuores-

cence microscope equipped with a mercury lamp and ﬁlter set

for DAPI (UV excitation (330-380 nm), emission ﬁlter (420

nm) is required, see Table 1.

Chemicals and consumables

The stain, Calcoﬂuor White M2R (Polysciences, Warrington

PA) is especially useful for qualitative as well as quantitative

analysis of thecate dinoﬂagellates because it stains the cellulose

in the thecal plates of dinoﬂagellates and not other plankton

organisms or detritus.

Calcoﬂuor White M2R is a ﬂuorescent brighter. The chemi-

cal formula of Calcoﬂuor White M2R is: C

Calcoﬂuor White M2R can be stored at room temperature.

Method

How to make the Calcoﬂuor working solution

Add approximatley 2 µg Calcoﬂuor to 10 mL of distilled wa-

ter in a clean acid rinsed (5% HCl) glass bottle. The Cal-

coﬂuor will dissolve immediately, and the working solution

is ready to use. If the glass bottle is not completely clean the

Calcoﬂuor may precipitate and the solution can not be used.

The working solution of Calcoﬂuor does not require preser-

vation. It can remain viable for a few days to several weeks at

room temperature in the laboratory.

Introduction

Identiﬁcation and enumeration of thecate dinoﬂagella-

tes, which include several toxic species, is frequently a time

consuming exercise. Species from the genera Dinophysis and

Prorocentrum (the causative organisms of diarrhetic shellﬁsh

poisoning, DSP), Alexandrium and Pyrodinium (the causative

organisms of paralytic shellﬁsh poisoning, PSP), Ostreopsis

and Gambierdiscus (responsible for ciguatera ﬁsh poisoning,

CFP) may cause problems to the aquaculture and ﬁshing

industries even when they occur in low concentrations. Tra-

ditionally, quantitative analysis of phytoplankton samples is

performed using the well established Utermöhl sedimenta-

tion procedure (Utermöhl 1958). The Utermöhl procedure

involves sedimentation of the plankton sample (5-50 mL) for

a period of 8 -24 hours depending on the sample volume.

The long sample sedimentation time prior to analysis and the

complexities of identiﬁcation of thecate dinoﬂagellates means

that this method cannot provide a rapid result. Quantitative

epiﬂuorescence techniques, basically adapted from the acri-

dine orange technique by Hobbie et al. (1977), involves ﬁltra-

tion and staining of the organisms on polycarbonate ﬁlters.

This method, which was originally described for counting of

pelagic bacteria has been modiﬁed for counting of hetero- and

autotrophic nanoﬂagellates as well as larger phytoplankton

and protozooplankton organisms (see e.g. Haas 1982, An-

dersen and Sørensen 1986). Examples of ﬂuorochromes used

are acridine orange (Andersen and Sørensen 1986) or DAPI

(Porter and Feig 1980). Apart from the DNA, these ﬂuoro-

chromes also stain other compounds found in cells. When

working in coastal waters and shallow fjords where the pelagic

biomass is high and often dominated by diatoms, quantiﬁca-

tion of thecate dinoﬂagellates present in low concentrations

must be carried out on relative large water samples (50-100

mL). In such cases the analysis can be rendered practically im-

possible using either acridine orange or DAPI. This is because

thecate dinoﬂagellates must be identiﬁed among high con-

centrations of other organisms such as diatoms, which also

ﬂuorescence heavily.

In this chapter, a method is presented which is based upon

the quantitative epiﬂuorescence technique using Calcoﬂour

White M2R as a stain. The method has previously been des-

cribed in Andersen (1995), Andersen and Kristensen (1995),

Andersen and Throndsen (2004). Calcoﬂuor is a speciﬁc stain

for the cellulose in the thecal plates of the thecate dinoﬂagel-

lates (Lawrence and Triemer 1985). The stain does not stain

structures in most other pelagic organisms including the dia-

toms. Using this method it is possible to analyse sample volu-

mes from 10 to 500 mL, thereby obtaining reliable estimates

of thecate dinoﬂagellates present in low concentrations in the

presence of large concentrations of diatoms.

5 Filtering – calcoﬂuor staining – quantitative epiﬂuorescence

microscopy for phytoplankton analysis

Per Andersen*

Orbicon A/S, Johs. Ewalds Vej 42-44, DK-8230 Åbyhøj, DENMARK

*Author for correspondence e-mail: [email protected]

• Calcoﬂuor White M2R or a similar product

• A 10 mL glass bottle

• Polycarbonate membrane ﬁlters (pore size 5 µm)

• Parafﬁn oil

• Neutral Lugol’s iodine

• A ﬁltration unit

• A vacuum pump

• Glass microscope slides

• Cover slips (24x24 mm)

• An epiﬂuorescence microscope equipped with a mercury

lamp and ﬁlter set for DAPI (UV excitation, 330-380 nm,

emission ﬁlter, 420 nm)

Table 1. Equipment and consumables required for the quantitative

epiﬂuorescence method using Calcoﬂuor White M2R.

IOC Manuals & Guides no 55

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

Scope

Qualitative and quantitative analysis of thecate dinoﬂagellates.

Detection range

Detection range is dependent on the volume of sample ﬁltered.

Counting all of the cells from a 200 mL sample will give a de-

tection limit of 5 cells per Litre.

Advantages

Preparation time is short. Speciﬁc identiﬁcation of thecate di-

noﬂagellates is feasible as the staining makes the relevant morp-

hological features visible. Preparations can be stored for analysis

or re-examination for weeks/months.

Drawbacks

This method is limited to analysis of thecate dinoﬂagelllates

only. This means that other methods will have to be used if

the rest of the phytoplankton community needs to be studied.

Type of training needed

A basic knowledge of light and epiﬂuorescence microscopy is

needed. Analysis requires continuous training over years with

in-depth knowledge of taxonomic literature.

Essential Equipment

Epiﬂuorescence microscope with UV excitation

Filtration unit incl. pump.

Equipment cost*

20000-50000 US$ (see Appendix to this chapter for details).

Consumables, cost per sample**

Less than 1-2 €/$1-2.

Processing time per sample before analysis

App. 15 minutes for ﬁltration and mounting of ﬁlter.

Analysis time per sample

Depending on the number of species to be quantiﬁed and vo-

lume ﬁltered. A routine analysis of 5-10 species requires ap-

prox. 15 min. per sample, excl. reporting/database handling of

results.

Sample throughput per person per day

10-20.

No. of samples processed in parallel

One per analyst.

Health and Safety issues

Analysis sitting at the microscope is tiresome for eyes, neck and

shoulder. Frequent breaks are needed. The stain Calcoﬂour is

for laboratory use only. Caution: Avoid contact and inhalation!

The relevant health and safety guidelines should be followed.

*service contracts not included

**salaries not included

The fundamentals of

The ﬁltering - calcoﬂuor staining - quantitative ﬂuorescence microscopy

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

Sample preparation

A known volume of sample, ﬁxed using neutral Lugol’s iodi-

ne, is vacuum ﬁltered using a polycarbonate ﬁlter with a pore

size 5 µm (Fig. 1). Filters with larger or smaller pore sizes can

be used depending upon the organisms of interest.

When approximatley 1 mL of sample is left in the ﬁlter chim-

ney, 0.2 mL (3-4 drops) of Calcoﬂuor White M2R working

solution is added and ﬁltration is continued until the ﬁlter is

dry (Table 2).

Note that Calcoﬂuor White M2R will only work at a neutral

pH (= 7). In more acidic samples Calcoﬂuor will precipitate

which will interfere with the identiﬁcaiton and enumera-

tion of dinoﬂagellate cells. If the sample is ﬁxed using acidic

Lugol’s iodine, the sample must be neutralised prior to the ad-

dition of the Calcoﬂuor. To achieve this, the ﬁltration should

be stopped when there is approximatley 0.5 mL of sample left

in the ﬁltration chimney. The ﬁlter should then be washed by

adding approx. 2-5 mL of ﬁltered seawater ﬁxed in neutral

Lugol’s iodine. The ﬁltration procedure is then continued as

described as described in Table 3.

The dry ﬁlter should be mounted on top of a drop of parafﬁn

oil on a microscope slide. Another drop of parafﬁn oil is then

placed on top of the ﬁlter and a cover glass is mounted on

top of the parafﬁn oil (Fig. 2). The ﬁlter can then be analysed

using epiﬂuorescence microscopy, using UV excitation (330-

380 nm) and an appropriate emission ﬁlter (420 nm). For

routine use an OLYMPUS BH-2 microscope equipped with

a mercury lamp (100 W) or a similar microscope is appro-

priate.

Thecate dinoﬂagellates can be identiﬁed and enumerated,

either by counting the whole surface of the ﬁlter or selected

fractions of the ﬁlter surface. Large organisms (diameter >20

µm) such as cells from the genus Dinophysis, Prorocentrum

or Alexandrium, can be counted using 100X magniﬁcation.

Higher magniﬁcations can be used when it is necessary to see

the morphology of the thecal plates to allow identiﬁcation to

species level.

The prepared slides for quantitative analysis can be stored in

a refrigerator (< 5

C) for weeks with no/very little loss of cells

or ﬂuorescence.

Trouble shooting

The most frequent problems encountered when working with

the quantitative Calcoﬂuor method are:

1 The ﬁlter set on the epiﬂuorescence microscope does not

work with Calcoﬂuor White M2R;

2 The pH of the sample to be analysed is not 7;

3 The working solution of Calcoﬂuor has precipitated and the

solution looks milky.

Preservation and storage

Samples to be analysed with this method should preferably

be ﬁxed using neutral Lugol’s iodine. If samples are to be sto-

red for more then a few days the samples should be stored in

brown glass bottles and stored in the dark. Samples stored

according to these guidelines can be kept for months/years,

however, it must be ensured that there is sufﬁcient neutral

Lugol’s iodine in the sample to maintain the perservation.

If the sample has the colour of weak tea there is sufﬁcient

Lugol’s present. If the sample is clear, more neutral Lugol’s

iodine must be added.

Counting procedure and calculation of concen-

trations

Initial analysis of the sample can be performed using a low

magniﬁcation such as 100X. The performance of the Cal-

Figure. 1. The ﬁltration equipment used to concentrate phytoplank-

ton on polycarbonate membrane ﬁlters for quantiﬁcation using

epiﬂuorescence microscopy.

Figure. 2. How to mount the polycarbonate ﬁlter on a slide for

observation using epiﬂuorescence microscopy.

IOC Manuals & Guides no 55

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

coﬂuor stain must ﬁrst be assessed by establishing that the

thecate dinoﬂagellates light up blue on a dark background.

After this has been checked, the sample analysis can be per-

formed.

The counting strategy employed depends on the number of

cells on the ﬁlter. It is preferable to count the entire surface of

the ﬁlter. If there are a high number of cells on the ﬁlter, sub-

sampling or counting only a fraction of the ﬁlter (half of the

ﬁlter surface or diagonals), can be performed.

Calculation of cell concentrations:

To calculate the concentration (cells L

-1

) of the different spe-

cies in your preparation you must know:

V = Volume of sample concentrated on the ﬁlter (mL).

= Area of the ﬁlter (mm

= Area of the part of the ﬁlter counted (mm

N = Number of cells counted for the species of interest.

The conversion factor (CF):

The concentration of the species C (cells mL

-1

) is then:

The conversion factors must be calculated for each ﬁltering

unit and microscope as well as for each combination of sub-

sampling area and magniﬁcation (Table 4).

Examples of calculations of concentrations using the conver-

sion table are presented in Table 5.

1. Measure the required sample volume using a graduated

cylinder;

2. Add the sample to the ﬁltration unit;

3. Turn on the vacuum pump (maximum pressure = 200

mmHg);

4. Turn off the vacuum pump when there is approximately 1

mL left in the ﬁltration chimney;

5. Add 3-5 drops of CalcoFluor working solution (concentra-

tion 2 mg L

-1

);

6. Turn the vacuum pump on again and ﬁlter until the ﬁlter

goes dry;

7. Remove the ﬁlter from the chimney and dry the back of it

gently on a tissue to remove surplus water;

8. Mount the ﬁlter on a drop of parafﬁn oil on a slide, add

another drop of parafﬁn oil on top of the ﬁlter and put on the

cover slip (24 x 24mm);

9. Observe your preparation using an epiﬂuorescence micros-

cope.

Figure 3. Dinophysis sp. stained with calcoﬂuor as seen in the epi-

ﬂuorescence microscope. The cell height is approximately 50 µm.

1. Measure the required sample volume using a graduated

cylinder;

2. Add the sample to the ﬁltration unit;

3. Turn on the vacuum pump (maximum pressure = 200

mmHg);

4. Stop the ﬁltration when there is about 1-0.5 mL left in the

chimney;

5. Wash the ﬁlter by adding approx. 2-5 mL of ﬁltered seawa-

ter ﬁxed in neutral Lugol’s iodine and turn on the vacuum

pump;

6. Turn off the vacuum pump when there is approximatley 1

mL left in the ﬁltration chimney and add 3-4 drops of the

Calcoﬂuor working solution (concentration 2 mg L

-1

);

7. Turn on the vacuum pump again and ﬁlter until the ﬁlter

goes dry;

8. Remove the ﬁlter from the chimney and dry the back of it

gently on a tissue to remove surplus water;

8. Mount the ﬁlter on a drop of parafﬁn oil on a slide, add

another drop of parafﬁn oil on top of the ﬁlter and put on the

cover slip (24x24mm);

9. Observe your preparation using an epiﬂuorescence micros-

cope.

Table 3. Summary of how to prepare samples preserved with

acidic Lugol’s iodine, formaldehyde or gluteraldehyde for the

quantitative epiﬂuorescence method using Calcoﬂuor White M2R.

NC *=

Table 2. Summary of how to prepare samples preserved with

neutral Lugol’s iodine for the quantitative epiﬂuorescence method

using Calcoﬂuor White M2R.

Table 4. Example of a calibration table used for calculating con-

centrations of microalgae using epiﬂuorescence microscopy (ﬁlter

area = 189 mm

. Note: CF is the conversion factor to be applied

when the count of cells in e.g. one diagonal window is to be cal-

culated to the concentration on the total ﬁlter surface.

Magni-

ﬁcation

Window count

Window area (mm

)

Diagonal window count

Diagonal window area (mm

)

40X 4.08 31.52

100X 0.66 12.64

200X 0.16 6.2

CF: window CF: diagonal window

40X 46.3 6.04

100X 286 15.1

200X 1181 30.5

CF =

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

Discussion

The method described is excellent for the rapid processing of

samples, for example as required in many toxic phytoplank-

ton monitoring programmes. Low concentrations of thecate

dinoﬂagellates are easily detected in the presence of high pe-

lagic biomass (e.g. diatoms). This is particularly relevant for

concentrated net tow samples. It is possible to identify thecate

dinoﬂagellates to species level because of the easy recognition

of the thecal plates of taxonomical importance which can not

always be recognised using the Utermöhl procedure using

Lugol’s iodine preservation.

Samples preserved with neutral Lugol’s iodine produce excel-

lent slide preparations. There is no requirement to use more

toxic preservatives like formaldehyde or glutaraldehyde. The

ﬂuorochrome Calcoﬂour White MR2 is considered to be of

low toxicity.

Depending upon the organisms to be quantiﬁed, the descri-

bed method can easily be modiﬁed by using other ﬁlters such

as low cost glass ﬁber ﬁlters (GFC ﬁlters). Large and robust

species e.g. from the genus Ceratium, occurring in low con-

centrations, can be investigated using large volumes of water

(500 mL) and GFC ﬁlters with excellent results.

The procedure can be used to separate auto- from hetero-

trophic thecate dinoﬂagellates on the basis of the presence or

lack of chlorophyll and other pigments by switching between

appropriate ﬁlter sets (Lessard and Swift 1986, Hallegraeff

and Lucas 1988, Carpenter et al. 1991 ).

It has been observed that species from the genus Alexandrium

can break/implode (maximum 10% of the cells) during sam-

ple preparation. Breakage of cells appears to occur in cases

where the ﬁltration time is prolonged as a result of the ﬁlter

blocking due to high sample biomass. This problem may be

common to all ﬁltration based methods. It is strongly recom-

mended that this problem be investigated more fully. It is sug-

gested that the loss of cells can be minimised by adjusting the

combination of ﬁlter pore size and the volume of sample ﬁlte-

red thus reducing the total number of particles in the sample.

When counting, one has to be aware that empty thecae

(”ghost” cells) can be hard to distinguish from cells which

were viable when sampled. When counting viable cells, the

”ghost” cells can lead to a slight overestimation of the concen-

tration of viable cells. ”Ghost” cells can be conﬁrmed by swit-

ching between ﬁlter sets, e.g. to blue light excitation, which

will reveal the cell content of the thecae under examination.

The resulting high contrast between thecate dinoﬂagellates

and the dark background common in this technique as well as

the lack of interference from other organisms such as diatoms

means that this technique may be very useful in combina-

tion with image analysis to develop automated procedures for

computerised identiﬁcation and counting of speciﬁc thecate

dinoﬂagellates.

References

Andersen P (1995) Design and Implementation of Harmful Algal

Some Monitoring Systems. IOC Technical Series No. 44

Andersen P, Kristensen HS (1995) Rapid and presize identiﬁcation

and counting of thecate dinoﬂagellates using epiﬂuorescence mi-

croscopy. In Harmful Marine Algal Blooms (Lassus PG, Arzul P,

Gentian P. Marcaillou P eds) pp. 713-718. Lavoisier Publishing,

Paris

Andersen P, Sørensen HM (1986) Population dynamics and trophic

coupling in pelagic microorganisms in eutrophic coastal waters.

Mar. Ecol. Prog. Ser. 33: 99-109

Andersen P, Throndsen J (2004). Estimating cell numbers. In Hal-

legraeff, G. M., D. M. Anderson & A. D. Cembella (eds) Manual

on Harmful Marine Microalgae. Monographs on Oceanographic

Methodology no. 11. p. 99-130. UNESCO Publishing

Carpenter EJ, Chang J, Shapiro LP (1991). Green and blue ﬂuores-

cing dinoﬂagellates in Bahamian waters. Mar. Biol. 108: 145-149

Haas LW (1982). Improved epiﬂuorescence microscopy for ob-

serving planktonic microorganisms. Ann. L’Inst. Oceanogr.

58(s):261-266

Hallegraeff GM, Lucas I (1988). The marine dinoﬂagellate genus

Dinophysis (Dinophyceae): Photosynthetic, neretic and non-pho-

tosynthetic, oceanic species. Phycologia 27: 25-42

Hobbie J E Daley R, Jasper S (1977) Use of Nuclepore ﬁlters for

counting bacteria by ﬂuorescence microscopy. Appl. Environ. Mi-

crobiol. 33:1225-1228

Lawrence F, Triemer RE (1985). A rapid simple technique utilizing

Calcoﬂuor White M2R for the visualization of dinoﬂagellate the-

cal plates. J. Phycol. 21: 662-664

Lessard EJ, Swift E (1986) Dinoﬂagellates from the North Atlantic

classiﬁed as phototrophic or heterotrophic by epiﬂuorescence mi-

croscopy. J. Plankton. Res. 8: 1209-1215

Porter K, Feig YS (1980). The use of DAPI for identifying and coun-

ting aquatic microﬂora. Limnol. & Oceanogr. 25(5): 943-948

Utermöhl H (1958) Zur vervollkomnung der quantitativen phyto-

plankton metodik. Mitt. int. ver. Limnol. 9

Example 1 (counting the entire ﬁlter)

Volume of sample concentrated on the ﬁlter = 100 mL

Counts (entire ﬁlter area) = 50 Dinophysis acuminata

Calculating cell concentration

(50/100) = 0.5 cells mL

-1

= 500 cells L

-1

Example 2 (counting one diagonal window)

Volume of sample concentrated on the ﬁlter = 100 mL

Counts (Diagonal window 100X) = 50 D. acuminata

CF = 15.1

Calculating cell concentration

(50 x 15.1)/100 = 7.5 cells mL

-1

= 7500 cells L

-1

Table 5. Examples of calculations of cell concentrations using the

conversion table.

IOC Manuals & Guides no 55

Chapter 5 Filtering - Calcoﬂuor staining - quantitative epiﬂuorescence microscopy

Item Price

Epiﬂuorescence microscope with UV excitation 20000-50000 US $

Mercury burner 100 US $/approx. 1200 samples, 0,08 US $/sample

Filtration unit incl. pump. 1000-1500 US $

Polycarbonate ﬁlter 0,5-1 US $/sample

Calcoﬂuor (for a life time) 50-100 US $/approx 5.000 samples, 0,02 US $/sample

Neutral Lugol´s (1 Litre) approx. 50 US $ /approx. 500 samples, 0,10 US $/sample

Slides and coverslips 0,05 US $/sample

Parafﬁn oil 0,05 US $/sample

Table 1. Equipment and costs

Appendix

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 6 Filtering - semitransparent ﬁlters

Introduction

Phytoplankton species such as large dinoﬂagellates are often

present in the water column in low cell densities. Thus, samp-

les must ﬁrst be concentrated in order to accurately quantify

these species. One method for concentrating phytoplankton

cells in water samples is the use of semitransparent membrane

ﬁlters (Fournier 1978). In this method water samples are di-

rectly ﬁltered on a membrane ﬁlter which then is placed un-

der the microscope for the identiﬁcation and enumeration of

phytoplankton cells.

Materials

Laboratory facilities

No specialised laboratory facilities are necessary for this met-

hod. Water samples using preservatives with potential harm-

ful effects should be handled following the appropriate health

and safety procedures.

Equipment

The following equipment is required:

• Compound light microscope, with at least 100X and

200X times magniﬁcation.

• Filter manifold and vacuum pump (handpump or elec-

tric) with manometer to gauge the pressure.

• Forceps for handling ﬁlters.

Chemicals and consumables

The following consumables are required:

• Semitransparent membrane ﬁlter (e.g. PALL GN-6). The

ﬁlters are availaible in a variety of diameters and pore si-

zes. A ﬁlter diameter of 25 mm and a pore size of 0.45

µm is recommended for use in this method.

• Glass microscope slides and coverslips.

• Lugol’s iodine solution is recommended.

• Glass bottles (dark) for sampling, transport and storage

of water samples.

Methods

The recommended preparation procedure is as follows:

1 The ﬁltration manifold is assembled and the vacuum

pump tested (Fig. 1);

2 Using forceps, the ﬁlter is placed in the ﬁlter holder. The

funnel and holder are connected securely to prevent lea-

kage;

3 The preserved sample is gently agitated, by inverting the

sample bottle at least 10 times;

4 The volume to be ﬁltered is measured using a graduated

cylinder. The most appropriate volume for analysis may

vary with location and time of year. Too many organisms

6 Filtering – semitransparent ﬁlters for for quantitative

phytoplankton analysis

Einar Dahl & Lars-Johan Naustvoll*

Institute of Marine Research, Flødevigen Marine Research Station, N-4817 HIS, Norway

*Author for correspondence e-mail: [email protected]

on the ﬁlter will prevent the identiﬁcation of some spe-

cies. A guiding volume could be 25 mL for estuaries with

high production, 50 mL for inshore stations and 100 mL

for offshore stations;

5 The contents of the graduated cylinder are transferred

into the funnel above the ﬁlter;

6 A gentle vacuum is applied for ﬁltration, less than 1/6

atmosphere (125 mm Hg), using a hand or electric pump

with manometer. A low vacuum pressure prevents de-

struction of and minimises distortion of fragile cells.

7 Filtering continues until the ﬁlter is “dry”;

8 The ﬁlter is removed from the ﬁltration apparatus using

forceps and placed on a microscope slide;

9 The slide is analysed using a compound light microscope

at the desired magniﬁcation. The concentration of phy-

toplankton is calculated based on the number of cells

counted on the whole ﬁlter and the volume of sample

initially ﬁltered.

To calculate the concentration (cells L

-1

) of the different spe-

cies in your preparation you must know:

V = Volume of sample concentrated on the ﬁlter (mL).

= Area of the ﬁlter (mm

= Area of the part of the ﬁlter counted (mm

N = Number of cells counted for the species of interest

Figure 1. Filtration manifold and hand held vacuum pump.

IOC Manuals & Guides no 55

Chapter 6 Filtering - semitransparent ﬁlters

Scope

Concentration and enumeration of phytoplankton, particularly

large and more physically robust species.

Detection range

10-50 cells per Litre, depending on volume ﬁltered.

Advantages

This is a rapid and ﬂexible method for the concentration of

phytoplankton using standard easy to use laboratory equip-

ment. The volume to be analysed can be easily adjusted depen-

ding on cell density.

Drawbacks

Fragile cells can be destroyed during the ﬁltration process.

Type of training needed

No special training is required for the preparation of samples.

Taxonomic competence required for identiﬁcation and enume-

ration.

Essential equipment

Compound light microscope, ﬁltration manifold, vacuum

pump, forceps, semitransparent ﬁlters and microscope slides.

Equipment cost*

Microscope: Depending on manufacturer and type, from 4000

€ (6000 US $)

Filtration manifold: from 250 € (320 US $)

Vacuum pump: hand held from 180 € (240 US $)

The fundamentals of

The ﬁltering - semitransparent ﬁlters method

Consumables, cost per sample**

Cost for ﬁlters and glass is approximately 1 € (1.5 US $).

Processing time per sample

Preparation time for the ﬁlters is 5 to 10 minutes, depending

volume ﬁltered, amount of phytoplankton and ﬁltration vo-

lume.

Analysis time per sample

Analysis time is about 30 minutes for target species, depending

on skill, quantity of phytoplankton cells and number of target

species.

Sample throughput per person per day

10-12 samples per day.

Health and Safety issues

Some ﬁxatives that are used for phytoplankton preservation

could be potential harmful. Appropriate health and safety pro-

cedures must be followed at all times. Continual microscope

work could result in strain injury. It is important to incorporate

breaks into the daily analyses time. Ergonomic adjustments to

the microscope and the working place is recomended.

*service contracts not included

**salaries not included

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 6 Filtering - semitransparent ﬁlters

The conversion factor (CF):

The concentration of the species C (cells mL

-1

) is then:

The conversion factors must be calculated for each ﬁltering

unit and microscope as well as for each combination of sub-

sampling area and magniﬁcation.

Preservation and storage

Water samples can be preserved with neutralised formalde-

hyde or neutral Lugol’s iodine solution. Formaldehyde should

be used with care because of its toxicity and potential to trig-

ger allergic reactions. Neutral Lugol’s iodine solution is re-

commended for this method. If necessary, the brownish colo-

ration of the algae caused by this preservative can be removed

by oxidising the Lugol’s iodine using a few drops of sodium

thiosulfate per mL (3 g Na

in 100 mL water). When

using Lugol’s iodine solution the water samples should be sto-

red in dark glass bottles in the dark. Samples should retain the

colour of ‘weak tea’. The ﬁxation of archived samples should

be checked every third month and additional Lugol’s iodine

should be added if the sample has lost its colouration. Pro-

perly ﬁxed samples can last for years with appropriate main-

tenance.

Once a sample has been ﬁltered it should be analysed im-

mediately. It is possible to store samples for one day, but this

may impede taxonomic identiﬁcation. If samples are to be

stored the ﬁlter should be kept moist. A drop of ﬁltered sea-

water should be added to the ﬁlter on the microscope slide.

A cover slip should be placed on top of the ﬁlter and gently

pressed downward. The microscope slide is then placed in the

refrigerator to reduce any evaporation. If the ﬁlter has dried

a new drop of seawater should be added without lifting the

cover slip prior to analysis of the sample on the microscope.

Discussion

Filtering with semitransparent ﬁlters is a rapid method for

concentrating phytoplankton samples before enumeration us-

ing light microscopy. It is a ﬂexible method as the volume ﬁl-

tered can be easily altered depending on the target species for

analysis. The detection limit can be improved by increasing

the volume ﬁltered. The equipment needed for this concen-

tration method is standard laboratory equipment and rela-

tively inexpensive to purchase.

The main advantage of using the ﬁltration method is the

short handling time from when the water sample arrives in

the laboratory to when it is analysed. The pore size of the

ﬁlters determines the size of the cells retained. If only a de-

ﬁned size fraction of the phytoplankton is of interest, using

ﬁlters with a speciﬁc pore size could be advantageous as this

reduces the amount of background non target particles. The

equipment needed for the method is in most cases relatively

straightforward, compact and easy to use in the ﬁeld.

The method has some disadvantages compared to other

methods. Some difﬁculties may arise in the taxonomic char-

acterisation of phytoplankton cells on ﬁlters as they cannot be

physically manipulated to change their orientation to allow

better examination of morphological features. Any non ran-

dom distribution on the ﬁlter could interfere with the results

if only a portion of the ﬁlter is analysed. The main disad-

vantage of this method is that cells can become distorted, or

destroyed during the ﬁltration process. This method is not

recommended for fragile and delicate phytoplankton species,

e.g. Haptophytes and Chrysophytes, since these species would

be unidentiﬁable after the treatment. The semitransparent ﬁl-

ter method favours the more robust species, e.g. diatoms and

thecate dinoﬂagellates (Figs. 2 and 3).

References

Fournier RO (1978) Membrane ﬁltering. In: Sournia, A (ed)

Phytoplankton manual. Monographs on oceanographic method-

ology 6, Unesco, Paris, p. 108-112

Figure 2. Dinophysis sp. observed on a semitransparent ﬁlter.

Figure 3. Rhizosolenia sp. observed on a semitransparent ﬁlter.

NC *=

CF =

IOC Manuals & Guides no 55

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 7 Filter - transfer - freeze

Introduction

Precise and accurate identiﬁcation and enumeration of phy-

toplankton cells in ﬁeld samples is fundamental to the main-

tenance of time-series data on distribution and abundance

of taxa, as well as for biological oceanographic research and

plankton surveillance programmes, e.g. for harmful algal

blooms. Such counting methods are also required for cell enu-

meration in unialgal or multialgal cultures for laboratory or

mesocosm experiments on phytoplankton. In this sense, the

term “phytoplankton” is used loosely to include all eukaryo-

tic microalgae, protists, cyanobacteria and unicellular benthic

and epiphytic taxa, including cysts and other resting stages.

Both “classical” methods based upon microscopic observa-

tions of morphological features of whole cells and molecular

methods (nucleic acid hybridisation, antibodies, lectins, etc.)

are now available for comparison (Godhe et al. 2007).

Although the ﬁlter-transfer-freeze (FTF) technique is consi-

dered among the classical approaches for cell counting and

identiﬁcation, it is not speciﬁcally a cell enumeration met-

hod, but rather a means of cell concentration, collection,

and transfer for counting by alternative means. When app-

lied correctly, the accuracy and reproducibility of cell counts

performed by the FTF technique is more a function of the

subsequent counting and identiﬁcation methods than of the

FTF procedure itself. The FTF method was introduced more

than two decades ago (Hewes and Holm-Hansen 1983), but

in spite of its simplicity and proven effectiveness, the method

has not been widely employed. This is regrettable because the

FTF method can be applied for both critical taxonomy (with

some caveats) and for rapid but superﬁcial analysis of phy-

toplankton samples. The original method was designed with

respect to nanoplankton and indeed appears to work best for

taxa in the size range of ca. 5 – 200 µm diameter. Smaller cells

tend to get lost to the ﬁlter and larger organisms do not trans-

fer well. Nevertheless, this size-range embraces most of the

diatoms and nanoﬂagellates of interest and chain-formation

does not markedly decrease transfer efﬁciency from the ﬁlter

to slide. The method has been rigorously tested for use in

ﬁeld monitoring of phytoplankton in the Gulf of St. Lawren-

ce region of Atlantic Canada (J. Smith and K. Pauley, Dept.

of Fisheries and Oceans, Canada, unpublished manual) and

in comparison with alternative methods for counting cells in

cultures of the marine dinoﬂagellate genus Alexandrium (Ra-

fuse 2004). Consolidation of the methodological variants in

this chapter may assist in wider dissemination of this rather

neglected method for phytoplankton cell enumeration.

Materials

Equipment

• Electric or manual vacuum pump (preferably with va-

cuum gauge)

• Filtration apparatus (funnel, clamp, frit, rubber stopper,

connecting rubber or Tygon tubing) – single or multiple

as required

• Vacuum trap ﬂask (if connected to electric pump)

• Waste disposal ﬂask (for ﬁltered ﬁxatives/preservatives)

• Freezer (-20 ˚C), dry ice or cold block (prefrozen)

• Digital or mechanical cell counter (optional)

• Research microscope equipped with bright-ﬁeld, phase-

contrast or Normarski optics

• Standard glass microscope slides

• Glass cover slips (24 x 24 or 24 x 50 mm)

• Watch glass

• Polycarbonate membrane ﬁlters (25 mm); Poretics™ or

Nuclepore™ (pore size: 3 µm or dependent upon size

distribution of taxa of interest)

• Pasteur pipettes with rubber bulb

• 0.5 or 1.0 l plastic squirt bottle

• Millipore-type ﬂat forceps

• Grease pencil (optional)

• Source of ﬁltered (0.22 µm) seawater

• Fixatives/preservatives (if desired)

• 95% or 70% ethanol (for cleaning)

Laboratory facilities

The FTF method and subsequent microscopic analysis requi-

re no sophisticated facilities. The technique can be performed

even in a rudimentary “laboratory”, at dockside or on board

ship, if a means of quick freezing of the slide is available. In

the absence of a laboratory freezer, the slide can be frozen

upon dry ice, liquid nitrogen (carefully!) or upon an alumi-

num or plastic cold block that has been prefrozen at -20 to

-80 ˚C and maintained in a well-insulated container. The ba-

7 The ﬁlter - transfer - freeze method for quantitative

phytoplankton analysis

Allan Cembella*

and Cheryl Rafuse

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany

Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Canada

*Author for correspondence: [email protected]

Figure 1. Filtration and vacuum apparatus used in the FTF method.

IOC Manuals & Guides no 55

Chapter 7 Filter - transfer - freeze

Scope

Appropriate for ﬁxed, labelled, or unﬁxed planktonic chain-for-

ming or unicellular organisms.

Detection range

Detection range is dependent on the volume ﬁltered. Best pre-

cision and counting accuracy is achieved with transfer of 200

– 400 cells per taxon of interest to the slide surface.

Advantages

Can be applied for enumeration of cells prepared with a variety

of different preservation and labelling methods or from fresh

samples. The method can accommodate alternative microsco-

pic techniques: bright ﬁeld, phase-contrast, Normarski, and

epiﬂuorescence methods. The preparation is extremely simple

and rapid (minutes) and is limited only by the ﬁltration time.

Drawbacks

Potential loss of cells in the transfer process and some morpho-

logical distortion of delicate specimens is possible. The ﬁltration

and microscopic method is normally applied serially, i.e. one

sample at a time, although in principle multiple ﬁltration fun-

nels could be used to ﬁlter samples in parallel. Other limitations

of the method are generic to all optical microscopic techniques

(resolution limit, operator error in identiﬁcation, etc.).

Type of training needed

Only a simple practical demonstration of the ﬁltration and

transfer method is required – this method can be mastered in

<1 hour with an expected unsuccessful transfer occurring only

in the ﬁrst ﬁve attempts. Phytoplankton identiﬁcation requi-

res continuous training over years with in-depth knowledge of

taxonomic literature.

Essential Equipment

Vacuum ﬁltration apparatus with pump, optical microscope

and a source of rapid freezing (-20 ºC freezer, dry ice, or cold

block).

Equipment cost*

100 – 2000 € (excluding microscope). Estimated cost for accep-

table microscope ranges from 3,000 € for a simple bright ﬁeld

or student-grade microscope to >60,000 € for a fully equipped

research microscope with Normarski or epiﬂuorence capabili-

ties.

Consumables, cost per sample**

Less than 2 €/2 US $ (determined mostly by the cost of the

ﬁlter).

Processing time per sample before analysis

Varies with the volume of water ﬁltered, but typically <5 mi-

nutes.

Analysis time per sample

Limited by the degree of scrutiny required and the complexity

of the sample. For accurate and precise counts from monocultu-

res or simple mixtures <10 minutes would be expected, whereas

for complex plankton matrices from dense ﬁeld samples up to 1

hour per sample may be required. These timings are dependent

on the skill of the analyst.

Sample throughput per person per day

10 – 50 samples per day (8 working hours), depending upon

the cell concentration and sample complexity.

No. of samples processed in parallel

Up to 12 ﬁltrations may be carried out simultaneously, but mi-

croscopic observations must be in series.

Health and Safety issues

Determined only by the toxicity of the ﬁxative and labelling

components (if any) used for the preparation of the samples.

Appropriate health and safety procedures must be followed at

all times. Continual microscope work could result in strain

injury. It is important to incorporate breaks into the daily ana-

lyses time. Ergonomic adjustments to the microscope and the

working place is recomended.

*service contracts not included

**salaries not included

The fundamentals of

The ﬁlter - transfer - freeze method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 7 Filter - transfer - freeze

sic requirements are approximately 1 m

free bench space for

vacuum pump, ﬁltration apparatus, in addition to space for

mounting of a standard microscope.

Methods

Sample collection, ﬁxation, storage and preparation for mi-

croscopic analysis have been well described in the UNESCO

Manual on Harmful Marine Microalgae in the chapter by

Andersen and Throndsen (2004) on estimating cell numbers.

This reference should be consulted for general procedures.

Only speciﬁcs relevant to the FTF method are provided here

as follows:

1 Prepare a clean glass microscope slide by outlining a

circular ﬁlter-separator (25 mm) with a black grease pencil.

This step may be omitted as it does not always provide an

advantage for corralling the cells on the slide;

2 Mount a polycarbonate membrane ﬁlter (25 mm diame-

ter) onto the the ﬁltration apparatus and ensure that the

ﬁlter remains centred when clamping or threading the ﬁl-

tration funnel (Fig. 1);

3 Filter a suitable volume of plankton sample (must be deter-

mined empirically after initial trials and rough counting of

taxa of interest) under gentle vacuum (<200 mm Hg) but

at a suitable ﬂow rate (ca. 50 mL min

-1

);

4 Continue ﬁltration until a few drops are left in the fun-

nel, then turn off pump and allow the residual vacuum to

draw the ﬁlter just to the point of dryness. Release vacuum

slowly;

5 Remove the ﬁltration funnel and with ﬂat forceps (Mil-

lipore™- type) carefully lift off the ﬁlter, invert it onto the

centre of the prepared glass slide (in the centre of the grease

pencil ring if this step was followed);

6 Place the glass slide onto a cold ﬂat surface (dry ice or cold

block in the freezer) ﬁlter side up and allow the ﬁlter to

freeze completely to the slide (usually two minutes is suf-

ﬁcient time);

7 Remove the glass slide from the cold surface or from the

freezer, and place on a ﬂat counter area with the ﬁlter side up;

8 If the ﬁlter slide is crackling frozen, wait a few seconds

until the frost just begins to disappear. With a ﬂat forceps,

grasp the edge of the ﬁlter and with a smooth rolling mo-

tion of the hand, peel back the ﬁlter parallel to the speci-

men surface until the ﬁlter is free from the slide. A deposit

of material should be apparent at the centre of the slide

(Fig. 2). Retain the ﬁlter (shiny side up) in a watch glass or

Petri dish for further observation (Fig. 3);

9 Add a drop or two of ﬁltered seawater (0.22 µm) with a

Pasteur pipette to the sample at the centre of slide. Place

the glass coverslip (25 x 50 mm) carefully upon the glass

slide (Fig. 4);

10 Identify and count the cells by viewing the entire contents

trapped inside the grease chalk circle or under the entire

cover slip. Cells should be counted in zigzag transects un-

der the appropriate magniﬁcation for identiﬁcation;

11 Calculate cell numbers as the total cells counted on the

slide per unit volume of sample ﬁltered (assumes 100%

transfer efﬁciency from the ﬁlter to the slide).

Useful notes on the application of the method

1 The optimal ﬁlter pore size must be determined by the

cell size of the taxa of interest, the concentration of the

suspended particulates in the seawater sample and the vo-

lume of seawater to be ﬁltered. In general, use the largest

available pore size for the ﬁlter (to maximise ﬂow through

and minimise clogging) that will retain all of the key taxa.

For nanoplankton samples, 3 or 5 µm pore size is usually a

good compromise;

Figure 2. Removal of ﬁlter from microscope slide using forceps.

Figure 3. Microscope slide after the ﬁlter has been removed. The

ﬁlter is retained in the Petri dish

Figure 4. A drop of seawater is added to the microscope slide

containing the sample and a coverslip added.

IOC Manuals & Guides no 55

Chapter 7 Filter - transfer - freeze

2 A drop or two of ﬁltered seawater can be useful in seating

the ﬁlter on the ﬁlter apparatus. Make sure the ﬁlter is al-

ways mounted in the same orientation, usually shiny-side

up for most types (but check!);

3 It is not usually feasible to count more than a few hundred

cells of a given taxon on a single slide, but counts must be

sufﬁciently high (see Andersen and Throndsen 2004) to

avoid having to count many replicate slides for statistical

validity. The sample must be thoroughly homogenised in

the bottle by a gentle end-to-end and side-to-side rolling

motion between the hands before ﬁltration;

4 A wet ﬁlter will result in the loss or mobilisation of cells on

the ﬁlter as it is removed from the funnel. This is likely to

be the largest source of error in the method. On the other

hand, sucking the ﬁlter under high vacuum to complete

dryness or for a prolonged time will embed the cells in the

membrane and they will not transfer efﬁciently from the

ﬁlter;

5 Make sure that there are no pleats or folds in the ﬁlter and

that the entire surface is in good contact with the slide;

6 Perform a cursory examination of the upper ﬁlter surface

for residual cells that were not transferred. This can be

done quickly with a stereo-microscope or a standard mi-

croscope under low power (40X). If more than a few cells

are present, the ﬁltration procedure must be repeated;

7 This procedure is designed for immediate observation of

specimens without archiving. Techniques for preparing

semi-permanent mounting and embedding with various

preparations of glycerol and embedding medium for later

taxonomic analysis may be consulted in Hewes and Holm-

Hansen (1983);

8 Either of two techniques can be used for placing the cover

slip: 1) gently lowering one end of the coverslip until con-

tact is made with the water droplet, then letting surface

tension act as the cover slip is lowered at an angle; or 2) the

“bombardier principle”, whereby the cover slip is dropped

gently from just above the water droplet. Take care to avoid

bubbles under the coverslip.

Preservation and storage

The FTF method can be applied to microscopic analysis of

plankton samples directly from seawater, since ﬁltration ﬁrst

immobilises and quick-freezing kills the cells. Cells frozen to

the ﬁlter may be stored in the freezer for several hours (or even

overnight) without apparent damage or effect on subsequent

transfer. Nevertheless, for archival purposes, ﬁxation and/or

preservation of cells may be desired to reduce decomposition

and to maintain morphology for future identiﬁcation and

enumeration. Aldehyde preservatives affect steric conﬁgura-

tion by cross-linking of proteins via multiple interaction with

various amino acid residues and even peptide bonds (Shi et al.

2000), whereas ethanol is a coagulant ﬁxative causing limited

and unstable cross-linkages. Although exhaustive comparative

trials have not been conducted speciﬁcally for the FTF meth-

od, all commonly used preservatives and ﬁxatives (Keller and

Manak 1993, Amann 1995, Cañete et al. 2001), including

Lugol’s iodine solution, formulation of gluteraldehyde, for-

malin or paraformaldehyde (PFA) in various buffer solutions,

as well as saline-ethanol mixtures for application of molecular

probes are generally compatible with this method.

Details of ﬁxation and preservation are beyond the scope of

this chapter, but a few general observations here with respect

to the FTF method are relevant. Lugol’s iodine solution has

been traditionally used to preserve microalgae for counts via

Utermöhl settling chamber method (see Chapter 2, this vol-

ume) and as such can be employed also for the FTF tech-

nique. The disadvantages of this solution are that it strongly

colours cells so that autotrophic and heterotrophic cells can-

not easily be distinguished and cells cannot be readily stained

for epiﬂuorescence microscopy. Decolourisation (i.e. with

thiosulphate) is possible but often results in a loss of mor-

phological details and even cell lysis. Lugol’s solution is par-

ticularly effective with highly siliciﬁed structures, e.g. most

diatoms and silicoﬂagellates, but less so for naked and thecate

ﬂagellates for which details are often obscured.

Gluteraldehyde is excellent for ﬁxation and preservation of

structures, particularly for subsequent analysis by SEM or

TEM, but is relatively expensive and highly toxic. Use of

gluteraldehyde is therefore discouraged for the FTF method,

which does not usually yield the highest quality samples for

electron microscopy in any case. Other aldehydes ﬁxatives are

preferred, for a compromise preservation of siliceous, calcare-

ous and cellulosic structures of phytoplankton. Acidic forma-

lin preparations can be very destructive to calcareous struc-

tures, such as those of coccolithophorids.

A high quality universal preservative for most phytoplankton

samples based upon buffered paraformaldehyde (PFA) has

been extensively tested with the FTF method. This formula-

tion stabilises most cell structures and provide a robust re-

sistance to ﬁltration and freeze-thaw damage. The recipe is

therefore given here as follows:

Preparation of 10% PFA:

1 Add 100 g of PFA powder to 800 mL dH

O (or a suitable

buffer);

2 Heat to about 60-80

C with constant magnetic stirring;

3 Add NaOH (1N) gradually until PFA is just completely

dissolved (do not add too much!);

4 Let the solution cool to room temperature

5 Adjust to desired pH using NaOH or HCl (1N);

6 Add H

O (or buffer) to make up to 1000 mL.

For most plankton samples, acidity should be adjusted to pH

4 and ﬁnal concentration in the sample should be 1 – 2%.

Thereafter, samples can be archived for many months prior to

ﬁltration for FTF microscopic analysis.

Discussion

The FTF technique has some of the advantages and draw-

backs of other ﬁltration-based techniques. Nevertheless, the

problems cited for direct counting of cells upon ﬁlters (morp-

hological distortion of cells, poor contrast, low resolution,

difﬁculty in applying stains, etc.) (Hewes and Holm-Hansen

1983) are effectively eliminated in the FTF method. Rela-

tive to other classical counting methods, such as the Uter-

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 7 Filter - transfer - freeze

möhl settling chamber method, the FTF technique has the

advantage that there is no time-lag for sedimentation, with

attendant concerns about physical disturbance of the setting

regime (e.g., on board ship). Counts can be performed with a

standard research microscope rather than with a less common

inverted optical system. As a cell concentration and transfer

technique, the FTF method can be successfully combined

with other methods for critical taxonomy by epiﬂuorescence

microscopy, such as the calcoﬂuor staining method for the

cellulose plates of dinoﬂagellates, or application of ﬂuorescen-

ce probes for nucleic acids or antibody targets. In the ﬂuores-

cence approach, the appropriate stains can be applied at the

edge of the cover slip and drawn across the sample by apply-

ing a laboratory tissue at the opposite corner of the cover slip.

Furthermore, FTF preparation time is only a few minutes,

with ﬁltration as the rate-limiting step. This makes it easy to

adjust the cell concentration on the counting slide for opti-

mal accuracy and precision of counting (typically 200 - 400

cells per taxon of interest) by varying the volume ﬁltration or

by diluting the sample with ﬁltered seawater. The latter may

be important to avoid cell overlap, e.g. of diatom chains, or

clumping of aggregated cells, particularly of concentrated net

plankton hauls.

As with other ﬁltration-based techniques, the physical loss

or damage of cells must be carefully monitored during the

preparation procedures. Sloppy ﬁltration techniques, leakage

of the ﬁltration funnels, overloading the ﬁlter and failure to

thoroughly clean the ﬁltration apparatus with detergent bet-

ween usages, followed by rinsing with ethanol then deionised

water between each sample, can cause cell loss. Sometimes the

sample may leak outside of the grease chalk circle and there-

fore may not be counted; for this reason it is often preferable

to eliminate this circle and merely count the entire surface

area under the cover slip.

In comparative testing of the FTF method against whole-cell

ﬂuorescence in situ hybridization (FISH) and the Utermöhl

settling chamber methods for counting cultured cells of the

marine dinoﬂagellates Alexandrium tamarense and A. osten-

feldii (Rafuse 2004), the ﬁltration-based methods yielded a

consistently higher coefﬁcient of variation, most of which was

attributed to variable cell loss. Yet in a detailed comparison of

17 alternative cell counting methods for A. fundyense (Godhe

at al. 2007), the FTF method exhibited among the lowest

standard errors among replicate counts (n = 4 or 5) within the

concentration range of 102 to 105 cells L

-1

. During the ﬁltra-

tion step, cells can be lost within the ﬁltration apparatus, i.e.

between the funnel and base or via leakage from the interface

between the ﬁlter holder and the ﬁlter. All standard funnel ﬁl-

tration apparatus (Fig. 1), such as the common clamp-systems

obtainable from Millipore™, Gelman/Pall™, Sartorius™,

etc., are acceptable for this method, including the threaded

mounting (Radnoti™-type) systems for attaching the funnel

to the ﬁlter base. The clamp-type systems are preferable be-

cause they are simpler and do not permit the possible loss of

cells between the O-rings (Rafuse 2004) that may occur for

threaded-type ﬁlter systems. Evidence of this cell loss from

concentrated samples may be observed by visual inspection

of the funnels and other components after the ﬁlter is remo-

ved. Use of glass versus plastic (polystyrene) funnels does not

appear to be critical, but the funnels should be transparent

to observe the ﬁltration process. To maximize transfer of all

cells to the ﬁlter, the ﬁltration should proceed until the ﬁlter

surface is just dry (i.e. the upper surface is no longer shiny),

but not beyond this point, to preserve cell integrity. Dirty ap-

paratus and ultra-slow ﬁltration (<2 mL min

-1

) can also lead

to adherence of cells to the walls of the ﬁltration funnel; such

loss can be considerable – >5% of cells under a worst case sce-

nario and often taxonomically biased towards mucilaginous

cells or aggregates. In any case, this selective loss from the

harvested sample can be veriﬁed if necessary by thoroughly

washing down the walls of the funnel system with a high-

pressure stream of ﬁltered seawater from a squirt bottle and

recovering the cells on a fresh ﬁlter under gentle vacuum. If

few cells are found, loss of cells in the system can be assumed

to be minimal.

Breakage of cells upon contact with the ﬁlter surface, and es-

pecially sucking the ﬁlter beyond the dry point under high va-

cuum can also account for considerable cell loss or distortion

in all ﬁlter-based methods. For this reason, vacuum pressure

should be kept low (<15 mm Hg) but substantial enough to

ensure a steady ﬂow of ﬁltrate (a couple of drops per second

or about 50 mL min

-1

is acceptable). Correct operation of the

ﬁltration protocol can be veriﬁed by observation under a dis-

secting microscope of random or haphazard distribution cells

on the upper ﬁlter surface of unialgal cultures. For mixed algal

assemblages, evidence of clumping or patchy cell distribution

over the ﬁlter surface is a sign of poor ﬁltration technique.

The foregoing are generic strengths and weaknesses of all ﬁl-

tration-based counting methods. Surprisingly, two elements

that are speciﬁc to the FTF method – freeze-thaw and ﬁlter-

transfer – do not contribute in a major way to cell loss or

morphological distortions when the method is applied cor-

rectly. Quick freezing is preferable to slower methods since

the former leads to less crystal formation and hence less cell

breakage. Cells may also break when the ﬁlter is ripped off the

frozen sample that contains the cells, or if the cells are weak,

not completely frozen, or have thawed too quickly. Practice

will generally ensure excellent reproducibility.

Acknowledgements

The authors are indebted to J.C. Smith and K. Pauley, Gulf

Fisheries Centre, Dept. of Fisheries and Oceans, Canada for

collating details of the method in the unpublished ﬁeld and

laboratory manual for the collection, identiﬁcation and enu-

meration of toxic marine phytoplankton from southern and

eastern regions of the Gulf of St. Lawrence.

References

Amann R (1995) In situ identiﬁcation of micro-organisms by whole

cell hybridization with rRNA-targeted nucleic acid probes. Mol

Microb Ecol Manual 3.3.6: 1-15

Andersen P, Throndsen J (2004) Estimating cell numbers. In: Halle-

graeff GM, Anderson DM, Cembella AD (eds) Manual on harm-

ful marine microalgae, Monographs on oceanographic methodo-

logy. UNESCO Publishing, Paris, 99-129

Cañete M, Juarranz A, López-Nieva P, Alonso-Torcal C, Villanueva

A, Stockert C (2001) Fixation and permanent mounting of ﬂuo-

rescent probes after vital labelling of cultured cells. Acta Histo-

IOC Manuals & Guides no 55

Chapter 7 Filter - transfer - freeze

chem 103: 117-126

Godhe A, Cusack C, Pedersen J, Andersen P, Anderson DM, Bres-

nan E, Cembella A, Dahl E, Diercks S, Elbrächter M, Edler L,

Galuzzi L, Gescher C, Gladstone M, Karlson B, Kulis D, LeGres-

ley M, Lindahl O, Marin R, McDermott G, Medlin MK, Naust-

voll L-J, Penna A, Töbe K (2007) Intercalibration of classical and

molecular techniques for identiﬁcation of Alexandrium fundyense

(Dinophyceae) and estimation of cell densities. Harmful Algae 6:

56-72

Hewes D, Holm-Hansen, O (1983) A method for recovering na-

noplankton from ﬁlters for identiﬁcation with the microscope:

the ﬁlter-transfer-freeze (FTF) technique. Limnol Oceanogr 28:

389–394

Keller GH, Manak MM (1993) DNA probes. Second Edition,

Stockton Press, New York, USA

Rafuse C (2004) Effects of physiological and environmental condi-

tions on rRNA probes for two species of microalgae, Alexandrium

ostenfeldii and A. tamarense. MS dissertation, Dalhousie Univer-

sity, Halifax, Canada

Shi S-R, Gu J, Taylor CR (eds) (2000) Antigen retrieval techniques:

immunohistochemistry and molecular morphology. Eaton Pub-

lishing, Natick, MA, USA

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Introduction

In water quality analysis and monitoring the ability to count,

capture and save images of the particles or organisms in a sam-

ple is advantageous. Traditional “particle counting” methods,

such as microscope particle analysis, can be slow and tedious.

The FlowCAM, an imaging ﬂow cytometer developed by

Fluid Imaging Technologies, captures digital images of par-

ticles in a ﬂuid stream using laser light detection, enabling the

measurement of many cell parameters, such as length, width,

equivalent spherical diameter and ﬂuorescence (Sieracki et al.

1998). The captured images from a sample can be studied vi-

sually as well as post-processed by a computer to automatical-

ly search for particles of a certain type or class. The FlowCAM

detects and measures ﬂuorescence emissions at two different

wavelengths, typically red and orange ﬂuorescence, indicating

the presence of chlorophyll or phycoerythrin within the indi-

vidual particles/cells in a ﬁeld sample. This technology is very

similar to a ﬂow cytometer that captures only ﬂuorescence

and scatter properties of a particle. However, the FlowCAM

merges the technologies of a ﬂow cytometer and an imaging

microscope – resulting in the Flow Cytometer and Microsco-

pe (FlowCAM – Fig. 1). The FlowCAM automatically counts

and images particles or cells in a discrete sample. Using the

image analysis data generated during sample processing, the

FlowCAM software uses image libraries previously created by

the user of target groups or classes that can assist in analysis

and classiﬁcation. Overall, the FlowCAM can be customised

to accommodate most ﬁeld environments and can be used

to detect and quantify different algal groups within a sample

(Zarauz et al. 2009), including harmful algal species (Buskey

and Hyatt 2006).

Materials

Equipment

For quantitative microphytoplankton analysis (20-300 µm)

a Benchtop or Portable FlowCAM (VS IV) is required (Figs

2 & 3). The FlowCAM must be equipped with either a blue

(488 nm) or green laser (532 nm) for ﬂuorescent and par-

ticle detection. In addition the instrument should have and

be setup with, a 4X or 10X objective depending on the size of

the cells to be visualised and quantiﬁed. Each objective must

use a corresponding rectangular. tubular glass Flow Cell (100

or 300 µm depth) in which all cells are to be analysed. For dis-

crete volume sampling a funnel stand or pipet tip apparatus

must be used, as provided with the instrument.

Chemicals and consumables

• Filtered Seawater (FSW)

• Distilled water

• Calibration Beads (5, 10, 20, 50 & 100 µm)

• Disposable plastic pipets tips (1-10 mLs)

• Small funnels

• FlowCAM Flow Cells (100 or 300 µm depth)

• Silicone Tubing

• Nylon mesh (100 or 300 µm)

• Graduated cylinders to measure volumes to be processed

Solutions for preservation

• 10% buffered formalin solution (see Appendix 1 for pre-

paration instructions)

• Formalin: Acetic acid (see Appendix 1)

Methods

The FlowCAM was originally developed for phytoplankton

detection and quantiﬁcation and is ideally suited for analysis

of natural ﬁeld samples that contain speciﬁc groups or species

of microplankton that are usually counted using traditional

microscopic techniques. To begin, the FlowCAM (including

the integrated computer) and laser need to be turned on, and

the laser allowed to warm up for approximately 20 minutes

(according to FlowCAM manual). FlowCAMs are equipped

with either a green or blue laser; both can be used for algal

ﬂuorescence detection. As the FlowCAM laser is warming

8 Imaging ﬂow cytometry for quantitative

phytoplankton analysis — FlowCAM

Nicole J. Poulton*

and Jennifer L. Martin

J.J. MacIsaac Aquatic Cytometry, Facility Bigelow Laboratory for Ocean Sciences, 180 McKown Point Road, P.O. Box 475 West Boothbay

Harbor, ME 04575, USA

Fisheries and Oceans Canada Biological Station, 531 Brandy Cove Road, St. Andrews, NB E5B 2L9, Canada

*Author for correspondence: npoulton@bigelow.org

Figure 1. Illustration of the ﬂuidics and optics of the FlowCAM.

IOC Manuals & Guides no 55

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Scope

The FlowCAM method captures digital images of particles and/

or cells. This enables the instrument to measure cell abundance

and many cell parameters, such as length, width, equivalent

spherical diameter and ﬂuorescence.

Detection range

The detection range depends on the volume of sample analy-

sed.

Advantages

Manual labour required for processing and handling is mini-

mised, compared to traditional microscopy techniques. The

method produces a non-biased digital record and instantaneous

image analysis data on every particle or cell within a sample.

The captured images from a sample can be studied visually as

well as post-processed by a computer to search and quantify

cells of a certain type or class. Portable FlowCAMs are also av-

ailable for ﬁeld measurements.

Drawbacks

Cells are required to have a distinct morphology to be readily

identiﬁed by FlowCAM. Different objectives are needed for

different phytoplankton size ranges. Preservation of the sam-

ple is not recommended, live samples are optimal, as cell auto-

ﬂuorescence can decline or is removed with certain preservation

techniques (ie. Utermohls). Heavy particle loads (riverine or

estuaries) could interfere with image capture – dilution of the

sample for accurate abundances may be required.

Type of training needed

1. To use FlowCAM: Approximately one-two days of training

would be required with guidance from a trained individual and

follow-up support.

2. To troubleshoot and QC: A more experienced analyst with

up to 6 months experience would be more effective at troubles-

hooting the instrument. The company that manufactures the

FlowCAM, Fluid Imaging Technologies, provides on-site, web-

based, and over the phone customer assistance when needed.

3. Taxonomic expertise is required to interpret images and re-

sults.

Essential Equipment

FlowCAM VS IV with 4X & 10X objectives, associated Visu-

alSpreadsheet (ViSp) software, and Flow Cells.

Equipment cost*

$75,000-85,000 US (depends on the model – Benchtop or Por-

table), see Appendix 2.

Consumables, cost per sample**

1-5 US dollars.

Processing time per sample before analysis

Ranges from 15 minutes-1 hour depending on the volume run

and particle density.

Analysis time per sample

Ranges from 1 hour to minutes. The time consuming part is

identifying cells and developing image libraries for automated

sample analysis (hours of time upfront).

Sample throughput per person per day

4-36 depending on the time of run and analysis time.

No. of samples processed in parallel

One sample at at time.

Health and Safety issues

Minimal, caution and general safety practices must be used

when using the laser within the instrument and preserved sam-

ples.

*service contracts not included

**salaries not included

The fundamentals of

The FlowCAM - maging ﬂow cytometer method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 8 Imaging ﬂow cytometry - FlowCAM

up, the FlowCAM VisualSpreadsheet (ViSp) software can be

started.

Before running natural ﬁeld samples, the FlowCAM needs to

be ”setup”, meaning the values for triggering using ﬂuores-

cent detection (thresholds) and the cell size range of particles

to be collected need to be determined. The FlowCAM also

needs to be properly focused, similar to how a microscope

would work. In order to get good visualisation of the particles

in each sample optimal focus is required. The set up process

usually takes between 20-40 minutes. Once the settings for

microplankton detection are determined the settings do not

change between samples, unless the ecosystem or background

within a sample (such as particle load) changes. More details

on how to set up the instrument can be found in the Flow-

CAM Operators Manual (Anonymous 2009).

For analysing and counting microplankton in natural ﬁeld

samples 4X, 10X or 20X objective are usually used in com-

bination with an appropriate Flow Cell (Table 1). The Flow

Cell is a rectangular glass tube that can vary in size depen-

ding on the objective used. Silicone tubing is afﬁxed to both

ends and allows the sample to pass through the tube to a pe-

ristaltic pump downstream of the Flow Cell. The Flow Cell

mimics the glass side of a microscope. Note that the Flow

Cell is oriented vertically in the instrument differing from the

microscope mounted slide that is positioned horizontally. In-

stallation of a 100 µm depth Flow Cell is described in Fig. 4.

Data Acquisition

1 Each ﬁeld sample needs to be thoroughly mixed by gentle

rotation to resuspend any particles that may have settled;

2 Using a graduated cylinder, a predetermined volume of

each sample is dispensed into a funnel or pipet tip;

3 Set the ViSp FlowCAM software to start analysis and col-

lect the data (ﬁle folder name and location) in Trigger

mode;

4 Start the peristaltic pump, once data acquisition in the

ViSp FlowCAM software begins the sample will now be

analysed for ﬂuorescent events (phytoplankton) that pass

by the laser that is aligned with the Flow Cell.

For most ﬁeld samples, between 10-100 mL of each sample

is analysed and processed. Sample processing rate (ﬂow rate)

will depend on the density of cells in the ﬁeld sample, and

the dimensions of the Flow Cell used. As a starting point, set

the standard VWR pump to fast mode and the speed dial to

5. As the sample is ﬂowing through the Flow Cell, check the

ﬂuorescence trigger rate using ”Trigger Mode Setup”. If the

images are appearing at a rate more than one per second begin

to turn down the ﬂowrate of the pump until the optimum

level is attained. In somes cases the microphytoplankton con-

centration will be low and there will appear to be no trigger

events. The user should be aware that increasing the ﬂowrate

too much (PRIME mode) may cause image distortion and

prevent accurate image capture. To test the ﬂuorescence set-

tings (thresholds) of the FlowCAM it is recommended that a

diluted phytoplankton culture (100-1000 fold) be used ini-

tially to verify the settings being used. It is essential that the

Flow Cell and funnel are rinsed throughly afterwards to pre-

vent contamination of subsequent samples to be quantiﬁed

and analysed. Typically, a ﬁeld sample can be processed in

30-40 minutes.

During the run, the operator should occasionally mix the

sample in the funnel using a pipette to prevent particles or

cells from settling. The operator should also check the Flow

Cell for any potential clogs or blockages. If clogging occurs,

the user would typically observe particles sitting at the en-

trance to the glass tubing. If a clog occurs the operator should

end the run and restart the analysis. To prevent clogging from

occurring, screening or sieving the sample prior to analysis

is recommended using nylon mesh (100 or 300 µm) depen-

ding on the depth of the Flow Cell being used. During a run

the operator should also periodically pinch the tubing down

stream of the Flow Cell in order to prevent large particles

from blocking the entrance to the Flow Cell. Near the end

of a run the funnel should be rinsed with FSW to ensure all

particles are removed from the funnel (and have entered the

Figure 2. Benchtop FlowCAM.

Figure 3. Portable FlowCAM (VS IV).

Cell Size Range Objective Flow Cell Flow Cell

Dimensions

(Depth x Width)

10 µm – 100 µm 10 X FC100 100 µm x 2 mm

20 µm – 300 µm 4 X FC300 300 µm x 3 mm

60 µm – 600 µm 4 X FC600 600 µm x 6 mm

Table 1. Recommended cell size range (diameter) and objectives

for different FlowCAM Flow Cells.

IOC Manuals & Guides no 55

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Step 1. Remove the CH300 Flow Cell Holder from the Focu-

sing Collar by loosening the thumb screw and pulling the Flow

Cell Holder away from the focusing rails. Loosening the thumb

screw is accomplished by turning the screw counter-clockwise.

The 10X objective can be replaced with a 4X objective.

Step 2. Unscrew the retaining cap from the Flow Cell Holder.

This is accomplished by turning the cap counterclockwise until

it separates from the Flow Cell Holder.

Step 3. For some FlowCAM models the CH300 Flow Cell Hol-

der has a backer ring (previously installed) that needs to be

adjusted when using the 100 µm depth ﬂow cell. The backer

ring can be moved by crossing your index and middle ﬁngers

and inserting them into the inside diameter of the ring. Move the

ring in or out until it backs the ﬂow cell achieving a good ﬁt when

the Flow Cell Holder cap is tightened.

Step 4. Place a 100 µm Flow Cell into the pre-cut notches of the

Flow Cell Holder. Ensure that the inlet tube is at the same end

as the thumb screw. The silicone tubing has been previously

attached to the Flow Cell.

Step 5. Reinstall the retaining cap onto the Flow Cell Holder

by turning the cap counter-clockwise. The cap is signiﬁcantly

tightened when the Flow Cell cannot be moved. The user will

have to hold the Flow Cell straight as cap tightening will tend to

twist the Flow Cell. Caution – overtightening will break the 100

µm Flow Cell.

Step 7. Connect the outlet tubing to the ﬂuid pump (peristaltic

pump). Ensure the efﬂuent tube is connected to an appropriate

waste container. Once the 100 µm Flow Cell has been installed

the Flow Cell needs to be thoroughly rinsed with ﬁltered seawa-

ter (FSW) and the ﬂuid level brought to the neck of the funnel.

Step 6. Re-install the Flow Cell Holder onto the Focusing Collar

and tighten the thumb screw. Connect the inlet tube of the Flow

Cell to the sampling funnel using the funnel stand apparatus.

Figure 4. Installation of a 100 µm depth Flow Cell.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Flow Cell) before it runs dry. Preventing air from entering the

Flow Cell between samples will prevent tiny bubbles from

forming that could be detected as particles and imaged in the

next sample. Any clogs can be removed by reversing the pump

direction or inverting the Flow Cell and rinsing with FSW or

distilled water.

Data Analysis

Each ﬂuorescent particle/cell is digitally acquired and archi-

ved by the FlowCAM ViSp software (Fig. 5). Analysis of the

samples is done either immediately upon completion of the

sample acquisition or at a later time after many samples have

been acquired or archived. The ViSp software allows the ope-

rator to use previously acquired image libraries of target spe-

cies or organisms to assist in sample analysis. The libraries

are user generated and can be created from images obtained

from cultured organisms or from positively identiﬁed natural

ﬁeld images of the target organism. The libraries allow the

operator to pattern match each ﬁeld sample by ﬁltering and

sorting the data into user deﬁned catagories. This procedure

can be repeated for different target organisms within a single

sample or multiple samples. Once the analysis is completed,

the positively identiﬁed images are veriﬁed by the operator,

and a total count of each class or group is determined. The

ﬁnal cell concentration of each class is determined using an

equation that includes the cell count, volume analysed and

Flow Cell Factor – see Formula for Calculating Results below.

Calibration

Prior to microphytoplankton sample analysis, the FlowCAM

needs to be properly calibrated using an optical micrometer.

The micrometer is used to measure the ﬁeld-of-view of the

camera for each magniﬁcation objective (4x & 10x), enabling

an accurate size calibration for each pixel. This ”calibration

factor” becomes part of the context settings for each mag-

niﬁcation. Prior to shipment, a new FlowCAM is calibrated

to assure accurate measurements of particle concentration

(particles/mL) and for sizing particles. Although no in-ﬁeld

calibration is required, a FlowCAM user can analyse polysty-

rene or latex beads (of known size) for calibration assurance.

Calibration beads can be purchased at a variety of commercial

companies (10-100 µm, Duke Scientiﬁc, USA). To verify the

FlowCAM calibration refer to the FlowCAM Operators Ma-

nual for a detailed step by step protocol.

Preservation and storage

Unpreserved samples are ideal for quantifying and analysing

microphytoplankton in the ﬁeld, and is recommended. Ho-

wever, this is not always appropriate. Preservation of samp-

les should be done using a 10% buffered formalin solution

or formalin:acetic acid, such that the ﬁnal concentration is

approximately 1-2%. Preserved samples should be processed

as soon as possible, as ﬂuorescence detection is essential for

accurate cell detection and enumeration. Chlorophyll ﬂuo-

rescence within the cell decreases with prolonged storage.

Preserved samples are best analyzed using Auto Image Mode,

to assure accurate phytoplankton counts. However, more pro-

cessing time is required in this mode in order to analyze an

adequate sample volume. If longer preservation is required,

the effects of the storage length should be tested on the Flow-

CAM prior to processing samples on a routine basis.

Formulas for calculating results

To determine the concentration of the target microplankton

group or species within a sample three values are needed:

1 The volume processed by the FlowCAM;

2 The cell count of the target group (based on classiﬁca-

tion) (N);

3 The Flow Cell Factor.

Since the camera on the FlowCAM can only visualise a por-

tion of the Flow Cell (usually between 33-95% - depending

on the objective and Flow Cell used) the Flow Cell Factor

takes into account the portion of the ﬁeld of view that is not

visualised by the FlowCAM. The Flow Cell Factor will vary

depending on the objective/Flow Cell combination utilised.

The Flow Cell Factor is calculated automatically by the ViSp

software once the correct dimensions of the Flow Cell used

are provided by the user. Note that Fluid Imaging Technolo-

gies has recently developed a new Flow Cell design that elimi-

nates the need for Flow Cell Factor.

Discussion

The ability to automate the detection and counting of

micro phytoplankton in ﬁeld samples is a huge advantage

for monitoring aquatic systems. With the development of

more automated techniques, such as the FlowCAM there is

a movement to employing more remote automated monito-

ring techniques. The FlowCAM, with further development,

could assist in water quality monitoring for both marine and

freshwater environments. The FlowCAM is ideally suited for

quantifying microphytoplankton between 20-300 µm in size

(however, smaller and larger particles can also be detected).

Cell classiﬁcation using the FlowCAM ViSp software is best

used on species/groups of phytoplankton that have unique

cell characteristics, such as cell size, shape or colour. For ex-

Figure 5. Representative black and white FlowCAM images from a

ﬁeld sample containing the following genera, Dinophysis, Alexan-

drium, Protocentrum and Ceratium.

−

FactorCellFlow

sample of Volume

N count Cell

mLcells ion concentrat Cell

)(

) (













−

FactorCellFlow

sample of Volume

N count Cell

mLcells ion concentrat Cell

)(

) (













IOC Manuals & Guides no 55

Chapter 8 Imaging ﬂow cytometry - FlowCAM

ample, the genera Dinophysis, Ceratium, and Chaetoceros have

many distinctive morphological features which are easier to

classify than different species within the genus Alexandrium.

The FlowCAM is best used to examine a wide range of species

within one sample. It can be used as a monitoring tool for

coastal projects or in the laboratory, processing discrete samp-

les when needed. In order to best evaluate the instrument a

list of Advantages and Disadvantages of the FlowCAM met-

hod are provided below:

Advantages of the FlowCAM Method

1 The amount of manual labour required for sample pro-

cessing and handling is greatly reduced when using a

FlowCAM for microphytoplankton analysis. For discrete

sample analysis (as described in this chapter) the operator

is required to set up and begin a run, but will only oc-

casionally monitor the FlowCAM when it is running and

detecting particles;

2 The FlowCAM records a “non-biased” digital record of

every particle/cell within a speciﬁc size range (determined

by the operator) for further analysis using the FlowCAM

ViSp software. Using traditional microscope techniques,

the operator scans the slide and either seeks out the par-

ticles/cells of interest or identiﬁes all the observed cells on

the slide. This may result in a biased analysis and count of

a sample (which depends on the operator’s attention to de-

tail and identiﬁcation knowledge). The data generated by

the FlowCAM is archived and can be reanalysed by more

skilled individuals when problems arise or when different

analyses are required;

3 In addition to capturing an image for each particle detec-

ted by the FlowCAM, the software provides instant image

analysis on each particle , up to 30 different image para-

meters. For example, particle length, width, equivalent

spherical diameter (ESD), area-based spherical diameter

(ABD), ﬂuorescence, time of ﬂight and aspect ratio are

some of the the primary data measurements obtained. Gi-

ven the data provided, the operator can develop speciﬁc

algorithms or use previously determined algorithms from

the literature for values of interest such as bio-volume and

Carbon:Chlorophyll ratio depending on the needs of the

application or project;

4 The FlowCAM is portable. Even the benchtop model has

a relatively small foot-print (100 x 70 cm) and can be used

in the lab or at sea on board ships. The image capture sys-

tem prevents problems usually associated with vibration;

5 The FlowCAM allows for the visualisation and/or detec-

tion of a wide particle size range (1 µm – 1 mm equivalent

spherical diameter). To detect across this large size range a

series of objectives and ﬂow cells would need to be used.

Based on the size of the target organism to be detected

(for example, Alexandrium), a 10 X objective with a 100

µm depth ﬂow cell would be used. To examine smaller or

larger particles within a sample, other objectives and ﬂow

cell combinations may need to be used.

Disadvantages and Drawbacks

1 In terms of cell identiﬁcation it is essential to achieve the

best possible focus, otherwise the images will be blurry and

will be difﬁcult to analyse;

2 Depending on the ecosystem that is being analysed the

phytoplankton cell size range may vary greatly. Each ob-

jective and Flow Cell size that can be used has a minimum

and maximum cell size range that’s possible (similar to the

limitations of microscopy). Therefore, to best identify all

phytoplankton within a given sample, two FlowCAM runs

may be required at two different magniﬁcations. This de-

pends on what magniﬁcation is acceptable for cell iden-

tiﬁcation. If cells are too large to pass through the Flow

Cell clogging may occur. Therefore, method development

at the beginning of a particular project is important. Ho-

wever, if only one particle/cell size is required and cell

identiﬁcation is not necessary– one FlowCAM run may be

sufﬁcent;

3 A limited number of preservation techniques for phyto-

plankton detection using ﬂuorescent based triggering can

be used. To assure accurate cell concentrations using pre-

served samples Auto Image Mode is recommended, but

more processing time is required (1-2 hours). The best

method of preservation is 1-2% ﬁnal concentration for-

malin. Preserved samples should be stored cold and in the

dark. Prior to processing let the samples acclimate to room

temperature. Lugol’s iodine or other iodine based preserva-

tion methods are not recommended for use with the Flow-

CAM as the ﬂuorescence of the particles is lost and the

high contrast of the images makes it difﬁcult to identify

different phytoplankton groups/genera;

4 In situations where cold samples are being analysed in high

humid environments (for example, samples of deep water

being analysed on board ships), condensation on the ﬂow

cell may interfere with particle detection. The solution to

this problem is to allow the sample to attain room tempe-

rature prior to analysis. The sample should be preserved

prior to manipulation;

5 When the particle load is very high as in riverine samples

containing high concentrations of detritus, more than one

particle may be captured in a single ﬁeld of view on the ca-

mera using Trigger Mode. Although each particle will have

different image analysis values, such as particle length,

width, ESD etc., the ﬂuorescent value of both particles will

be the same. It is also possible to capture “non-ﬂuorescent”

material if the particle load (sediment) is too high and the

instrument is detecting particles in ﬂuorescent detection

mode. In these cases, Auto Image Mode could be utilised;

6 The operator or user should have some knowledge of phy-

toplankton identiﬁcation for analysis of the FlowCAM

data.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Type of training required to operate a FlowCAM

1 To perform sample analysis: Approximately one-two days

of training would be required with guidance from a trained

individual and follow-up support;

2 To troubleshoot and QC: A more experienced analyst

with up to 6 months experience would be more effecti-

ve at troubleshooting the instrument. The company that

manufactures the FlowCAM, Fluid Imaging Technologies

provides on-site, web-based, and over the phone customer

assistance when needed.

Acknowledgements

We would like to thank the support of Fluid Imaging Tech-

nologies who kindly provided assistance and pictures, speciﬁ-

cally, Harry Nelson, Brian Thompson, Jonathan Villeneuve,

Gerald Brown and Doug Phinney. We also thank Murielle

LeGresley for comments and suggestions to the manuscript.

References

Anonymous (2009). FlowCAM Operators Manual, Fluid Imaging

Technologies, Inc., March 2009 Revision 0109.

Buskey, E . and Hyatt CJ (2006) Use of the FlowCAM for semi-

automated recognition and enumeration of red tide cells (Karenia

brevis) in natural plankton samples. Harmful Algae 5:685-692

Sieracki CK, Sieracki M and Yentsch CS (1998) An imaging-in-ﬂow

system for automated analysis of marine microplankton. Marine

Ecology Progress Series 168: 285-296

Zarauz L, Irigoien X, and Fernandes JA (2009) Changes in plank-

ton size structure and composition, during the generation of a

phytoplankton bloom in the central Canatabrian sea. Journal of

Plankton Research 2: 193-207

Materials

Paraformaldehyde powder

1N NaOH

Stirring/hot plate

Chemical fume hood

pH meter

Phosphate buffer ed saline (PBS) or ﬁltered seawater

Distilled H

G/F ﬁlters

1 Mix 900 mL distilled water and 100 g paraformaldehyde powder;

2 Set up on a stirring/hot plate under hood. Heat to approximately 60

C. Do not boil;

3 Stir for approx 1 hour. Turn off heat;

4 Add 100 µL 1N NaOH to “clear” solution. Cool to room temperature. Note: In some cases, not all the paraformaldehyde will go into

solution;

5 Add 100 mL phosphate buffered solution or ﬁltered seawater. , depending on whether the samples are freshwater or marine;

6 Filter through GF/F ﬁlter to remove precipitate;

7 Test pH. Should be 7.4 - 8.0 (approx. equal to seawater);

8 If necessary, add more NaOH.

This yields a 10% solution (approximately).

Preparation of a Formalin: Acetic Acid Solution

Materials

Formaldehyde (37%)

Concentrated acetic acid

1 Mix equal parts of formaldehyde and acetic acid;

2 Using this solution, add 2.5 mL per 100 mL of sample.

Appendix 1

Preparation of a Buffered Paraformaldehyde/Formalin Solution

IOC Manuals & Guides no 55

Chapter 8 Imaging ﬂow cytometry - FlowCAM

Equipment Supplier Cat. Number

€

US $

FlowCAM



VS IV Fluid Imaging Technologies

(FIT), USA

VS IV or portable VS IV 53,760 80,000

50um Flow Cell FIT, USA FC50 28 41

100um Flow Cell FIT, USA FC100 19 28

300um Flow Cell FIT, USA FC 300 19 28

Nylon Mesh Wildco, USA www.wildco.com 24-C27 (53 µm)

24-C34 (100 µm)

24-C48 (300 µm)

89 132

Silicone Tubing (1.6

mm ID)

Cole Palmer, USA K-06411-62 12 18

Calibration Beads Duke Scientiﬁc, USA

www.dukesci.com

4205A (5 µm diameter)

4210A (10 µm diameter)

4220A (20 µm diameter)

4250A (50 µm diameter)

4310A (100 µm diameter)

623 927

Plastic Pipets VWR, USA 14670-103 36 53

Mini-funnel Hutzler Manufacturing

www.usplastic.com

801 0.67 1

Paraformaldehyde VWR, USA MK262159 38 56

Sum approx.

54,536 81,284

Appendix 2

Table 1. Equipment and costs..

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

Introduction

Fluorescence in situ Hybridisation (FISH)

Ribosomes are the sites for protein synthesis in all cells. All

cells are packed with many ribosomes because protein synthe-

sis is an on-going cellular process. Each ribosome is composed

of ribosomal RNAs (rRNAs) and accompanying proteins.

The RNAs within the ribosome fold into a shape that permits

the synthesis of the proteins and this folding of the molecule

is maintained for proper functioning of the ribosome. If mu-

tations occur that violate the folding of the molecule, then the

molecule is non-functional. Within the interior of the folded

RNA is the conserved sequence regions. These cannot change

otherwise the molecule will not fold properly. The more vari-

able regions of the RNA molecule are found on the surface of

the molecule which do not interfere with the folding of the

molecule. Thus, based upon conserved and variable regions of

the rRNA, signature base sequences of varying taxonomic spe-

ciﬁcity can be found (Fig. 1). In other words, regions can be

identiﬁed in the rRNAs that will recognise all members from

as broad a group as a kingdom of organisms or be so selective

to identify only a species or cluster of strains in that species.

These short sequences have been used to develop probes for

the identiﬁcation of organisms at various taxonomic levels.

Given the vast amount of rapidly accumulating sequence data

for all kinds of organisms, it is now possible to develop these

probes for a broad spectrum of taxa. When these probes are

coupled with a ﬂuorescent marker, the target organism can

be easily identiﬁed by a technique known as Fluorescence in

situ Hybridisation (FISH). Fluorescence in situ hybridisa-

tion enables the rapid detection of different species or strains

(Amann 1995). This technique has been successfully applied

for the detection of harmful algae (Anderson 1995, Miller

and Scholin 1996, 2000, John et al. 2003, Anderson et al.

2005) as well as algal classes (Simon et al. 1997, 2000, Rhodes

et al. 2004a, 2004b, Eller et al. 2007) and other taxonomic

hierarchies (Groben et al. 2004, Groben and Medlin 2005,

Töbe et al. 2006).

Basic Principles of FISH

The target algal cells are hybridised with ﬂuorescently (e.g.

the ﬂuorochrome ﬂuorescein isothiocyanate; FITC) labelled

oligonucleotide probes, which bind to the complementary

target sequence of the rRNA in the ribosomes (Fig. 2). This

results in a bright labelling of the entire algal cell because of

the high target number of ribosomes in cells (Figs. 2-4). A list

of all algal probes can be found on the EU PICODIV (Di-

versity of Picoeukaryotic Organisms) website (http://www.

sb-roscoff.fr/Phyto/PICODIV/). In each RNA molecule the

more conserved positions can be used to develop taxonomic

probes e.g. on a class level, whereas the variable regions are

used for lower taxonomic levels.

Initially, the cells are ﬁxed with a preservative that actually

makes the cell membrane permeable for the entry of the

probe into the cell. The ﬂuorescently labelled probe ﬁnds its

way to the ribosome and binds to the region of the rRNA

to which it is complementary, forming a duplex. When the

sample is viewed under a ﬂuorescent microscope using light

of the correct wavelength, the ﬂuorochrome is excited and

the cell of interest can easily be visualised. The three methods

described in this chapter all incorporate these basic principles

of FISH; however, there are slight variations in their protocols

9 Detecting intact algal cells with whole

cell hybridisation assays

Kerstin Töbe*

, David Kulis

, Donald M. Anderson

, Melissa Gladstone

, and

Linda K. Medlin

Alfred Wegener Institute for Polar and Marine Research Am Handelshafen 12. D-27570 Bremerhaven, Germany

Biology Department, MS #32, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

Cawthron Laboratory Services, Cawthron University, Private Bag 2, Nelson, New Zealand

Observatoire Océanologique de Banyuls-sur-Mer, Laboratoire Arago, 66651 Banyuls-sur-Mer, France

*Author for correspondence e-mail: Kerstin.T[email protected]

Figure 1. Variability map of the 18S rRNA molecule. Source:

Bioinformatics.psb.ugent.be.

IOC Manuals & Guides no 55

Chapter 9 Whole cell hybridisation assays

Scope

Detection and quantiﬁcation of target phytoplankton species.

Detection range

Detection of microalgal RNA by FISH is very sensitive. The

number of cells that can be detected depend on the sample vo-

lume. High biomass can obscure the view of target cells.

Advantages

Relatively inexpensive if appropriate ﬂuorescence micrsocopy

facilities are available. Visible observation of cells is possible.

Simple and easy to use. Sample volumes can easily be adjusted.

Simultaneous labeling and detection of multiple species is pos-

sible.

Drawbacks

Probes are only available for a limited number of target species.

Rigorous optimisation and speciﬁcity testing on local strains is

required before the method can be implemented. Finite storage

time for samples. Processing procedure may result in cell loss.

Relatively expensive start-up costs. Intensity of the positive

reaction may vary with cell conditions. Access to molecular ex-

pertise is essential. Appropriate laboratory facilities for storage

and processing of probes and reagents are necessary.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of mole-

cular biology. Approximately one week of supervised training

required to properly perform the method.

Essential Equipment

Epiﬂuorescence microscope, ﬁltration unit, hybridisation oven,

various air displacement pipettes, vacuum pump.

Equipment cost*

Total set-up cost = 20000 € (30000 US $)

See Appendix, Table 1 for details.

Consumables, cost per sample**

Cost per sample = 1.67 € (2.50 US $). See Appendix, Table 2

for details.

Processing time per sample before analysis

In total ca. 4 hours per sample. This excludes ﬁxation time

which is 1-24 hours.

Analysis time per sample

Microscopical analysis 45 minutes to 2.5 hours/sample.

Sample throughput per person per day

A trained person can process and count 16 or more samples/

day.

No. of samples processed in parallel

Number of samples processed in parallel: 14-24 (dependent

upon manifold capacity).

Health and Safety issues

Formamide, formalin, methanol and DAPI are hazardous

chemicals. Proper safety guidelines should be employed when

using them - lab coat, eye protection and gloves are required.

Disposal of all waste products should follow prescribed labo-

ratory procedures. Continual microscope work could result in

strain injury. It is important to incorporate breaks into the daily

analyses time. Ergonomic adjustments to the microscope and

the working place is recommended.

*service contracts not included

**salaries not included

The fundamentals of

The ﬂuorescent in situ hybridisation (FISH) method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

that offer the user various advantages depending upon their

speciﬁc sample requirements. Optimisation of the FISH assay

is required for each new probe/probe set and should involve

testing a matrix of conditions including:

• Hybridisation buffer - 1X, 5X, respectively, depending on

the protocol used. Set buffer and if required, 5% intervals

of formamide concentrations. See Appendix, Table 3 for

more details on how to make up the different buffers

• Hybridisation length - longer hybridisation times can re-

sult in non-speciﬁc binding

• Hybridisation temperature - low temperatures can cause

non-speciﬁc binding and temperatures that are too high

may inhibit binding

• Hybridisation wash buffer and wash duration – these

steps inﬂuence the intensity of the target label as well the

degree of non-speciﬁc labelling

The strongest positive signal from the target organism and

lowest cross reactivity from non-target organisms will deﬁne

the optimal assay conditions for the probe.

Materials

Laboratory Facilities

This method can be used in laboratories and on board re-

search vessels.

Required Equipment (essential)

The whole cell method requires the following equipment:

• Epiﬂuorescence microscope

• Filtration manifold

• Hybridisation oven or water bath with thermometer

• Air displacement pipettes (10 µL – 10 mL)

Equipment, Chemicals and Consumables

The equipment, chemicals and consumables used in this

method are presented in the Appendix, Tabels 1-2 at the end

of this chapter. Suppliers, catalogue numbers and estimated

cost in Euros and US Dollars for the year 2007 are also listed

in Appendix.

Method

Three different ﬂuorescence in situ hybridisation

(FISH) methods are presented. Method 1 and 3 use

the LSU probe, NA1, recognising the North Ameri-

can Alexandrium ribotypes (Alexandrium fundyense

/catenella/tamarense) 5’-Fluor/AGTGCAACACTCCCACCA

-3’ (Anderson et al. 1999). In method 2 another LSU probe,

NA2, which also discriminates North American ribotypes

was used: 5’-Fluor/ AACACTCCCACCAAGCAA -3’ (John

et al. 2003). This probe has a 5 bp (base pair) shift of NA1

sequence to avoid the hair pin loop that this probe makes

with itself that might reduce its hybridisation ability at low

temperature.

Methods 1-3 below are variations of the FISH assay using Al-

exandrium fundyense as the target organism. Each method dif-

fers slightly according to different personal preferences with

regards to some of the equipment used, preservation methods

and the hybridisation protocol (see Table 1 for more details).

With the appropriate ﬁltration set, many samples can be

processed in parallel. Samples in methods 1 and 2 can be eas-

ily ﬁltered overnight for processing in the morning with probe

hybridisation. It is preferable that no signiﬁcant delays oc-

cur during the sample processing steps when using method

3. With any of the methods, the hybridisation takes approxi-

mately 2 hours and the ﬁlters can be enumerated thereafter.

The number of samples to be processed and counted will vary

with each worker; the majority of time is devoted to micro-

scopic examination of the sample. The time spent on each

sample varies depending upon the target cell density, the con-

centration of non- target cells, detrital matter and counting

method employed. The reader is referred to chapter 14 on

the laser scanning cytometer to avoid personal examination

of the ﬁlters.

Figure 2. Micrographs of Alexandrium fundyense cells using the

FISH method: A FISH with FITC-labelled probe NA1 (Miller and

Scholin 1998); B Negative control, no probe. 400X.

Figure 3. Fluorescence in situ hybridisation with Alexandrium

fundyense cells with Cy3-labelled probe NA1. Note the autoﬂuo-

rescence of the dinoﬂagellate cells of the genera Dinophysis and

Ceratium in the background (arrows) 200X.

Figure 4. In-Stu Hybridisation with Alexandrium ostenfeldii cells: A

FISH with FITC-labelled probe Aost (John et al. 2003). B Negative

control, no probe. 400X. Cawthron laboratory.

A B

IOC Manuals & Guides no 55

Chapter 9 Whole cell hybridisation assays

Method 1

Materials

Solutions for Fixatives

• Modiﬁed Saline Ethanol Solution (Miller and Scholin

2000; see Appendix, Table 3 for details).

This ﬁxative is stable at room temperature for several months

without precipitate formation. Note: 300 mL of ﬁxative is

sufﬁcient for approximately 70 reactions. The modiﬁed sa-

line ethanol does not form precipitates and can be made in

advance, whereas the unmodiﬁed version with a higher etha-

nol concentration forms precipitates and should be made just

before use.

• 25X SET buffer (discard after 12 months; see Appendix,

Table 3 for details)

Solutions for Fluorescence in situ Hybridisation

The hybridisation buffer concentration needs to be optimised

for each probe set (see the discussion in ‘Basic Principles of

FISH’).

• 1X SET buffer (used for dinoﬂagellates from the genera

Karenia and Alexandrium; see Appendix, Table 3 for de-

tails)

• 5X SET buffer (used for diatoms from the genus Pseudo-

nitzschia; see Appendix, Table 3 for details)

• Mix solutions into a baked 500 mL Duran bottle and

ﬁlter through a 0.45 µm ﬁlter. Add 1 mL Polyadenalyic

acid (12.5 mg mL

-1

, Sigma Chemical, P-9403)

• Wash buffer: 1X SET buffer (see Appendix, Table 3 for

details) in sterile Milli-Q water

Probes

Probes are received as a dried powder and need to be made up

to a concentration of 200 ng µL

-1

i.e. 50 µg of probe in 250

µL of 1X TE buffer, pH 7.8- 8.0. Make sure all equipment

is RNase free and the area is in dim light. Divide into 50 µL

aliquots and store at 4 ºC. Probes stored this way will remain

stable for 4 months, otherwise, samples need to be lyophilised

and stored at -80 ºC for long term storage.

Additional Equipment

• Milleriser Rig (includes tubes, bases, o-rings, lids)

• Water bath with heater and thermometer

• Hand held vacuum pump and trap

• 2-20 µL micropipette + sterile tips

• 100 - 1000 µL micropipette + sterile tips

• 1-10 mL pipette + sterile tips

• Glassware (including Duran bottles) baked for 4 hours at

160 °C

• Latex disposable gloves

• Glass slides

• 22 mm x 22 mm glass cover slips

• Fine tip tweezers

• 500 mL volumetric ﬂasks, acid washed in 3N HCl

• Poretics Polycarbonate ﬁlters; 3.0 µm pore size; 13 mm

diameter (Osmonics Inc.). Note that 5-8 µm pore size

ﬁlters can be used for larger cells

• Slowfade® Gold antifade reagent (Molecular Probes, In-

vitrogen Detection Technologies, S36936)

Fixation

1 Gloves must be worn at all times to avoid contamination

of samples with human derived RNAases;

2 Switch water bath heater on, set at 45 ºC;

3 Set up Milleriser Rig (Fig. 5). Assemble one ﬁlter set for

each species speciﬁc probe, plus a positive control, SSU-

targeted universally conserved sequence (Embley et al.

1992, Field et al. 1988), a negative control (the comple-

ment of UniC) and a ‘no probe’ control;

4 Using tweezers, place the o-ring into the tube. Place a

ﬁlter onto the o-ring ensuring that the shiny side faces

the sample. Screw the base into tube and tighten until

ﬁnger tight. Do not over tighten. Place assembled tube

into Milleriser Rig. Attach tube of vacuum trap ﬂask to

the Milleriser Rig and tube of vacuum pump to outside

ﬂask outlet;

5 Ensure valves are closed (i.e. horizontal);

Differences between methods Advantages Disadvantages

Method 1 Method 2 Method 3

Fixative Saline ethanol Saline ethanol Formaldehyde

with methanol

extraction

Formaldehyde/methanol

allows long term storage

of samples (> 1 year @

-20 ºC)

Use of hazardous

chemicals and an extra

centrifugation step

Saline ethanol is not

hazardous

Hybridisation buffer 5X or 1X Set

Buffer

5X SET Buffer with

Formamide

5X SET Buffer with

Formamide

Use of an hazardous

chemical

Wash buffer 1X Set Buffer 1X Set Buffer 0.2X Set buffer Method 1 does not require

a separate wash solution

for dinoﬂagellates.

Equipment Water bath Hybridisation oven Hybridisation oven Water bath is not very

expensive

A water bath is

cumbersome to work with

A hybridisation oven is

easy to use

A hybridisation oven is

more expensive

Table 1. Summary of the differences, advantages and disadvantages between the three methods discussed in this chapter.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

6 Live ﬁeld samples: Add 5 mL saline ethanol ﬁxative to

each tube. Add live sample to the ﬁxative. Use 5-10 mL

of ﬁeld sample (the volume can be varied according to

cell numbers present). Filter down to 3 mL and add a

further 3 mL of saline ethanol ﬁxative;

7 Lugol’s iodine treated samples: add 5 mL saline etha-

nol ﬁxative to each tube. Add 10 mL of Lugols treated

sample to the saline ethanol ﬁxative. Add 3 drops of 3%

Sodium thiosulphate (decolouriser) to each tube using a

Pasteur pipette;

8 Cap tubes and tap gently to ensure no air bubbles are sit-

ting on the ﬁlter. Let the sample stand for 1 to 24 hours,

occasionally tapping gently to remove any air bubbles;

9 Filter ﬁxed samples ensuring the solution level does not

drop below the level of the gasket. Do not let pressure

gauge read over 10 mgHg. Fixed samples can be kept for

up to 4 weeks when stored at 4 ºC.

Fluorescence in situ hybridisation

10 Add 2 mL 1X SET hybridisation buffer to each tube;

11 Filter samples as above - do not let ﬁlters dry out;

12 Add 0.5 mL 1X SET hybridisation buffer to each tube;

13 Darken room as much as possible for following steps as

probes are light sensitive;

14 Add 12 µL (ﬁnal concentration in hybridisation 4.8 ng

µL

-1

) probe to tubes. Gently mix the pipette tip in the

hybridisation buffer taking care not to touch the ﬁlter;

15 Put lids on tubes. Place the Milleriser Rig and tubes in

a water bath, ensuring that the solution in the tubes is

covered by the water level. Cover water bath with lid (or

aluminium foil) to prevent light exposure. Leave in water

bath for 1 hour;

16 Remove from water bath and ﬁlter as above;

17 Add 2 mL 1X SET hybridisation buffer and leave at

room temperature for a few minutes;

18 Gently ﬁlter samples until dry;

19 One by one disassemble tubes, remove ﬁlters using twee-

zers and place on labelled slides;

20 Pipette 12 µL of Slowfade® Gold antifade reagent onto

each ﬁlter and add coverslip;

21 Keep slides covered (i.e. in the dark) until they are ready

to be viewed. Colour reaction is enhanced if slides are left

to sit in the dark for about 30 minutes before analysing;

22 Analyse slides using an epiﬂuorescence microscope (exci-

tation 490 nm; emission 520 nm) at 200X magniﬁcation

(Fig. 4);

23 Cells ﬂuoresce brightly if the probe hybridises with tar-

get rRNA. This deﬁnes a positive result. Species-speciﬁc

probe ﬁlters need to be compared with the positive, nega-

tive and ‘no probe’ controls to discriminate positive ﬂuo-

rescence from autoﬂuorescence exhibited by non-target

cells;

24 Results may be recorded as a comment based on the inten-

sity of colour and the pattern of ﬂuorescence. For example:

”++” indicates a strong positive result, where cells

are very brightly coloured with the ﬂurochrome

”+” indicates a positive result or positive cells present.

”-” indicates no cells are showing a positive result, cells

may have a natural orange autoﬂuorescence;

25 Cells should be counted after an initial examination of

each ﬁlter. Positive cells on the positive control ﬁlter must

be counted as well as cells on all the species ﬁlters;

Method 2

Materials

Solutions for ﬁxation

Saline ethanol (Scholin et al. 1996; see Appendix, Table 3 for

details), prepared freshly for each experiment because of the

formation of precipitates or modiﬁed saline ethanol (Miller

and Scholin 2000; see Appendix, Table 3 for details) as de-

scribed in method one.

Solutions for Fluorescence in situ hybridisation

• Hybridisation buffer

• 5X SET buffer (see Appendix, Table 3 for details)

• 0.1 % (v/v) Nonidet-P40

• x % (v/v) Formamide*

*Note: Formamide concentrations must be determined for

every single probe by performing FISH assays with forma-

mide concentrations in 5 % intervals and microscopically ver-

iﬁed. The appropriate formamide concentration will brightly

label all of the target cells tested with no cross hybridisation.

The formamide addition will reduce the binding temperature

of the probe so that it can hybridise speciﬁcally at 50 ºC. The

hybridisation buffer should be ﬁlter sterilised if stored for a

longer time.

Wash buffer

• 1X SET buffer (see Appendix, Table 3 for details) in ste-Table 3 for details) in ste-for details) in ste-

rile Milli-Q water

Probes

Fluorescently labelled probes purchased from Thermo Scien-

tiﬁc, Germany are delivered lyophilised. Stock solution of 1 µg

µL

-1

should be prepared and working solutions of 500 µL

-1

and

50 ng µL

-1

in 1X TE buffer, pH 7.8- 8.0. Probe stock solution

should be stored at -80°C and working solutions at -20°C.

Additional Equipment

• Filter vacuum manifold (Millipore, Bedford, USA) or

glass ﬁlter equipment

• Hybridisation oven

• Vacuum pump

• Pipettes 1-20 µL, 100-100 µL + sterile tips

• 1-50 mL pipette + sterile tips

• Autoclaved glassware

• Disposable gloves

• Glass slides

• Coverslips

• Tweezer

• White polycarbonate ﬁlter membranes: 47 mm or 25

mm diameter, pore size depending on cell size (Millipore,

Bedford, USA)

• DAPI (4’-6-Diamidino-2-phenylindole, Invitrogen,

Karlsruhe, Germany)

• Citiﬂuor (Citiﬂuor Ltd., Cambridge, UK)

• Colourless nail varnish

Fixation

1 Filter approximately 5 mL sample down onto a polycar-

bonate ﬁlter with the lowest possible vacuum and incuba-

te in the ﬁxative for at least 1 hour at room temperature

or overnight at 4 ºC;

2 Incubate the ﬁlter for 5 minutes with hybridisation buffer

at room temperature to avoid precipitation. Air dry the

IOC Manuals & Guides no 55

Chapter 9 Whole cell hybridisation assays

ﬁlter and use directly for FISH. Alternatively the ﬁlter can

be stored at room temperature for at least 1 month.

Filters can also be cut in several pieces and treated individu-

ally for the detection of additional algal species in one sample.

This has to be taken into account when calculating the ﬁnal

cell density.

Fluorescence in situ Hybridisation

3 Apply 60 µL of hybridisation buffer containing the la-

belled probe onto the ﬁlter and hybridise for 2 h in the

dark at 50°C. The ﬁnal probe concentration in the buffer

should be 5 ng µL

-1

. Controls are espeically recommen-

ded for newly designed probes. These should include (1)

A ’no probe’ control: the same procedure without the ad-

dition of a probe. (2) A ’positive control’ where a known

working probe is used. (3) A ’negative hybridisation con-

trol’ where a ’nonsense’ probe is used which would not

bind to any of the cells because of its target sequence;

4 Place the slide in a darkened box with moistened ﬁlter

paper to provide a humid chamber for the hybridisation

to take place. The hybridisation temperature is kept at

50°C for all probes as the thermal melting point of the

probe is compensated by the addition of formamide to en-

hance the speciﬁcity of each probe. Readers are referred to

Amann (1995) and Groben and Medlin (2005) for a full

description of how probes should be developed. It is im-

portant to ensure that the entire ﬁlter piece is covered with

liquid and if necessary, more buffer-probe mixture should

be used. The ﬂuorescently labelled probe is light sensitive,

so the ﬁlters should be kept in the dark for the rest of the

procedure, i.e. cover them during incubation times and

minimise exposure to light when they are handled;

5 Terminate hybridisation by washing the ﬁlters in pre-

warmed (50 ºC) 1X SET wash buffer for 10 minutes

at 50 ºC. After washing, dry the ﬁlters by blotting onto

Whatman ﬁlter paper.

Counterstaining and Validation using Microscopy

6 Mix one mL of Citiﬂuor antifade + 0.5 mL Milli-Q water

+ 1.5 µL DAPI (1 µg µL

-1

);

7 Place the ﬁlters on a glass slide; two ﬁlters, 25 mm in dia-

meter, can be placed side by side on one glass slide. Add 60

µL DAPI/Citiﬂuor mixture to the ﬁlters, place a cover slip

over the ﬁlters and seal edges with nail varnish. Store slides

in the dark until examined. Slides can be kept frozen for

several months without loosing ﬂuorescence signal;

8 Analyse by epiﬂuorescent microscopy using the appro-

priate ﬁlter set for the ﬂuorochrome attached to the probe

(Fig. 2). When bound to the rRNA of target cells, positive

labelled cells ﬂuoresce bright and are counted as positive

signal. Also the intensity of the given ﬂuorescence has to

be recorded. The presumed positive results must be com-

pared with positive, negative and ’no probe’ controls to

differentiate between a real signal from the bound probe

and nonspeciﬁc binding of probes or autoﬂuoresence.

Method 3

Materials

Solutions for Fixation

• Formaldehyde, 37% (Fisher Scientiﬁc, F79P-4)

• 100% ice-cold methanol (Fisher Scientiﬁc, A452SK-4)

Solutions for Fluorescence in situ hybridisation

• 25X SET buffer (as described in the Appendix, Table 3)

• 5X SET Hybridisation buffer (to process 14 samples)

20.4 mL Milli-Q water

6.0 mL 25X SET buffer

300 µL Igepal CA-630 (Sigma Chemical, I 3021)

300 µL Polyadenylic acid (poly A) 10 mg mL L

-1

(Sigma Chemical, P 9403)

3.0 mL Super Pure Formamide

(Fisher Scientiﬁc, BP228-100)

Probes

For the North American Alexandrium ribotypes (Alexandrium

fundyense/catenella/tamarense) the NA1 oligonucleotide probe

can be used: 5’-Fluor/ AGTGCAACACTCCCACCA -3’

(Anderson et al. 1999). Probe stocks should be stored in dry

100 µg aliquots at -80 ºC. Resuspend probe stocks in 500

µL 1x TE [TE-Tris-EDTA buffer 100X concentrate (Sigma

Chemical, T-9285); working stock 200. Add 360 µL of resus-

pended probe to 15 mL hybridisation buffer for incubations.

• Wash buffer (0.2X SET)

120 µL 25X SET buffer

14.880 mL Milli-Q water

Additional Equipment

• Filter vacuum manifold (Promega Corp., A7231) with

custom made 25 mm ﬁlter funnels, (modiﬁcation of the

ones described by Scholin et al. (1997) and are similar to

those in Figure 5)

• Filter membranes: 25 mm Cyclopore membrane (What-

man Inc., 5 mm pore size, 09-930-14E)

• ProLong Gold antifade reagent (Molecular Probes, Invi-

trogen Detection Technologies, P36930)

Fixation

1 Preserve a 14 mL sample with 0.75 mL formaldehyde

(5% v/v, ﬁnal concentration = 1.9% formaldehyde) in a

disposable 15 mL centrifuge tube;

2 The 14 mL volume may be a raw water sample or a con-

centrated sample generated using ﬁltration;

3 In the ﬁeld, 2-4 lof water is typically ﬁltered through a 20

µm Nitex sieve (Nitex mesh, Sefar America, Inc., ﬁtted at

the end of a 3 in. diameter PVC tube) and the collected

Figure 5. Custom made Milleriser Rig.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

cell material is resuspended to the 14 mL mark and pre-

served with 5% v/v formalin;

4 Store the samples at 4 ºC for a maximum of 36 hours

until they can be centrifuged at 3000 x g, for 5 minutes

at room temperature;

5 Aspirate the supernatant and resuspend the cell pellet in

14 mL of ice-cold methanol to extract the chlorophyll

and to stabilise the rRNA. Samples are required to stand

in methanol for at least 1 hour prior to hybridisation but

are stable for many months when stored at -20 ºC.

Fluorescence in situ Hybridisation

6 Add a volume of sample to a ﬁlter funnel containing a

Cyclopore ﬁlter. Filter the sample to near dryness using

the lowest possible vacuum;

7 Add 1 mL of pre-hybridisation buffer and incubate for

5 minutes at room temperature. Note: The ﬁlter funnel

valves should only be open when ﬁltering; they should

remain closed at all other times;

8 Filter the pre-hybridisation buffer and add 1 mL hybridi-

sation buffer with added probe. Cap the ﬁlter tubes and

place the manifold in a black plastic bag. The bag should

contain several wet paper towels to provide a humid envi-

ronment. Seal the bag and place the samples in a dry heat

incubator at 50 ºC (±2 ºC ) for 1 hour;

9 Complete the hybridisation reaction by ﬁltering the

hybridisation buffer plus probe and adding 1 mL pre-

warmed (50 ºC) 0.2X SET wash buffer. Allow this to

incubate for 5 minutes at room temperature;

10 Filter the wash buffer. Keep the ﬁlter funnel valve open

and continue to apply vacuum to the sample until the

ﬁlter has been placed onto a microscope slide. Add 25

µL ProLong Gold antifade reagent and a coverslip to the

ﬁlter. Samples can be stored at 4 ºC in the dark for several

weeks prior to observation, but, best results are obtained

by viewing the sample immediately after hybridisation;

11 Samples can be observed and counted with epiﬂuores-

cence microscopy, at 100X using the appropriate ﬁlter

set. For Cy3, Chroma 41032 or FITC, Chroma 41012

(Chroma Technology Corp.) ﬁlter sets are recommended

(Fig. 3).

Formulas for Calculating Results

Ideally the entire ﬁlter is examined and positive cells enu-

merated.; hence, the cell number is reﬂective of amount of

original sample that was processed and preserved as well as to

the volume of the preserved sample that was placed onto the

ﬁlter for hybridisation. It should be noted, that only the ﬁlter

area is counted and not the entire area under the coverslip,

which is usually larger than that of the sample ﬁlter. If target

cell densities are high and warrant only counting a portion of

the ﬁlter, then a decision has to be made on how to proceed

with the count to afford a statistically reliable value. Andersen

and Throndsen (2004) provide a good review on this in their

chapter in the Manual on Harmful Marine Microalgae.

A generic formula to calculate cells L

-1

for all 3 methods:

where N is the number of positive cells on the whole ﬁlter and

V (mL) is the volume of sample used.

To calculate species composition of a bloom

The FISH assay can be used as a secondary test to light mi-

croscopy analysis when a designated cell count for a particular

genus (e.g. Pseudo-nitzschia) has been exceeded and speciation

is required to determine risk associated with toxicity. In cases

where the cell count has been determined previously for the

target genus, the percentage that each species comprises in

the sample (i.e. the total species positive counts) can be deter-

mined using the following formula:

where S is the number of positive cells on a species-speciﬁc

ﬁlter and T is the total number of genus positive counts.

Percentages calculated from the FISH data can be applied to

the original cell count to get an approximate density of each

species in the sample. Pieces of a ﬁlter can also be analysed.

Counts on the portion of the ﬁlter can be calibrated in a simi-

lar fashion to those of the Utermöhl method (chapter 2). This

removes the requirement for analysing the entire ﬁlter, pro-

vided that a sufﬁcient proportion of a ﬁlter is analysed for

statistical signiﬁcance and that the proportion is the same for

each species-speciﬁc ﬁlter.

Discussion

Whole cell, ﬂuorescently labelled oligonucleotide enumera-

tion-based assays for HAB species can be a simple, effective

and efﬁcient tool for counting natural phytoplankton sam-

ples. Empirical trials with more traditional counting methods,

as discussed in this manual and other studies (e.g. Anderson

et al. 2005), need to be conducted to determine the speciﬁ-

city of the probe with the target species. This is because not

all species-speciﬁc probes will label morphologically identical

organisms because of their genetic dissimilarities. If a suitable

probe has been shown to hybridise with the target organism,

and the required equipment to perform the hybridisation

and subsequent microscopic analysis are available, then this

technique can easily be used to enumerate numerous samples

by researchers with limited laboratory and microscope experi-

ence. Automated counting systems, such as the ChemScan

(Chemunex, France), make this operation much faster (Töbe

et al. 2006).

The speciﬁcity that each probe binds to the target species

rRNA needs to be fully evaluated in optimisation and cross

reactivity trials. To optimise the FISH hybridization param-

eters for each probe, assay conditions such as reagent con-

centrations, hybridisation temperature and time can be ma-

nipulated to produce stronger epiﬂuorescent signals. Methods

described in this manual (such as light microscopy and Cal-

coﬂuor staining), and in other studies, (e.g. Anderson et al.

2005) must be conducted to determine the extent (if any) of

cross reactivity with non-target species.

1000*

)(

) (















−

mLsampleofVolume

NfilterwholeoncountcellPositive

LcellsionconcentratCell

1000*

)(

) (















−

mLsampleofVolume

NfilterwholeoncountcellPositive

LcellsionconcentratCell

100 *

) (















T filter whole on count cell genus Positive

S filter whole on count cell species Positive

ncompositiospeciesPercentage

100 *

) (















T filter whole on count cell genus Positive

S filter whole on count cell species Positive

ncompositiospeciesPercentage

IOC Manuals & Guides no 55

Chapter 9 Whole cell hybridisation assays

Careful consideration must be given when determining the

assay conditions to balance signal intensity against cross reac-

tivity. Positive, negative and ‘no probe’ controls are critical for

this. Signal intensity of the target species compared with the

positive control will give an indication of optimal assay condi-

tions. The negative control will indicate non-speciﬁc binding

caused by sub-optimal conditions. The ‘no probe’ control is

critical for assessing the amount of autoﬂourescence exhibited

by the cells, which must not be confused as a positive signal.

Consideration also needs to be given to the choice of sample

preservation as two of the methods described herein utilise

the relatively safe ethanol/SET preservative, but, these do not

afford the luxury of long-term sample storage. Formalin ﬁxa-

tion followed by methanol extraction and -20 ºC storage al-

lows for long-term sample archiving and subsequent hybridi-

sation, but, involves the use of hazardous chemicals and an

extra centrifugation step to remove the formalin seawater su-

pernatant from the cell pellet. Both ﬁxation methods will pro-

vide the user with good labelling intensity of the target cells.

In New Zealand, the FISH assay is used as a supporting tool

to light microscopy providing additional information to regu-

lators. Multiple species-speciﬁc probes can be run as a screen

to determine the composition of a bloom when a sample has

returned elevated cell counts of potential species of concern.

The FISH assay enable rapid identiﬁcation to the species level

in such cases; a task that would be both time consuming and

require a signiﬁcant level of experience to achieve the same

results using light microscope techniques.

To increase cell detection limits for ﬁeld samples, large vol-

umes of water (1-8 l) can be ﬁltered through an appropriate

mesh size for concentration purposes, if the target organism

is amenable to this type of concentration (e.g. Anderson et al.

2005). The captured cell material can then be washed into a

centrifuge tube, preserved and an aliquot of the sample slurry

can then be processed for the FISH assay. One of the beneﬁts

of using FISH is that samples with high biomass as well as

difﬁcult to identify species can be easily examined and enu-

merated by an inexperienced microscopist. A 2 Litre sample

of ﬁeld material can be concentrated down to a volume of 15

mL, of which, 7.5 mL is then processed for FISH affording a

limit of detection of 1 cell L

-1

. One Litre is more than sufﬁ-

cient, unless the study, e.g. picoplankton, calls for greater vol-

umes. The sample volume used in the assay can be adjusted

based on cell counts derived from light microscope analysis

on test samples.

This method is suitable for all species for which a probe has

been designed and are amicable to the preservation methods

outlined here. The detection limit of one cell per ﬁlter can

easily be achieved by manual counts or if automatic counting

devices are used, such as the ChemScan solid phase cytometer

(see chapter 14 and Töbe et al. 2006). The accuracy of manual

counting depends on the amount of debris in the sample and

the skill of the microscopist to discriminate the positive signal

against the weak autoﬂuorescence of other cells. In order to

ensure that the method works well, it is important that good

temperature control is achieved during the hybridisation step.

It is also imperative that the excess probe is washed off the

ﬁlter in order to reduce background ﬂuorescent interference.

Acknowledgements

The work done by LKM and KT was funded by the Stif-

tung Alfred-Wegener-Institut für Polar und Meeresforschung

in the Helmholtz-Gesellschaft, Bremerhaven, Germany and

in part by EU DETAL, Project QLRT-1999-30778. Support

was provided by the following grants to DMA: the Univer-

sity of New Hampshire Cooperative Institute for Coastal and

Estuarine Technology (CICEET) as a subcontract through a

National Oceanic and Atmospheric Administration (NOAA)

Grant NA05NOS4191149; NSF (National Science Founda-

tion) Grant OCE-0136861 and the ECOHAB (The Ecol-

ogy and Oceanography of harmful Algal Blooms) program

through NOAA Grant NA16OP1438. This effort was sup-

ported in part by the U.S. ECOHAB program sponsored by

NOAA, the U.S. EPA (Environmental Protection Agency),

NSF, NASA (National Aeronautics and Space Administra-

tion) and ONR (Ofﬁce of Naval Research) - ECOHAB con-

tribution number 323. Attendance at the workshop for Melis-

sa Gladstone was funded through the Technical Participation

Programme, New Zealand, and the New Zealand Food Safety

Authority.

References

Amann RI (1995) In situ identiﬁcation of micro-organisms by whole

cell hybridisation with rRNA-targeted nucleic acid probes. In:

Akkermans ADL, van Elsas JD, de Bruijn FJ (eds) Molecular mi-

crobial ecology manual 336. Kluwer Academic Publishers, Dor-

drecht, NL, p. 1-15

Andersen P, Throndsen J (2004) Estimating cell numbers. In: Halle-

graeff GM, Anderson DM, Cembella A (eds), Manual on harmful

marine microalgae Intergovernmental Oceanographic Commis-

sion, UNESCO, Paris, France, p. 99-129

Anderson DM (1995) Identiﬁcation of harmful algal species using

molecular probes: an emerging perspective. In: Lassus P, Arzul

Erard G, Gentien P, Marcaillou C, (eds) Harmful marine algal

blooms. Lavoisier, Intercept Ltd, p. 3-13

Anderson DM, Kulis DM, Keafer BA, Berdalet E (1999) Detection

of the toxic dinoﬂagellate Alexandrium fundyense (Dinophyceae)

with oligonucleotide and antibody probes: variability in labeling

intensity with physiological condition. J Phycol 35:870-883

Anderson DM, Kulis DM, Keafer BA, Gribble KE, Marin R, Scho-

lin CA (2005) Identiﬁcation and enumeration of Alexandrium

spp. from the Gulf of Maine using molecular probes. Deep-Sea

Res II 52:2467-2490

Eller G., Töbe, K, and Medlin, L.K. (2007) A set of hierarchical

FISH probes for the Haptophyta and a division level probe for the

Heterokonta. Journal of Plankton Research, 29:629-640

Embley TM, Finlay BJ, Thomas RH, Dyal PL (1992) The use of

rRNA sequences and ﬂuorescent probes to investigate the phylo-

genetic positions of the anaerobic ciliate Metopus palaeformis and

its archaeobacterial endosymbiont. J Gen Microbiol 138:1479-87

Field KG, Lane DJ, Giovanni SJ, Ghilselin MT, Raff EC, Pace NR,

Raff RA (1988) Molecular phylogeny of the animal kingdom. Sci-

ence. 239:748-53

Groben R, John U, Eller G, Lange M, Medlin LK (2004) Using ﬂuo-

rescently-labelled rRNA probes for hierarchical estimation of phy-

toplankton diversity-a mini review. Nova Hedwigia 79:313-320

Groben R, Medlin LK (2005) In Situ Hybridisation of phytoplank-

ton using ﬂuorescently labelled rRNA probes. In: Zimmer EA,

Roalson E (eds), Methods in enzymology. Elsevier, San Diego,

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

CA. 395:29-310

John U, Cembella A, Hummert C, Ellbrächter M, Groben R, Med-

lin LK (2003) Discrimination of the toxigenic dinoﬂagellates

Alexandrium tamarense and A. ostenfeldii in co-occurring natural

populations from Scottish coastal waters. Eur J Phycol 38:25-40

Miller PE, Scholin CA (1996) Identiﬁcation of cultured Pseudo-

nitzschia (Bacillariophyceae) using species speciﬁc LSU rRNA

targeted ﬂuorescent probes. J Phycol 32:646-655

Miller PE, Scholin CA (1998) Identiﬁcation and enumeration of

cultured and wild Pseudo-nitzschia (Bacillariophyceae) using

species-speciﬁc LSU rRNA-targeted ﬂuorescent probes and ﬁlter

based whole cell hybridization. J Phycol 38: 371-382

Miller PE, Scholin CA (2000) On detection of Pseudo-nitzschia

(Bacillariophyceae) species using whole cell hybridization: Sample

ﬁxation and stability. J Phycol 36:238-250

Rhodes L, Holland PT, McNabb P, Adamson J, Selwood AR (2004a)

Production of isodomoic acid C by Pseudo-nitzschia australis, as

determined by DNA probe and LC-MS technologies. In: Pro-

ceedings of HABTech03 Workshop, Nelson, November (2003)

Holland P, Rhodes L, Brown L (eds) Cawthron report No. 906,

p127. ISBN 0-476-00622-8

Rhodes L, Haywood A, Adamson J, Ponikla K, Scholin C (2004b)

DNA probes for the detection of Karenia species in New Zea-

land’s coastal waters. In: Steidinger K, Landsberg J, Vargo G (eds)

Harmful algae. (2002) Florida Fish and Wildlife Cons Comm,

Florida Inst Oceanography and IOC of UNESCO, p. 273-275

Scholin CA, Buck KR, BritschigT, Cangelosi G, Chavez FP (1996)

Identiﬁcation of Pseudo-nitzschia australis (Bacillariophyceae) us-

ing rRNA-targeted probes in whole cell and sandwich hybridiza-

tion formats. Phycologia 35:190-197

Scholin C, Miller P, Buck K, Chavez F, Harris P, Haydock, P, Howard

J, Cangelosi G (1997) Detection and quantiﬁcation of Pseudo-

nitzschia australis in cultured and natural populations using LSU

rRNA-targeted probes. Limnol Oceanogr 42: 1265–1272

Simon N, Brenner J, Edvardsen B, Medlin LK (1997) The identiﬁ-

cation of Chrysochromulina and Prymnesium species (Haptophyta,

Prymnesiophyceae) using ﬂuorescent or chemiluminescent oligo-

nucleotide probes: a means of improving studies on toxic algae.

Eur J Phycol 32:393-401

Simon N, Campbell L, Örnólfsdóttir E, Groben R, Guillou L, Lange

M, Medlin LK (2000) Oligonucleotide probes for the identiﬁca-

tion of three algal groups by dot blot and ﬂuorescent whole-cell

hybridisation. J Euk Micro 47:76-84

Töbe K, Eller G, Medlin LK (2006) Automated detection and enu-

meration of Prymnesium parvum (Haptophyta: Prymnesiophyc-

eae) by solid-phase cytometry. J Plankton Res 28:643-657

IOC Manuals & Guides no 55

Chapter 9 Whole cell hybridisation assays

Table 1. Equipment and Suppliers

Table 2. Chemicals and Suppliers

Appendix

Equipment Supplier Cat. Number € US $

Method

Filter Manifold Millipore XX2702550 400 587 2

Vacuum pump Omnilab 9.881 391 574 2,3

Incubator ”Shake’n’Stack” VWR 7996 2310 3387 2,3

Epiﬂuorescence microscope, Eclipse

E800

Nikon MAA600BA 25000 36.654

Epiﬂuorescence microscope,

Axioimager A1

Carl Zeiss 4300050000000000 16000 25000 1,3

Milleriser Rig Custom made 55 80 1

Waterbath Grant JBAqua-26 1130 1650 1

Filter Manifold Promega A7231 76 114 3

Pipette 2 µL – 20 µL Fisher Scientiﬁc 05-402-87 193 289 1,2,3

Pipette 20 µL – 200 µL Fisher Scientiﬁc 05-402-89 193 289 1,2,3

Pipette 200 µL – 1000 µL Fisher Scientiﬁc 05-402-90 193 289 1,2,3

Pipette 1 mL – 10 mL Fisher Scientiﬁc 05-403-121 193 289 1,2,3

Equipment Supplier Cat. Number

€

US $ Method

Fixation

Ethanol, absolute,1l Merck 1.009.832.500 73 107 1,2,3

Methanol, 100% Fisher Scientiﬁc A452SK-4 61 90 3

Milli-Q water Most labs have an in-house

supply

1,2,3

Sodium chloride, 1 kg Sigma-Aldrich S9888 23 34 1,2,3

Tris/HCl, 500 g Sigma-Aldrich T3253 96 141 1,2,3

Nonidet-P40, 100 mL Roche 11754599 74 109 2

EDTA, 500 g Sigma-Aldrich E7889 56 82 1,2,3

Formaldehyde, 37% Fisher Scientiﬁc F79P-500 27 40 3

Isopore white polycarbonate membrane ﬁlter, 3 µm pore size, Qty. 100 Millipore TSTP02500 130 191 2

Whatman,Cyclopore, 5 µm pore, 25 mm dia., Qty. 100 Whatman 7062-2513 67 100 3

Hybridisation

FITC-labelled probe Thermo Scientiﬁc - 65 95 2

FITC-labelled probe Oligos Etc. - 0.7* 1* 3

Igepal CA-630, 50 mL Sigma Chemical I3021 16 23 1,3

Polyadenylic acid (poly A) 10 mg/mL Sigma Chemical P-9403 29 42 1,3

Deionized formamide, 100 mL Sigma-Aldrich F 9037 46 67 2,3

Nonidet-P40, 100mL Roche 11754599 74 109 2

Counterstaining and microscopical validation

DAPI (4’-6-Diamidino-2-phenylindole) Invitrogen, Karlsruhe, Germany D1306 97 142 2

Slowfade® Gold antifade reagent Molecular Probes, Invitrogen

Detection Technologies

S36936 82 120 1

Citiﬂuor antifade Citiﬂuor Ltd., Cambridge, UK X506 117 172 2

ProLong Gold antifade reagent Molecular Probes, Invitrogen

Detection Technologies

P36930 71 104 3

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 9 Whole cell hybridisation assays

Solution/Reagent Purpose Contents

Modiﬁed Saline Ethanol Solution Fixative

(after Miller and Scholin 2000)

To make up 300 mL:

220 mL 22 vol. 95% ethanol

50 mL 5 vol. Milli-Q water

30 mL 3 vol. 25X SET buffer

Saline Ethanol Solution Fixative

(after Scholin et al. 1996)

To make up 300 mL:

250 mL 25 vol. 95% ethanol

20 mL 2 vol. Milli-Q water

30 mL 3 vol. 25X SET buffer

25X SET buffer Fixative

(discard after 12 months)

To make up:

219.15 g 3.75 M NaCl

157.60 g 0.5 M Tris/HCl , pH has to be near 8.0, before

adding EDTA

9.3 g 25 mM EDTA, pH 7.8, ﬁlter sterilised, 1 Litre

5X SET buffer Hybridisation buffer

(used for diatoms from the genus Pseudo-nitzschia)

To make up 400 mL:

316 mL Milli-Q Water

80 mL 25X SET buffer

4 mL 10% Igepal, CA-630 (Sigma Chemical, I 3021)

1X SET buffer Hybridisation buffer

(used for dinoﬂagellates from the genera Karenia

and Alexandrium)

To make up 400 mL:

380 mL Milli-Q Water

16 mL 25X SET buffer

4 mL 10% Igepal, CA-630

(Sigma Chemical, I 3021)

1X SET buffer, other use Wash buffer 1X SET buffer in sterile Milli-Q water

0.2X SET Wash buffer To process 14 samples:

120 µL 25X SET buffer

14.880 mL Milli-Q water

Table 3. Buffers and solutions

IOC Manuals & Guides no 55

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 10 Electrochemical detection of toxic algae with a biosensor

Introduction

DNA-biosensors (devices that use molecular probes to detect

target nucleic acids in a sample) are utilised in a number of

different scientiﬁc ﬁelds. Glucose detection was one of the

ﬁrst applications developed using biosensors (Clark 1956).

Electrochemical biosensors combine a biochemical recogni-

tion with signal transduction for the detection of speciﬁc mol-

ecules. The detection component such as a probe sequence,

an antibody, an enzyme or other biomolecule, catalyzes a re-

action with, or speciﬁcally binds to, the target of interest. A

transducer component then transforms this detection event

into a measurable signal. Different types of biosensors can use

optical, bioluminescent, thermal, mass and electrochemical

recognition (Gau et al. 2005). Currently, biosensors are used

in many different applications, such as the identiﬁcation of in-

fectious organisms, hazardous chemicals and the monitoring

of metabolites in environmental samples (Hartley & Baeum-

ner 2003). Biosensors can be produced very cheaply for mass

production. A new detection method for the identiﬁcation of

harmful algae is currently being developed using a hand held

device and biosensors (Fig. 1 and 2). The ﬁrst prototype was

used to identify the toxic dinoﬂagellate Alexandrium ostenfel-

dii (Metﬁes et al. 2005). The second prototype, manufactured

by Palm Instruments BV (Houten, Netherlands) (Fig. 1), has

been extensively used to improve the biosensors. The hand

held device “PalmSens” is a compact portable potentiostat for

all kinds of electrochemical sensors and cells. It was designed

for applications in the ﬁeld as well as laboratories. It can be

conﬁgured for a single application but can also be used as a

generic electrochemical instrument.

Basic principles of electrochemical de-

tection of toxic algae with a biosensor

Molecular probes

Identiﬁcation of toxic algae is based on oligonucleotide probes

that speciﬁcally target ribosomal RNA. Targets for the probes

are the small and large subunit rRNA genes in the ribosomes

of the cells. The conserved and variable regions in these genes

make it possible to develop probes speciﬁc for different taxo-

nomic levels (Groben et al. 2004). The ARB (latin, “arbor”

= tree) software package can be used for probe development

(Ludwig et al. 2004). Theoretical probe speciﬁcity is depend-

ent on the number of sequences of the targeted gene available

in the databases. If molecular probes are designed from only

a few sequences, there is a danger of cross hybridisation to

non target species and organisms whose sequences are not in

the GENBANK database. Prior to the analysis of ﬁeld sam-

ples, molecular probes have to be tested for speciﬁcity with

cultivated target species as well as closely related species as in

silico (calculated by means of a computer simulation) and in

situ results can show different speciﬁcity signals. Nucleic acid

probes have been developed for toxic micro-algal taxa includ-

10 Electrochemical detection of toxic algae with a biosensor

Sonja Diercks

*, Katja Metﬁes

and Linda Medlin

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven , Germany

Observatoire Océanologique de Banyuls-sur-Mer, Laboratoire Arago, 66651 Banyuls-sur-Mer, France

*Author for correspondence: E-mail: sonja.dierc[email protected]

Figure 2. Sensorchip of original prototype with counter and refe-

rence electrode and the reaction layer.

Figure 1. PalmSens device from Palm Instruments.

Figure 4. Principle of redox-reaction presenting the electron trans-

port from the surface of the sensor.

Figure 3. Principle of Sandwich Hybridisation with capture and

signal probe.

IOC Manuals & Guides no 55

Chapter 10 Electrochemical detection of toxic algae with a biosensor

Scope

The hand held device can be used for the rapid detection of

phytoplankton in water samples. The device is a prototype and

has been further automated in an EU-funded project (Project

ALGADEC).

Detection range

The detection limit has to be determined for each probe set.

The calculated detection limit with the hand held device for

Alexandrium ostenfeldii is ~ 800 cells.

Advantages

Several probe sets have already been developed for this met-

hod.

Drawbacks

At the time of publication of this Manual, the hand held device

is still a prototype and not available for general purchase. Probes

for only a limited number of phytoplankton exist. Probes must

be validated for each region where they are applied. Genera-

tion of calibration curves are required for each probe set. High

sample volume is required if the cell densities are expected to

be relatively low. Manual RNA isolation should be done by a

trained molecular scientist. The isolation of a sufﬁcient amount

of target rRNA from the sample to be tested is required for

this assay. A validation of probe signals against total rRNA and

over the growth cycle of the algae under different environme-

ntal conditions has to be carried out before the method can be

applied to ﬁeld samples.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of mole-

cular biology.

a) perform task: approximately ﬁve days are necessary to train

an individual in this method.

b) troubleshoot and quality control: a skilled molecular biolo-

gist should be available to solve any problems that may arise

using this method.

Essential Equipment

Incubator

Thermoheater

Vacuumpump

Washbottle

Mini-Centrifuge

Beadbeater

RNeasy Mini Plant Kit (QIAGEN)

Nanodrop Spectrophotometer

Handheld Device

Equipment cost*

€27350, $38400, for details see Table 4

Consumables, cost per sample**

0.24 € , $0.35 not including probes

Processing time per sample before analysis

Concentration of cells by centrifugation ~20 minutes, coating

of electrodes ~ 2 hours and RNA isolation ~1 hour.

Analysis time per sample

Analysis time per sample: Sandwich hybridisation ~ 2 hours.

Sample throughput per person per day

12 to 16 samples per day

No. of samples processed in parallel

Six to eight

Health and Safety issues

Relevant health and safety procedures must be followed. Read

Material Data Safety Sheets for all chemicals.

The following three chemicals are particularly hazardous

• Hydrogen peroxide 30 % (H

)

• N-Phenyl-1,4-benzenediamine hydrochloride (ADPA)

• β-Mercaptoethanol

*service contracts not included

**salaries not included

The fundamentals of

Electrochemical detection of toxic algae with a biosensor

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 10 Electrochemical detection of toxic algae with a biosensor

ing diatoms and dinoﬂagellates such as Pseudo-nitzschia and

Alexandrium (Scholin et al. 1996, Simon et al. 1997, Miller &

Scholin 1998, John et al. 2003).

Disposable sensor-chip and detection principle

The disposable biosensor chip is composed of a carrier mate-

rial on which a working electrode is printed and the detection

reaction takes place, a reference electrode and an auxiliary

electrode (Fig. 2).

The working electrode has a diameter of 1 mm and is made

of a carbon paste. A biotinylated probe is immobilised on the

reaction layer of the working electrode via avidin. The nucleic

acids are detected on the sensor chip via a sandwich-hybrid-

isation (Zammatteo et al. 1995, Rautio et al. 2003) (Fig. 3).

The underlying principle of the sandwich hybridisation is

that the target speciﬁc probe (capture probe) is immobilised

via avidin on the surface of the working electrode. If a target

nucleic acid is bound to the immobilised probe on the work-

ing electrode, the detection of the nucleic acid takes place

via a hybridisation reaction to a second target speciﬁc probe,

the signal probe that is coupled to digoxigenin (Metﬁes et al.

2005).

The digoxigenin speciﬁc antibody coupled to horseradish-

peroxidase is added to the sensor chip. Horseradish-peroxi-

dase catalyses the reduction of hydrogen peroxide to water.

Reduced peroxidase is regenerated by p-aminodiphenylamine

(ADPA), which functions as a mediator. The oxidised me-

diator is reduced at the working electrode with a potential

of -150 mV (versus Ag/AgCl) taking an electron from the

surface of the sensor (Fig. 4). A potential is applied between

the working and the reference electrode. The hand held de-

vice measures the resulting current activated through the ﬂow

of the electrons from the surface. An electrochemical signal

can only be measured if the target nucleic acid binds to both

the capture and signal probes (Metﬁes et al. 2005). For each

target species the RNA concentration per cell has to be deter-

mined. A calibration curve must be developed for each new

probe set in order to determine the signal intensity at different

RNA concentrations. Using the information on the curve, the

electrical measurement of the hand held device can be related

to cell numbers in a ﬁeld sample.

Materials

Laboratory facilities

Fume hood for RNA isolation

Equipment, Chemicals and Consumables

Information on the equipment, chemicals and consumables

used in this method are presented in the Appendix, Tables

1-2, at the end of this chapter (Tables 1-2). Suppliers, Cata-

logue numbers and estimated cost in Euros and US Dollars

are also listed in Tables 1-2.

Required Equipment (essential)

• Centrifuge

• Filter, 0.5 µm, ISOPORE™, membrane ﬁlters, Milli-

pore, Ireland

• Frit, ﬂask and funnel, Millipore, Ireland

• Mini-Beadbeater™, Biospec products, Biospec products

Inc, USA

• Mini-Centrifuge

• Thermoshaker

• Incubator

• Vacuum pump with wash bottle

• Biosensors, Gwent Electronic Materials, Pontypool, UK

• Freezer -80 ºC

Solutions for preservation of microalgal cells

RNAlater, Ambion, Huntingdon, UK

Method

Concentration of cells

Harvesting of cells can be performed by either centrifugation

and the supernatant discarded or ﬁltration using a ﬁltration

device and a hand held vacuum pump (Fig. 5). A maximum

of ~ 1 x 10

cells can be processed with the RNeasy Plant

Mini Kit.

Preservation and storage

After collecting water samples, the algae cells can be stored at

room temperature for several days using RNALater from Am-

bion, Huntingdon, UK for a later RNA isolation. Follow the

instructions that come with RNALater carefully. The cells can

also be frozen for long term storage by ﬂash-freezing in liquid

nitrogen and immediately transferred to -70

RNA Isolation with the RNeasy Plant Mini Kit

(QIAGEN) (modiﬁed protocol)

General handling of RNA: Ribonucleases (RNases) are very

stable, active enzymes and are difﬁcult to inactivate; even

minute amounts are sufﬁcient to destroy RNA. All glassware

must ﬁrst be cleaned with a detergent, thoroughly rinsed, and

oven baked at 240 ºC for four or more hours before use to

avoid any RNase contamination. Gloves must always be worn

while handling reagents and RNA samples to prevent con-

tamination from the surface of the skin or from dusty labora-

tory equipment. Isolated RNA should be stored on ice while

being processed.

RNA-Isolation

1 Add 450 µL buffer RLT with ß-ME (ß-Mercaptoethanol)

to the cells;

2 Pipette the lysate onto the glass beads and disrupt the

lysate in a bead beater for 2x 20 seconds;

3 Pipette the lysate directly onto a QIAshredder spin co-

lumn (lilac) placed in a 2 mL collection tube, and centri-

fuge for 15 minutes at maximum speed. Carefully trans-

fer the supernatant of the ﬂow-through fraction to a new

microcentrifuge tube without disturbing the cell debris

pellet in the collection tube. Use only this supernatant in

subsequent steps;

4 Add 0.5 volume (usually 225 µL) ethanol (96–100 %) to

the cleared lysate and mix immediately by pipetting. Do

not centrifuge. Continue without delay;

5 Apply sample (usually 650 µL), including any precipi-

tate that may have formed, into an RNeasy mini column

(pink) placed in a 2 mL collection tube. Close the tube

gently and centrifuge for 15 seconds at 8000 x g. Discard

the ﬂow-through. Reuse the collection tube in the next

step;

IOC Manuals & Guides no 55

Chapter 10 Electrochemical detection of toxic algae with a biosensor

6. Add 700 µL buffer RW1 to the RNeasy column. Close

the tube gently and wait for ca. 45 seconds, then centri-

fuge for 15 seconds at ≥8000 x g to wash the column.

Discard the ﬂow-through and collection tube;

7 Repeat step 6;

8 Transfer the RNeasy column into a new 2 mL collection

tube (supplied). Pipette 500 µL buffer RPE onto the

RNeasy column. Close the tube gently, and centrifuge

for 15 seconds at 8000 x g to wash the column. Discard

the ﬂow-through. Reuse the collection tube in step 9.

9 Repeat step 8;

10 Add another 500 µL buffer RPE to the RNeasy column.

Close the tube gently and centrifuge for 2 minutes at

8000 x g to dry the RNeasy silica-gel membrane;

11 To elute DNA, transfer the RNeasy column to a new 1.5

mL collection tube. Pipette 30-50 µL RNase-free water

directly onto the RNeasy silica-gel membrane. Close the

tube gently and centrifuge for 1 minute at 8000 x g to

elute;

12 To obtain a higher total RNA concentration, a second

elution step may be performed by using the ﬁrst eluate

(from step 11);

13 Measure the RNA concentration by using a spectropho-

tometer e.g. Nanodrop spectrophotometer.

The isolated RNA should be ﬂash-frozen in liquid nitrogen

and immediately transferred to -70 ºC.

Sandwich Hybridisation

A. Coating of Sensor chips

1 The sensor chips are moistened with 50 µL of carbonate

buffer (pH 9.6) (Table 1, Fig. 6), which is aspirated off

with a vacuum pump (Figs. 7-8);

2 Incubate over night in a moisture chamber at 4 ºC with 2

µL NeutrAvidin in carbonate buffer (500 µg mL

-1

) (Table

3). Store the electrodes during this period in Petri dishes

with moist Whatman ﬁlters to protect the solutions from

evaporation (Fig. 9);

3 Remove excess NeutrAvidin by washing the chips in PBS

(pH 7.6) (Fig. 10, Table 1). Then dry the chips using a

vacuum pump attached to a wash bottle;

4 Block the sensors for one hour at room temperature with

20 µL 3 % casein in PBS. Remove the casein by washing

with PBS;

Buffer Compound Concentration

Carbonate buffer (pH 9.6) NaHCO

50 mM

10x PBS (pH 7.6) NaH

* H

O 0.5 M

NaCl (pH 7.6) 1.54 M

“bead buffer” NaCl 0.3 M

Tris (pH 7.6) 0.1 M

4x hybridisation buffer NaCl 0.3 M

Tris (pH 8.0) 80 mM

SDS 0.04%

10x POP buffer (pH 6.45) NaH

* H

O 0.5 M

NaCl (pH 6.45) 1 M

PBS-BT (pH 7.6) PBS 1x

BSA 0.1 % [w/v]

TWEEN 20 (pH

7.6) 0.05 % [v/v]

5 The NeutrAvidin coated electrodes can be stored in a

fridge for at least 1 year after incubation with 2 % Treha-

lose in PBS (pH 7.6). The electrodes are coated with 15

µL of Trehalose solution and dried at 37 ºC in an incuba-

tor. Before use the electrodes should be washed with PBS

(pH 7.6) to remove the Trehalose;

B. Immobilisation of biotinylated DNA-probe

6 Coat the sensor chips with 2 µL of the biotinylated probe

[10 pmol µL

-1

in bead buffer (0.3 M NaCl, 0.1 M Tris)]

(Table 1) and incubate for 30 minutes at room temperature;

7 Add 50 µL of 1X hybridisation buffer (0.3 M NaCl, 80

mM Tris, 0.04 % SDS) onto the sensors and directly as-

pirate off to remove excess unbound probe;

8 The coated electrodes can be stored in a fridge for at least

1 year after incubation with 2 % Trehalose on PBS (pH

7.6). The electrodes are coated with 15 µL of Trehalose

solution and dried at 37 ºC in an incubator. Prior to use

the electrodes should be washed with PBS (pH 7.6) to

remove the Trehalose;

C. Sandwich Hybridisation of immobilized DNA probe, RNA

and dioxigenin labelled DNA probe

9 Fragment the RNA by using a fragmentation buffer (200

mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM

MgOA). Add 10 µL of rRNA to 2.5 µL fragmentation

buffer, heat for 5 minutes at 94 ºC in a thermoshaker

(Fig. 11) and immediately chill on ice;

10 Hybridisation preparation details are presented in Table

2. The positive control ensures that the probes are wor-

king and the negative control shows the detection of the

used compounds that do not contain any target RNA;

11 Heat the preparation for 4 minutes at 94 ºC in a ther-

moshaker to denature the RNA target strands. Immedia-

tely chill on ice:

12 Apply 2 µL of the hybridisation solution onto each sen-

sor. Note that each sample is applied in triplicate;

13 Incubate the chips for 30 minutes at 46 ºC in an incu-

bator then cool the incubator down to room temperature

for 5 minutes;

Figure 5. Filtration equipment and hand pump.

Table 1. Buffers for sandwich hybridisation on carbon electrodes.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 10 Electrochemical detection of toxic algae with a biosensor

D. Detection

14 Wash the sensors in 1X POP buffer (pH 6.45) (Table 1)

to remove excess RNA;

15 Incubate the sensors with 1.5 µL Anti-Dig-POD [7.5

U mL

-1

in PBS-BT] at room temperature for 30 minutes

(Table 1);

16 Wash the sensors separately in 1x POP buffer to remove

excess Anti-Dig-POD and dry with a vacuum pump.

17 Add 20 µL of POD substrate onto the electrode (POD

substrate contains 1.1 mg N-Phenyl 1,4-phenylenedia-

mine hydrochloride (ADPA) dissolved in 110 µL etha-

nol, 250 µL of 100 mM H

are added and ﬁlled up to

25 mL with 1X POP buffer);

18 Plug the chip into the hand held device and record the

measurement (Fig. 12). A summary of the buffers used

during the sandwich hybridisation process are presented

in Table 1.

Formulas for calculating results

A calibration has to be determined for each probe set to ﬁnd

the signal intensity (nA) for 1 ng RNA. For each target spe-

cies, the RNA concentration per cell has to be investigated.

Subsequently the cell concentration of the target species in a

water sample can be calculated from the electrochemical sig-

nals:

Let then

Quality control

When developing this method it is useful to conﬁrm the re-

sult signals and calculated cell counts with real cell count re-

sults. This QC system will also determine if the user training

provided is sufﬁcient. A molecular biologist should be able to

solve any problems that arise during the experiments. False

negative signals can result for a number of reasons such as

degradation of antibodies or buffers.

Discussion

The electrochemical detection method with the hand held

device and biosensors is a rapid method to detect target algae

in a water sample. Electrodes can be mass produced. Proto-

cols and electrochemical readings of the hand held device are

simple and easy to use, read and interpret. This is useful for

people with limited experience with the method.

The hand held device has a standard operating procedure

(SOP) with many manual steps. This has now been reﬁned

with many improvements resulting from the EU-project AL-

GADEC. Today, most of the steps are automated (an auto-

mated ﬂow and heating chamber for the biosensors) which

now allows the detection of 14 species in parallel. The initial

sampling, ﬁltering and RNA extraction steps remain the same.

The present biosensor consists of a disposable sensor chip

with 16 electrodes (Diercks et al. 2008a). The redox reaction

takes place between the substrate probe and the signal probe

to yield a ﬂow of electrons. This allows for a electrochemi-

Figure 8. Drying of chips using a vacuum pump and a wash bottle.

Figure 7. Pump and washbottle for exhausting of buffers from the

electrodes.

Figure 6: Applying of buffer onto the electrodes.

Figure 9. Petri dish with Whatman ﬁlter and electrodes.

cell)(per ngRNA

) signal (probenA

numberCell

sample

)

in the(present RNA ng total ) signal (probe

A 

IOC Manuals & Guides no 55

Chapter 10 Electrochemical detection of toxic algae with a biosensor

cal detection proportional to the RNA of the target captured

on the chips and thus to the number of cells in the water

sample tested. Probes for other toxic algae (e.g., Alexandrium

minutum and Gymnodinium catenatum) have been developed

for their detection with this hand held device (Diercks et al.

2008b). About 17 different toxic algae can now be detected,

along with the negative and positive controls. These newly

developed probes are regularly reviewed for their speciﬁcity,

since new sequences are added to the available online genetic

databases daily. This cross check will help to determine if

there is any cross reactivity with other marine organisms.

The current detection limit of the hand held device can op-

erate with sample volumes of up to 8-10 Litres which is ad-

vantageous if only low cell densities of the target organism

are present in the sample. In order to isolate target RNA, an

appropriate cell density is required. The detection limit of the

hand held device for Alexandrium ostenfeldii is ~ 16.00 ng

µL

-1

, with an average yield of ~ 0.02 ng cell

-1

. This equates to

ca. 800 cells. A sampling volume of 6.4 l is required to ob-

tain a detectable amount of RNA when the target organism is

present at a cell density of 250 cells L

-1

(Metﬁes et al. 2005).

The manual isolation of RNA is currently the limiting fac-

tor of the system. This is because a certain quantity of high

quality RNA is required for the assay. It has been found that

separate users can isolate different qualities of rRNA from the

same sample with an equal number of algae cells present. The

resulting signal intensities cannot be compared to cell counts

determined using another enumeration technique. Genera-Genera-

tion of calibration curves is required for each probe set. A

validation of probe signals against total rRNA over the growth

cycle of the target microalgae under different environmen-

tal conditions has to be conducted to verify the calibration

curves. This will allow the extrapolation of the electrochemi-

cal readings into more accurate values of cells per Litre. A high

sample volume is required if the cell densities are low. The

automated RNA isolation developed under the ALGADEC

project will overcome some of these difﬁculties.

Acknowledgements

Sonja Diercks was supported by the EU-project ALGADEC

(COOP-CT-2004-508435-ALGADEC) of the 6th Frame-

work Program of the European Union and the Alfred Wege-

ner Institute for Polar and Marine Research.

Table 2. Hybridisation preparation.

Figure 11. Schutron Thermoshaker for the fragmentation and

denaturation of the sample.

Figure 10. Washing of chips in a petri dish with wash buffer.

Detection of the species Negative control Positive control

3.5 µL 4X Hybridasation buffer 3.5 µL 4X Hybridasation buffer 3.5 µL 4X Hybridasation buffer

7.5 µL rRNA 1 µL Herring DNA (3480 ng µL

-1

) 1 µL Herring DNA (3480 ng µL

-1

)

1 µL Herring DNA (3480 ng µL

-1

) 1 µL DIG marked DNA probe (1.4 pM µL

-1

) 1 µL Test DNA (36 bases, 1.4 pM µL

-1

)

1 µL DIG marked DNA probe (1.4 pM µL

-1

) 8.5 µL milliQ water 1 µL DIG marked DNA probe (1.4 pM µL

-1

)

1 µl milliQwater 7.5 µl milliQwater

Figure 12. Measuring of chips with Handheld device.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 10 Electrochemical detection of toxic algae with a biosensor

References

Clark LC (1956) Monitor and control of blood and tissue oxygena-

tion. Trans. Am. Soc. Artif. Intern. Organs 2:41-48

Diercks S, Metﬁes K, Medlin, LK (2008a): Development and adap-

tation of a multiprobe biosensor for the use in a semi-automated

device for the detection of toxic algae. Biosensors & Bioelectronics

23: 1527-1533

Diercks S, Metﬁes K, Medlin LK (2008b) Molecular probes for the

detection of toxic algae for use in sandwich hybridization formats.

J. Plankton Res. 30, 439 - 448

Gau V, Ma S, Wang H, Tsukuda J, Kibler J, Haake DA (2005) Elec-

trochemical molecular analysis without nucleic acid ampliﬁcation.

Methods 37:78-83

Groben R, John U, Eller G, Lange M, Medlin LK (2004) Using ﬂuo-

rescently-labelled rRNA probes for hierarchical estimation of phy-

toplankton diversity – a mini-review. Nova Hedwigia 79:313-320

Hartley HA, Baeumner AJ (2003) Biosensor for the speciﬁc detec-

tion of a single viable B. anthracis spore. Anal. Bioanal. Chem

376:319-327

John U, Cembella A, Hummert C, Elbrächter M, Groben R, Medlin

LK (2003) Discrimination of the toxigenic dinoﬂagellates Alexan-

drium tamarense and A. ostenfeldii in co occurring natural popula-

tions from Scottish coastal waters. Eur. J. Phycol. 38:25-40

Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar,

Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber

S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König

A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow

R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T,

Bode A, Schleifer K-H (2004) ARB: a software environment for

sequence data. Nucleic Acids Res 32:1363-1371

Metﬁes K, Huljic S, Lange M, Medlin LK (2005) Electrochemical

detection of the toxic dinoﬂagellate Alexandrium ostenfeldii with a

DNA-biosensor. Biosens. Bioelectron 20:1349-1357

Miller P, Scholin CA (1998) Identiﬁcation and enumeration of cul-

tured and wild Pseudo-nitzschia (Bacillariophyceae) using species-

speciﬁc LSU rRNA-targeted ﬂuorescent probes and ﬁlter-based

whole cell hybridization. J. Phycol 34:371-382

Rautio J, Barken KB, Lahdenpera J, Breitenstein A, Molin S, Neu-

bauer P (2003) Sandwich hybridisation assay for quantitative de-

tection of yeast RNAs in crude cell lysates. Microb. Cell Fact 2:4

Scholin CA, Buck KR, Britschgi T, Cangelosi G, Chavez FP (1996)

Identiﬁcation of Pseudo-nitzschia australis (Bacillariophyceae)

using rRNA-targeted probes in whole cell and sandwich hybridi-

zation formats. Phycologia 35:190-197

Simon N, Brenner J, Edvardsen B, Medlin LK (1997) The iden-

tiﬁcation of Chrysochromulina and Prymnesium species (Hyptop-

hyta, Prymnesiophyceae) using ﬂuorescent or chemiluminescent

oligonucleotide probes: a means for improving studies on toxic

algae. Eur. J. Phycol. 32:393-401

Zammatteo N, Moris P, Alexandre I, Vaira D, Piette J, Remacle J

(1995) DNA probe hybridisation in microwells using a new bio-

luminescent system for the detection of PCR-ampliﬁed HIV-1

proviral DNA. Virol. Methods 55:185-197

IOC Manuals & Guides no 55

Chapter 10 Electrochemical detection of toxic algae with a biosensor

Equipment Supplier Cat. Number

€

US $

Incubator ”Shake’n’Stack” VWR 7996 2310 3189

Thermoheater ”Comfort” Eppendorf 5355R 2545 3512

Vacuumpump Omnilab 9.880540 391 539

Washbottle (250 mL) Omnilab 5051436 20 25

Mini-Centrifuge Hereaus

”Biofuge pico”

Omnilab 7025235 938 1294

Mini-Beadbeater™ Biospec products, Biospec

products Inc, USA

3110BX 715 986

Nanodrop ND1000

Spectrophotometer

Peqlap Biotechnology 91-ND-1000 8995 12418

Handheld Device Palm Instruments BV,

Houten, Netherlands

PalmSens with PC software,

Without Pocket PC

3450 4739

Sum approx. 19364 26702

Table 1. Equipment and suppliers.

Appendix

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 10 Electrochemical detection of toxic algae with a biosensor

Chemical Supplier Cat. Number

€

US $

NeutrAvidin™, biotin binding protein PIERCE, Perbio, Germany 31000 90.00 125.00

D(+)-Trehalose, ≥ 99.5 % HPLC Fluka BioChemika, Switzerland 90208 31.00 42.64

Biotin-labelled probe (18 bases) Thermo Electron

Digoxigenin-labelled probe (18 bases) Thermo Electron

Herring-Sperm DNA Roche Applied Science 10223646001 101.00 138.94

1x PBS PIERCE, Perbio, Germany 28372 83.65 115.00

Tween 20 Sigma-Aldrich Chemie GmbH, Germany P1379 26.30 36.18

N-Phenyl 1,4-phenylenediamine hydrochloride C

HCl (ADPA,

N-Phenyl-1,4-benzenediamine hydrochloride, 1,1-Diphenylhydrazin-

hydrochlorid) ADPA

MERCK KGaA, Germany 814648 13.10 18.00

Anti-Digoxigenin-POD fab fragments Roche Applied Science 11207733910 171.50 235.92

Hydrogen peroxide solution H

, 30% (w/w) Sigma-Aldrich Chemie GmbH, Germany H1009 19.40 26.69

Ethanol MERCK KGaA, Germany 100983 72.50 99.73

Sodium hydrogencarbonat NaHCO

Riedel-de Haën®, RdH, Laborchemikalien, GmbH

& CoKG, Germany

15.80 21.74

NaH

* H

O MERCK KGaA, Germany 567545 29.75 40.92

NaCl Sigma-Aldrich Chemie GmbH, Germany S9888 24.80 34.11

Casein Sigma-Aldrich Chemie GmbH, Germany C5890 38.00 52.28

Tris (pH 8.0) Sigma-Aldrich Chemie GmbH, Germany T1503 101.50 139.63

SDS Sigma-Aldrich Chemie GmbH, Germany L4390 18.40 25.40

BSA Sigma-Aldrich Chemie GmbH, Germany 128910 11.40 15.74

-Mercaptoethanol MERCK KGaA, Germany 444203 60.00 82.83

RNeasy Plant Mini Kit Qiagen, Hilden Germany 74904 268.00 370.00

Whatman Chromatography Paper Whatman, Brentford, United Kingdom 3MM CHR 199.00 274.35

Glass beads (212 – 300 µm, 425 – 600 µm) Sigma-Aldrich Chemie GmbH, Germany G1277 99.00 136.19

Table 2. Chemicals and suppliers.

IOC Manuals & Guides no 55

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 11 Hybridisation and microarray ﬂuorescent detection

Introduction

The introduction of DNA microarray technology in 1995 was

one of the latest and most powerful innovations in the ﬁeld

of microbiology. This technique allows the rapid acquisition

of copious data (Schena et al. 1995). It is a new experimen-

tal approach in molecular biology (Blohm and Guiseppi-Elie

2001), which offers the possibility to analyse a large number

of samples using a range of different probes in parallel under a

diverse spectrum of applications (Ye et al. 2001).

Microarray technology was launched with a publication by

Schena et al. (1995). Many functional genomic methods

proﬁt from microarrays, such as genome expression proﬁling,

single nucleotide polymorphism detection and DNA rese-

quencing (Lipshutz et al. 1999, Kauppinen et al. 2003, Ji and

Tan 2004, Yap et al. 2004, Al-Shahrour et al. 2005, Broet et

al. 2006, Gamberoni et al. 2006). Thus, DNA microarrays

are a powerful and innovative tool that can facilitate monitor-

ing in the marine environment.

A microarray consists of DNA sequences that are applied to

the surface of a glass slide with special surface properties in

an ordered array. It is based on a minimised form of a dot-

blot (Gentry et al. 2006, Ye et al. 2001). A DNA microarray

experiment involves microarray production, sample isolation

and preparation, hybridisation and data analysis. Prior to the

hybridisation, the target nucleic acid is labelled with a ﬂuo-

rescent dye, which can be incorporated directly to the nucleic

acid or via indirect labelling of other substances (Cheung et

al. 1999, Southern et al. 1999, Metﬁes et al. 2006). The hy-

bridization pattern is captured via ﬂuorescent excitation in a

special device, the microarray scanner (Ye et al. 2001).

The application of DNA microarrays for the identiﬁcation of

marine organisms is a relatively new and innovative ﬁeld of

research. It provides the possibility to analyse a large number

of targets (species or other taxa) in one experiment (Ye et al.

2001), but is not yet widely applied to marine biodiversity

and ecosystem science. For the use of microarray technology

as a standard tool with rapid and simple routine handling,

further research into methodical optimisations is required

(Peplies et al. 2003).

A number of European research groups utilise DNA micro-

arrays for the identiﬁcation of marine organisms. The DNA

microarrays, or the so called “phylochip”, have been used to

identify phytoplankton (Metﬁes and Medlin 2004, Ki and

Han 2006, Medlin et al. 2006, Gescher et al. 2007), bacte-

ria (Loy et al. 2002, Peplies et al. 2003, Peplies et al. 2004a,

Peplies et al. 2004b, Lehner et al. 2005, Loy et al. 2005, Pe-

plies et al. 2006) and ﬁsh (Kappel et al. 2003). Speciﬁc probes

11 Hybridisation and microarray ﬂuorescent

detection of phytoplankton

Christine Gescher*

, Katja Metﬁes

and Linda K. Medlin

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven , Germany

Observatoire Océanologique de Banyuls-sur-Mer, Laboratoire Arago, 66651 Banyuls-sur-Mer, France

*Author for correspondence e-mail: Christine.Gescher@web.de

initially developed for other hybridisation assays (e.g. whole

cell) have been successfully modiﬁed and employed by the

microarray detection method.

Basic principles of hybridisation, micro-

array ﬂuorescent detection

Hybridisation refers to the reannealing of two single strands

of nucleic acids that contain complementary sequences. It uti-

lises the basic physical structural property of DNA. DNA has

the structure of a double helix with hydrogen bonds binding

the two strands of DNA together. When the DNA is heat-

ed at a temperature above 90°C, the hydrogen bonds break

and the DNA is subdivided in two separate complementary

strands. When the temperature is decreased, the two sepa-

rate strains will reanneal. Complementary nucleic acids with

a high degree of similarity anneal easier and will bind together

more ﬁrmly. The probes on the DNA microarray represent

one strand of the DNA and if there are complementary se-

quences in the examined sample, both stands will hybridise

together. This event can be detected by a ﬂuorescence label on

one of the two strands that have bound together.

The microarray experiment can be accomplished with ribos-

omal RNA (rRNA) or DNA, e.g. DNA-fragments generated

by a Polymerase Chain Reaction (PCR) from genomic DNA.

When using this technique to identify microorganisms rRNA

is advantageous over DNA because the cell contains rRNA in

a high number that can be easily extracted using commercial

kits. In contrast, if the number of copies of ribosomal genes in

the genomic DNA is too low then a PCR is needed to amplify

the target sequences.

The utilisation of PCR-fragments can introduce a bias to

the analyses and it has been shown frequently that microbial

communities may not be reﬂected correctly (Suzuki and Gio-

vannoni 1996, Wintzingerode et al. 1997, Simon et al. 2000,

Speksnijder et al. 2001, Kanagawa 2003, Medlin et al. 2006).

The hybridisation of RNA theoretically offers the possibility

of quantiﬁcation and delivers a less biased view of true com-

munity composition (Peplies et al. 2006). Possible disadvan-

tages are low yields of RNA from environmental samples and

inhibition of extraction by complex organic molecules (Alm

and Stahl 2000, Peplies et al. 2006). Furthermore, the RNA

content can vary over the cell cycle, especially in prokayotes

(Medlin 2003, Countway and Caron 2006).

The method requires the use of a molecular laboratory. A clean

fume hood should be available because of ß-mercaptoethanol.

DNA microarrays consist of glass microscope slides with par-

ticular surface properties (Metﬁes and Medlin 2005). The

IOC Manuals & Guides no 55

Chapter 11 Hybridisation and microarray ﬂuorescent detection

Scope

Detection and quantiﬁcation of target phytoplankton species.

At the time of publication of this Manual, this method is relati-

vely new and currently under development.

Detection range

The detection range depends strongly on the sensitivity of the

chosen probes. Calibration curves are required to correlate cell

counts with signal point intensities.

Advantages

Allows identiﬁcation of target phytoplankton to the species le-

vel.

Drawbacks

At the time of publication of this Manual, this microarray is still

a prototype and not available for general purchase. Probes for

only a limited number of phytoplankton exist. Probes must be

validated for each region where they are applied. The prepara-

tion of a calibration curve is required for each probe used.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of mole-

cular biology.

a) perform task: approximately ﬁve days are necessary to train

an individual in this method.

b) troubleshoot and quality control: a skilled molecular biolo-

gist should be available to solve any problems that may arise

using this method.

Essential equipment

A well equipped molecular biology laboratory, see Appendix,

Table 1 for details of instrumentation.

Equipment cost*

Total set-up cost = approx. €55,000 or approx. $75,000 US

See Appendix, 2 Table 1 for details.

Consumables, cost per sample**

38 € (50 US $).

Processing time per sample before analysis

A trained person can process up to 8 samples in 6 hours:

RNA Isolation = 1 hour;

Labelling of RNA= 1 hour;

Hybridisation= 4 hours.

Analysis time per sample

A trained person can analyse up to 8 samples in 1 hour.

Sample throughput per person per day

Four samples (with duplicates of each sample) or eight single

samples.

No. of samples processed in parallel

Four samples (including duplicates) or eight single samples

Health and Safety issues

Relevant health and safety procedures must be followed.

The following chemical is particularly hazardous: β-Mercaptoe-

thanol.

*service contracts not included

**salaries not included

The fundamentals of

The hybridisation, microarray ﬂuorescent detection method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 11 Hybridisation and microarray ﬂuorescent detection

glass slide is coated with a special chemical surface e.g., with

aminosilane, an epoxygroup or an aldehydegroup. The probes

should be ordered with the appropriate chemical group to be

linked to the slide surface. Figure 1 shows a light microscope

picture of spots on a glass slide. Furthermore, the probes are

immobilised as spots on a glass slide in a deﬁned pattern. Each

spot consists of many copies of an oligonucleotide probe that

is complementary to a speciﬁc target DNA sequence (Graves

1999). The target (RNAs or DNAs) hybridises to the capture

oligonucleotide probe on the microarray. The hybridisation is

detected via a ﬂuorescent label that is attached to the target

sequence during PCR or directly to the RNA (Metﬁes and

Medlin 2004). The ﬂowchart of a microarray hybridisation is

shown in Figure 2.

It may be necessary to design new probes or to choose probes

from other applications that are speciﬁc for the target taxo-

nomic group or species. If the 18S rRNA gene sequence is

used to design the probe, then it is important that the probe

be designed using only the ﬁrst 1000 base pairs (bp) of the

gene.

Materials

Laboratory Facilities

This method should be carried out in a molecular laboratory

with clean fumehood facilities.

Required Equipment (essential)

Microarray production requires the following equipment:

A spotter and an oven that can be heated to 60°C. Ordered

probes can be spotted onto a glass slide using a commercial

supplier. It is more ﬂexible and convenient to have a spot-

ter in the laboratory. However, these machines are expensive

to purchase with prices ranging from €50,000-100,000 (US

$67,000-134,000). It is advisable to outsource the spotting

procedure at the initial stage of use.

RNA isolation requires the following equipment:

• Mini-Beadbeater (e.g. BioCold Scientiﬁc Inc., USA)

used to homogenise the algal cells with glass-beads

• Conventional Mini-Centrifuge for small eppendorf tubes

(1.0 mL and 1.5 mL)

Figure 1. Microscopical view of a part of one glass slide with a grid

of 12 spots of probes diluted in 3x SSC salt buffer.

Figure 2. Flowchart of sample handling and hybridisation.

IOC Manuals & Guides no 55

Chapter 11 Hybridisation and microarray ﬂuorescent detection

The hybridisation step requires the following equipment:

• Thermoheater (Fig. 3)

• Incubator

• Bellydancer or shaker (Fig. 4)

• Microarray scanner with software (Fig. 5)

Chemicals and consumables

• RNA isolation, labelling and puriﬁcation:

• Isolation: RNeasy Plant Mini Kit

• Labelling of RNA: Biotin-ULS-Kit

• Puriﬁcation of labelled RNA: RNeasy MinElute Cleanup

Kit

• Removal of RNAse in fume hood and labware: RNase-

Zap (Ambion Inc., Austin, USA)

Information on the equipment, chemicals and consumables

used in this method are presented in the Appendix (Tables

1-2) at the end of this chapter. Suppliers, Catalogue numbers

and estimated cost in Euros and US Dollars are also listed in

Tables 1-2.

Method

Sample Preservation and Storage

1 It is essential to begin with the correct amount of algal

material to obtain optimal RNA yield and purity with

the RNeasy columns. The required amount depends on

the target phytoplankton species and can range from 1 x

to 1 x 10

cells L

-1

;

2 Fresh or frozen tissue can be used. To freeze tissue for

long-term storage, the material should be ﬂash-frozen

in liquid nitrogen and transfered immediately to -70 ºC

where it can be stored for several months. When pro-

cessing, the tissue should not be allowed to thaw during

weighing or handling prior to disruption in Buffer RLT.

Homogenised lysates, in Buffer RLT, can also be stored at

-70 ºC for several months;

3 To process frozen lysates, thaw samples and incubate for

15-20 minutes at 37 ºC in a water bath to dissolve salts;

4 It is possible to store the hybridised microarrays for at

least one year at -20 ºC, although it is usually unnecessa-

ry to keep them once they have been scanned. The reuse

of “phylochips” for new hybridisations has been tested

for expression analysis experiments where the microar-

rays were reused 5 times (Dolan et al. 2001, Bao et al.

2002);

RNA Isolation with the RNeasy Plant Mini Kit

(Qiagen)

5 Ribonucleases (RNases) are very stable and active enzy-

mes that break down RNA. They generally do not re-

quire cofactors to function. As RNases are difﬁcult to

inactivate, even minute amounts are sufﬁcient to destroy

RNA. Plasticware or glassware, therefore, should not be

used without ﬁrst eliminating any possible trace of RNase

contamination. Care should be taken to avoid inadver-

tently introducing RNases into the RNA sample during

or after the isolation procedure. Latex or vinyl gloves

should always be worn when handling reagents and RNA

samples to prevent RNase contamination from the skin

surface or dusty laboratory equipment. Gloves should be

changed frequently and tubes kept closed whenever pos-

sible. Samples should be kept on ice, particularly isolated

RNA, especially when aliquots are being pipetted. The

use of sterile, disposable polypropylene tubes is recom-

mended throughout. These tubes are generally RNase-

free and do not require a pre-treatment to inactivate

RNases. Glassware used for RNA work should be cleaned

with a detergent, thoroughly rinsed and oven baked at

240 ºC for 4 or more hours before use;

Important notes before getting starting

6 Beta-Mercaptoethanol (ß-ME) must be added to Buffer

RLT before use. Beta-Mercaptoethanol is toxic so dis-

pense in a fume hood and wear appropriate protective

clothing. Add 10 µL ß-ME per 1 mL Buffer RLT. Buffer

RLT is stable for 1 month after the addition of ß-ME;

7 Buffer RPE is supplied as a concentrate. Before using for

the ﬁrst time, add 44 mL of ethanol (96-100 %), as indi-

Figure 4. Washing of the microarrays on a belly dancer.

Figure 3. Thermoheater for denaturation of the labelled RNA.

Figure 5. Microarray scanner GenePix 4000B.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 11 Hybridisation and microarray ﬂuorescent detection

cated on the bottle, to obtain a working solution.

8 All steps of the RNeasy protocol should be performed

at room temperature (~20 ºC). Work quickly during the

procedure;

9 All centrifugation steps are performed at 20-25 ºC in a

standard microcentrifuge;

Harvesting of phytoplankton cells

10 Harvest the cells by centrifugation or ﬁltration;

11 Discard the supernatant and process the cell pellet;

RNA-Isolation

12 Add 450 µL Buffer RLT with ß -ME to the cell pellet;

13 Pipette the cells into an eppendorf cup containing glass

beads (212 µm- 300 µm and 312-600 µm) and homoge-

nise the cells in a bead beater for 20 seconds;

14 Pipette the lysate directly onto a QIAshredder spin co-

lumn (lilac colour) placed in 2 mL collection tube and

centrifuge for 2 minutes at maximum speed;

15 Carefully transfer the supernatant from the ﬂow-through

fraction to a new microcentrifuge tube without distur-

bing the cell debris pellet in the 2 mL collection tube.

This supernatant/lysate is used in all subsequent steps;

16 Add 0.5 volume (usually 225 µL) ethanol (96-100 %) to

the clear lysate, and mix immediately by pipetting. Do

not centrifuge. Continue without delay;

17 Apply the sample (usually 650 µL), including any preci-

pitate that may have formed, to an RNeasy mini column

(pink colour) placed in a 2 mL collection tube. Close the

tube gently and centrifuge for 15 seconds at ≥8000 x g

(≥10000 rpm). Discard the ﬂow-through;

18 Reuse the collection tube in the next step;

19 Add 700 µL Buffer RW1 to the RNeasy column. Close

the tube gently and centrifuge for 15 seconds at ≥8000 x

g (≥10000 rpm) to wash the column. Discard the ﬂow-

through and collection tube;

20 Transfer the RNeasy column into a new 2 mL collection

tube (supplied with the kit). Pipette 500 µL Buffer RPE

onto the RNeasy column. Close the tube gently and cen-

trifuge for 15 seconds at ≥8000 x g (≥10000 rpm) to

wash the column. Discard the ﬂow-through;

21 Add another 500 µL Buffer RPE to the RNeasy column.

Close the tube gently, and centrifuge for 2 minutes at

≥8000 x g (≥10000 rpm) to dry the RNeasy silica gel

membrane;

22 To elute the RNA, transfer the RNeasy column to a new

1.5 mL collection tube. Pipette 30-50 µL RNase-free wa-

ter directly onto the RNeasy silica gel membrane. Close

the tube gently and centrifuge for 1 minute at ≥8000 x g

(≥10000 rpm) to elute;

23 To obtain a higher total RNA concentration, a second

elution step may be performed by using the ﬁrst elua-

te (from the previous step). Pipette the eluate back on

the column and centrifuge for 1 minute at ≥8000 x g

(≥10000 rpm) to elute once again;

24 Measure the concentration of the RNA with a Spectrop-

hotometer (e.g. Nanodrop Spectrophotometer, Peqlab,

Erlangen, Germany).

Labelling of RNA with the Biotin-ULS-Kit

Background: This Labelling Kit uses the Universal Linkage

System (ULS) technique, which is based on the stable co-

ordinative binding of a platinum complex to nucleic acids.

The platinum complex acts as a linker between a detect-

able marker (label) molecule, i.e., ﬂuorescein or biotin, and

DNA or RNA. The marker is coupled directly to the nucleic

acid without any signiﬁcant interference. Universal Linkage

System consists of a Pt complex stabilised by a chelating di-

amine. It has two binding sites, one of which is used to bind a

marker. The other binding site is used to link the complex to

the aromatic nitrogen atoms of nucleobases and one nitrogen

atom of guanine is strongly preferred (Fig. 6). The resultant

Pt-N bond is very stable both chemically and thermally.

Features of the Biotin-ULS-Kit

• One-step reaction.

• Fast - only 30 minute to label the target.

• Universal - any nucleic acid, independent of size or

structure can be labelled.

• Easy to scale up and down. It allows labelling of as little

as 25 ng or as much as 10 µg of nucleic acid in a single

reaction

25 Add 1 µL (= ½ U) of Biotin ULS reagent to 500 ng of

nucleic acid template;

26 Adjust volume with labelling solution to 20 µL and mix

well;

27 Incubate for 30 minutes at 85 ºC;

28 Add 5 µL Stop solution and mix well;

29 Incubate for 10 minutes at room temperature;

30 Purify the solution with the RNeasy MinElute Cleanup

Kit before hybridisation;

Puriﬁcation of labelled RNA with the RNeasy MinElute

Cleanup Kit (Qiagen)

31 Adjust sample to a volume of 100 µL with RNase-free

water. Add 350 µL of Buffer RLT and mix thoroughly;

32 Add 250 µL of 96-100 % ethanol to the diluted RNA

and mix thoroughly by pipetting. Do not centrifuge.

Continue the next step immediately;

33 Apply 700 µL of the sample to an RNeasy MinElute Spin

Column in a 2 mL collection tube. Close the tube gent-

ly and centrifuge for 15 seconds at ≥8000 x g (≥10000

rpm). Discard the ﬂow-through;

34 Transfer the spin column into a new 2 mL collection

tube. Pipette 500 µL Buffer RPE onto the spin column.

Close the tube gently and centrifuge for 15 seconds at

≥8000 x g (≥10000 rpm) to wash the column. Discard

the ﬂow-through;

35 Add 500 µL of 80 % ethanol to the RNeasy MinElute

Spin Column. Close the tube gently, and centrifuge for 2

Figure 6. Labelling of nucleic acids with Biotin-ULS (source:

www.fermentas.com)

IOC Manuals & Guides no 55

Chapter 11 Hybridisation and microarray ﬂuorescent detection

minutes at ≥8000 x g (≥10000 rpm) to dry the silica gel

membrane. Discard the ﬂow through and collection tube;

36 Transfer the RNeasy MinElute Spin Column into a new

2 mL collection tube. Open the cap of the spin column

and centrifuge in a microcentrifuge at full speed for 5

minutes. Discard the ﬂow through and collection tube;

37 To elute the RNA, transfer the spin column to a new

1.5 mL collection tube. Pipette 14 µL RNase-free water

directly onto the centre of the silica-gel membrane. Close

the tube gently and centrifuge for 1 minute at maximum

speed to elute;

38 To obtain a higher total RNA concentration, a second

elution step may be performed by using the ﬁrst eluate

(from the previous step);

39 Measure the concentration of the RNA with a Spectrop-

hotometer;

Microarray Hybridisation

40 The positive control in the microarray hybridisation ex-

periment is a probe (ATGGCCGATGAGGAACGT)

speciﬁc for a 250 bp fragment of the TATA-box binding-

protein (TBP) gene of Saccharomyces cerevisiae (Metﬁes

and Medlin 2004);

41 The gene is ampliﬁed with the primers TBP-F (5’-ATG

GCC GAT GAG GAA CGT TTA A-3’) and TBP-R-

Biotin (5’-TTT TCA GAT CTA ACC TGC ACC C- 3’)

and is added to the hybridisation solution;

42 A negative control probe that has no match to any se-

quence found in the NCBI (National Center for Biotech-

nology Information) database should be used e.g. TCC-

CCCGGGTATGGCCGC (Metﬁes and Medlin 2004);

43 Pre-hybridisation step: incubate the microarrays in a mi-

croarray box containing ~ 20 mL pre-hybridisation buf-

fer [1X SET / 1 mg mL

-1

BSA] for 60 minutes at the

predetermined hybridisation temperature (58 ºC). Sub-

sequently wash the microarrays brieﬂy in deionised water

and by centrifugation;

44 Apply 30 µL of the 40 µL hybridisation solution to the

microarray. The solution contains labelled target nucleic

acid dissolved in hybridisation buffer. The ﬁnal concen-

tration of the target nucleic acid should be ~ 10 ng µL

-1

The solution should also contain the positive control

(i.e. 250 bp PCR-fragment TBP from S. cerevisiae with

biotin-labelled primers) in a ﬁnal concentration of 4.7 ng

µL

-1

;

45 Incubate the hybridisation solution for 5 minute at 94 ºC

in a thermoheater (Fig. 3) to denature the target nucleic

acid. The use of a special kind of coverslip, the Lifter Slip

coverslip (Implen, München, Germany) is recommended

to ensure an even dispersal of the hybridisation mixture

onto the microarray;

46 Place the cover slip on the slide and pipette 30 µL of the

hybridisation mixture under the cover slip (Fig. 7);

47 Hybridise at 58 ºC for 1 hour in a wet chamber. A 50 mL

Falcon-tube with a wet Whatman paper functions very

well as a moisture chamber. Apply approximately 1 mL of

hybridisation buffer onto the Whatman paper to obtain

enough humidity in the chamber;

48 After the hybridisation is complete, remove excessive

target nucleic acid and unspeciﬁc bindings by stringent

washing of the microarrays. Remove the cover slip from

the array and put the microarray into a Falcon-tube with

50 mL wash buffer 1. Place the Falcon-tube on a belly

dancer and shake for 10 minutes (Fig. 4). Repeat this step

with wash buffer 2. Wash-buffer 1 contains: 2X SSC / 10

mM EDTA / 0.05 % SDS; wash-buffer 2: 1X SSC / 10

mM EDTA;

49 Wash again for 5 minutes with increased stringency

[wash-buffer 3: 0.2X SSC / 10 mM EDTA]. Whereas

the ﬁrst washing buffer contains SDS, it is recommended

that the next 2 washing buffers do not contain SDS. This

is because residual SDS will generate high background

intensities on the microarray;

50 Remove the last wash buffer and dry the microarrays by

centrifugation in the falcon-tube (approx. 3 minutes at

approx. 2000 rpm). There are also special microarray

centrifuges with only the optimal speed;

Fluorescent Staining of the microarrays

51 Visualise the hybridised biotinylated target nucleic acids

by staining the microarray for 30 minutes at room tem-

perature in a wet chamber containing 50 µL Streptavidin-

Cy5 [0.1µg mL

-1

Streptavidin / 1X hybridisation buffer];

52 Remove excess stain by washing the microarrays twice

for 5 minutes with wash-buffer 1 and once for 5 minu-

tes with wash-buffer 2. Carry out the washing steps at

room temperature and place the microarrays in a 50 mL

Falkon-tube;

53 Dry the microarrays by centrifugation (approx. 3 minu-

tes at approx. 2000 rpm);

Analysis

54 Scan the microarray using the GenePix Axon 4000B

scanner at 635 nm;

55 Analyse the obtained signal intensities with the GenePix

6.0 software;

56 Calculate the signal to noise-ratios according to Loy et al.

(2002);

57 All calculated ratios should be normalised to the signal

of the TBP positive control. A schematic picture of the

excitation is shown in Figure 8.

Figure 7. Pipetting of hybridisation solution on one microarray.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 11 Hybridisation and microarray ﬂuorescent detection

Formula for calculating results

Discussion

The utilisation of molecular methods has increased the po-

tential for investigating the biodiversity of phytoplankton in

the marine environment. The identiﬁcation of phytoplank-

ton, especially of harmful algal species, is important from

an ecological and economic point of view. Microorganisms

dominate global biological diversity, in terms of their species

numbers, but their small size and lack of morphological fea-

tures make it difﬁcult to assess their overall biodiversity.

In the past, regular monitoring of phytoplankton has been

hampered by the lack of reliable morphological features in

some groups of species. Even with the introduction of elec-

tron microscopy, it is difﬁcult to make the correct classiﬁca-

tion, especially in picoplanktonic taxa or with hidden genetic

diversity in morphological indistinguishable species (Scholin

1998, Zingone et al. 1999, Massana et al. 2002, Janson and

Hayes 2006). As a result knowledge of the complexity of the

phytoplanktonic ecosystems is still limited. Many species are

sensitive to sample ﬁxation (Gieskes and Kraay 1983) and

some possess different life stages with varying morphological

properties (Partensky et al. 1988). The expertise of the scien-

tist may also inﬂuence the identiﬁcation (Bornet et al. 2005,

Godhe et al. 2007).

The utilisation of microarrays for the detection and moni-

toring of marine microalgae, although a relatively new tech-

nique, has already undergone several trials (Metﬁes and Med-

lin 2004, Ki and Han 2006, Medlin et al. 2006, Gescher et

al. 2007). Previous work has shown that microarrays can be

used for the identiﬁcation of phytoplankton using 18S rDNA

probes at the class level (Metﬁes and Medlin 2004, Medlin

et al. 2006). Ki and Han (2006) and Gescher et al. (2007)

have also demonstrated the speciﬁcity of 18S and 28S rDNA

probes for the detection of harmful algae at the species level.

One drawback of the method is the dependency on the se-

quence database for probe design. It is estimated that the

known rRNA sequence database is only a very small frac-

tion of the overall biodiversity present in the environment.

The number of 18S rRNA sequences in public databases is

constantly growing. Developed probes should be regularly

checked against all known sequences to ensure cross reactivity

with a non target organism does not occur.

The the detection limit of the microarray depends on the

sensitivity of the chosen probes. In general, a high sampling

volume of up to several Litres of seawater is required. This is

especially true if the number of cells present in the sample is

low. Another limiting factor of the microarray method lies in

the manual isolation of RNA. The manual isolation of RNA

from a set number of target cells can vary depending on the

skill of the operator. This can result in different signal intensi-

ties and so the resulting cell counts cannot be compared. The

utilisation of an automatic device (e.g. pipetting robot) for

RNA isolation may resolve this problem. The isolation of a

sufﬁcient amount of RNA is very important because the tar-

get RNA presents only a small fraction of the RNA isolated

from a wild sample.

A calibration curve must be developed for each new probe to

monitor individual algal species. The signal must be validated

with cell numbers. A good correlation of cell counts and RNA

concentration per cell with signal intensity is prerequisite for a

reliable analysis of ﬁeld samples. Different physiological con-

ditions may have an inﬂuence on the RNA content per cell.

The development and evaluation of microarrays is time-con-

suming and costly, but once a microarray is well developed,

it is a cost-effective, trusted and efﬁcient tool to detect the

target organism. Limitations and cost aside, the use of micro-

arrays to answer ecological and biodiversity questions offers

for the ﬁrst time the possibility to analyse samples with a large

number of different targets (species or other taxa) in parallel

(Ye et al. 2001). One of the most likely future uses of microar-

rays in the ﬁeld of phytoplankton ecology is to monitor the

biodiversity of phytoplankton over long-time scales (Medlin

et al. 2006, Gescher et al. 2007c).

Acknowledgements

Christine Gescher was supported by the EU-project

FISH&CHIPS (GOCE-CT-2003-505491).

1000 *

)(

) (













−

mLsampleofVolume

N filter whole on count cell Positive

Lcells ion concentrat Cell

1000 *

)(

) (













−

mLsampleofVolume

N filter whole on count cell Positive

Lcells ion concentrat Cell

Figure 8. Microarray detection.

IOC Manuals & Guides no 55

Chapter 11 Hybridisation and microarray ﬂuorescent detection

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IOC Manuals & Guides no 55

Chapter 11 Hybridisation and microarray ﬂuorescent detection

Appendix

Equipment Supplier Cat. Number

€

US $

Mini-Beadbeater™ Biospec products, Biospec

products Inc, USA

3110BX 715 986

Mini-Centrifuge Hereaus

”Biofuge pico”

Omnilab, Germany 7025235 938 1294

Nanodrop ND1000

Spectrophotometer

Peqlap Biotechnology,

Germany

91-ND-1000 8995 12418

Incubator ”Shake’n’Stack” VWR, Germany 7996 2310 3189

Thermoheater ”Comfort” Eppendorf, Germany 5355R 2545 3512

Shaker (Bellydancer) Sigma Aldrich, Germany Z36, 761-3 1400 1800

Scanner and software

(GenePix 4000B device and

GenePix Pro.6.0 software)

Molecular Devices

Corporation, USA

97-0002-00 40000 51000

Sum approx.

56903 74199

Chemical Supplier Cat. Number

€

US $

Spotting (per slide) Commercial supplier or with

own spotting device

- 75 100

RNeasy Mini Plant Kit Qiagen Inc., USA 74904 268 370

Biotin-ULS Kit Fermentas Inc., USA K0631 500 640

RNeasy MinElute Cleanup Kit Qiagen Inc.,USA 742043 235 300

EDTA Sigma Aldrich, Germany E5134 80 102

Citric Acid Merck KGaA, Germany 231211 25 32

Sodium chloride NaCl Sigma Aldrich, Germany S9888 23 29

SDS Merck KGaA, Germany L4390 40 51

Bovine Serum Albumin BSA Sigma Aldrich, Germany A2153 30 38

Trizma Base Sigma Aldrich, Germany T_1503 80 102

Triton x-100 Merck KGaA, Germany 648466 33 42

Ethanol, pro Analysis Merck KGaA, Germany 1,009,832,500 73 93

1 probe (18 bases, with

Aminolink) + Positive control,

Negative control

Thermo Electron, Gemany - 100 127

Herring-Sperm DNA Roche, Germany 223646 91 116

Streptavidin-CY5 KPL, USA 079-30-00 250 319

Table 1. Equipment and suppliers.

Table 2. Chemicals and suppliers.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 12 Semiautomated sandwich hybridisation

Introduction

The Sandwich Hybridisation Assay (SHA) provides a sim-

ple and rapid means to detect and estimate cell density of a

variety of algal species associated with harmful algal blooms

(HABs). Results from the SHA system can discriminate to

species level using both cultured and natural samples. Hav-

ing this level of discrimination without the need for micro-

scopy and advanced training in taxonomy, gives researchers,

public health ofﬁcials and water quality managers a powerful

tool to rapidly assess changing HAB communities. The SHA

system uses species-speciﬁc, ribosomal RNA (rRNA) targeted

DNA probes that are applied using a semi-automated robotic

processor (Scholin et al. 1996, 1997, 1999, Greenﬁeld et al.

2008). Currently, DNA probes for Pseudo-nitzschia spp., Al-

exandrium spp., Heterosigma akashiwo, Chattonella spp., and

Fibrocapsa japonica are available (Scholin et al. 2004). Others

probes include those for Coccholodinium polykrikoides (Mikul-

ski et al. 2008), a variety of Karenia spp., Karlodinium ven-

eﬁcum and Gymnodinium aureolum (Haywood et al. 2007).

In New Zealand the SHA method has gained international

accreditation and is used to regulate shellﬁsh harvests (e.g. Ay-

ers et al. 2005 and references therein). Recent progress made

on assays for invertebrates (Goffredi et al. 2006), including

the invasive European green crab (Jones et al. 2008), and ma-

rine bacteria (Preston, 2009) offer opportunities for use of the

SHA format to detect many other organisms as well.

Basic Principles of Sandwich Hybridisa-

tion

The SHA referred to here (after Scholin et al. 1996, 1999,

Greenﬁeld et al. 2008) employs two DNA probes that target

ribosomal RNA (rRNA) sequences. Assays for both the large

and small subunit (LSU, SSU) rRNA have been implement-

ed. Assays are performed using pre-ﬁlled 96 well microplates

and a robotic processor supplied by Saigene Biotech Inc. A

capture probe complementary to a variable sequence is at-

tached to a mechanical solid support (prong in a sandwich

hybridisation machine), which is then submerged into the

prepared sample and hybridizes with the target molecule if

present. Captured molecules are then washed to remove any

unbound material. To detect the captured molecules, a second

hybridisation step is initiated using a DNA probe conjugated

to a signal probe. This probe is targeted to a more conserved

region of the captured fragment. The resulting “sandwich” of

capture probe/target molecule/signal probe is detected using

an enzymatically-driven colorimetric reaction. Figure 1 pro-

vides a schematic view of the sandwich hybridisation chem-

istry described above (for details see Greenﬁeld et al. 2008).

The basic steps of the SHA method are:

1 Collect sample onto a ﬁlter,

2 Lyse sample using a chaotropic buffer (disrupts and dena-

tures the 3-D structure of macromolecules) and heat,

3 Filter lysate,

4 Load sample lysate into 96-well plate,

5 Run SHA processor (automated),

6 Record colour development (O.D. 650 and 450nm),

7 Compare results against standard curves to estimate

abundance of target species.

Laboratory Facilities

The SHA system requires a typical laboratory setting that is

protected from direct sunlight, excessive dust, and tempera-

ture extremes.

Essential Equipment

(for more details see Appendix, Table 1 at the end of this chap-

ter)

• Bench top processor (after Scholin et al. 1999)

• 96-well Microplate reader that can read wavelengths 650

nm and 450 nm

• Software to record microplate data and apply data con-

version algorithm

• Refrigerated storage (2

to 8

• Vacuum ﬁlter manifold

• 85

C heat block

• 12-channel multiple pipette (30-300 µL)

12 Toxic algal detection using rRNA-targeted probes in a semi-

automated sandwich hybridization format

Roman Marin III* and Christopher A. Scholin

Monterey Bay Aquarium Research Institute (MBARI) 7700 Sand-holdt Rd., Moss Landing, CA 95039-0628, USA

*Author for correspondence e-mail: maro@mbari.org

Figure 1. Schematic view of the sandwich hybridisation chemistry

BACK

Well A

FRONT

Well H

IOC Manuals & Guides no 55

Chapter 12 Semiautomated sandwich hybridisation

Scope

Detection and quantiﬁcation of a target phytoplankton species

using ribosomal RNA-targeted, DNA probe-based assays.

Detection range

Detection performance is unique to each probe set used in the

SHA system.

Advantages

The SHA system provides a robust and simple semi-automa-

ted method to detect and estimate cell abundances of target

species.

Drawbacks

Probes are only available for a limited number of target species.

Speciﬁcity of probes must be established on a regional basis.

This system may not be suitable for detection of very rare target

sequences.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of mole-

cular biology. A minimum of three days are needed for to cover

theory, operation, data processing, etc. A skilled molecular bio-

logist should be available to solve any problems that may arise

using this method.

Essential Equipment

SHA semi-robotic processor, microplate reader, heating block,

ﬁltration manifold, 12-channel and single channel micropipet-

tors.

Equipment cost*

SHA semi-robotic processor, €5139 (US $7500)

Total set-up cost = €14197 (US $20666).

See Appendix, Table 1 for details

Consumables, cost per sample**

€5-7 (US $7-10).

Processing time per sample before analysis

15-20 minutes (hands-on)

Analysis time per sample

75 minutes (hands-off).

Sample throughput per person per day

30-40 samples in an 8 hour day given 1 processor.

No. of samples processed in parallel

6, 8, or 12 samples with replicates 4, 3, or 2, respectively.

Health and Safety issues

Relevant health and safety procedures must be followed. The

lysis and signal probe buffers contain guanidine thiocyanate,

which can damage skin and eyes.

*service contracts not included

**salaries not included

The fundamentals of

The sandwich hybridisation method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 12 Semiautomated sandwich hybridisation

• 100-1000 µL single channel pipette

• Cold storage for samples: liquid nitrogen is preferred,

-80

C and –20

C (e.g., freezer or dry ice) can also be ef-

fective for storing archival samples (ﬁlters)

•

Chemicals and Consumables

(for more details see Appendix, Table 2 at the end of this

chapter)

• 25mm hydrophilic Durapore ﬁlter

• 2mL cryovial

• 13mm syringe ﬁlter

• 5cc syringe

• Polypropylene tubes 12X 75mm

• 10% H

• ﬁlling trays/pipette tips

Probes

The suite of SHA probes currently available for HAB spp.

include the following:

Diatoms

Pseudo-nitzschia australis (and other diatoms below, Scholin

et al. 1999)

Pseudo-nitzschia multiseries

Pseudo-nitzschia pungens

Pseudo-nitzschia pseudoelicatissima/multiseries complex

Dinoﬂagellates

Alexandrium tamerense/catenella/fundyense (North American

ribotype, Matweyou et al. 2004, Anderson et al. 2005)

Cochlodinium polykrikoides (Mikulski et al. 2008)

Gymnodinium aureolum (and other dinoﬂagellates below,

Haywood et al. 2007)

Karenia brevis

Karenia mikimotoi

Karenia selliformis

Karenia papilionacae

Karlodinium veneﬁcum

Raphidophytes

Heterosigma akashiwo (and others below, Tyrrell et al. 2001,

2002, Scholin et al. 2004)

Fibrocapsa japonica

Chattonella antiqua/subsalsa

Method

Preparation to run samples

1 Turn on heating blocks for sample lysis (Fig. 2) and pro-

cessor and check that desired temperatures are at their

proper values. Lysis is carried out at 85

C. The processor

plate should provide a temperature of 28-30

2 Obtain necessary lysis tubes for runs or alternatively label

2 mL cryovials for storage of sample ﬁlters in liquid nitro-

gen for later analysis (see below).

3 Start the microplate reader.

4 If using pre-made plates, remove seal and let it reach room

temperature protected from light and dust. If required,

dispense reagents into 96-well microplate as shown in the

instruction booklet that comes with materials supplied

by Saigene Corporation (see also Greenﬁeld et al. 2006).

When dispensing reagents to a microplate use barrier tips

Figure 2. Heating block set to 85ºC used for sample lysis.

Figure 3. Three-position vacuum ﬁlter manifold used to

collect particulate fraction of water sample.

Figure 4. Filter, sample side in, being placed in cryovial for

archival and/or lysis.

Figure 5. Addition of lysis buffer to cryovial with ﬁlter.

Figure 6. After lysis, lysate is syringe ﬁltered into clean tube.

IOC Manuals & Guides no 55

Chapter 12 Semiautomated sandwich hybridisation

to minimise cross-well contamination. Dispense 0.25 mL

per well. Do not blow out small amounts of ﬂuids fol-

lowing primary delivery of reagents and samples to the

plate or excessive bubbles will form in the well; bubbles

can interfere with the assay.

Sample, plate and prong handling

5 Protect plate and sample from sunlight and excessive

heat. Samples should be ﬁltered and lysed as soon as pos-

sible, or ﬁlters can be archived for later analysis by rolling

the ﬁlter into a cryovial (particles away from the tube wall

and freezing the ﬁlters in liquid nitrogen or alternative

cold storage). Use plate within 1 hour after removing

seal and/or dispensing reagents. Prongs should remain in

package until used. Handle prongs with forceps touching

only the strip, or backbone, that connects the 12 prongs;

avoid touching the prongs themselves. Store prongs at

4-8

C with packaging and desiccant provided.

Sample ﬁltration

6 Samples should be collected using a vacuum manifold

at a vacuum pressure of approximately 100-150 mmHg

(Fig. 3). Filter samples onto hydrophilic Durapore ﬁl-

ters (generally 0.65-0.45 µm pores size; Millipore). The

volume ﬁltered should be no more than what can pass

through the ﬁlter in about 20 minutes. Typical sample

volumes are 200 to 400 mL of whole water. Samples that

have been pre-concentrated (e.g. net tow or sieve) can

also be collected on the Durapore ﬁlter as well.

Archival and Lysis of Sample

7 After ﬁltering, place ﬁlter membrane into a 2 mL cryo-

vial. Place the ﬁlter with the sample side facing away from

the tube wall. Do not crumple the ﬁlter and be sure to

push the ﬁlter to bottom of the tube (Fig 4). If sample is

to be archived, cap the 2 mL cryovial and store in liquid

nitrogen without lysis buffer.

8 To process, add 1-2 mL of lysis buffer to cryovial with

ﬁlter and cap it tightly (Fig 5). Place cryovial in heating

block with wells half ﬁlled with water to enhance heat

transfer (Fig. 2). Heat for 5 minutes total with a brief

ﬁnger vortex after 2.5 minutes.

9 After heating, allow lysate to cool for 5 minutes. Use ly-

sate within 20 minutes of preparation.

10 Remove plunger from a 5 cc disposable syringe (Becton

and Dickinson).

11 Install a 13 mm, 0.45 µm Millex-HV (Millipore) ﬁlter

onto the syringe.

12 Place tip into a clean polypropylene collection tube, add

lysate to syringe barrel and push lysate through ﬁlter until

foam appears at the tip of ﬁlter (Fig. 6). Lysates from re-

plicate samples may be combined to yield a larger volume

of lysate from a given sample.

13 The lysate is now ready to be loaded onto the plate sam-

ple well (row H).

Processing

14 Load 0.25 mL ﬁltered lysate per sample well (Fig. 7).

15 Load prong onto processor arm and secure spring clip.

Do not handle prong with bare hands. Hold prong by the

backbone with tweezers and avoid scraping the prongs

against the processor arm.

Figure 7. Lysate (0.25 mL) is added to appropriate sam-

ple wells on microplate.

Figure 8. Microplate being placed on processor after

prongs added to processor arm.

Figure 9. Processor being started after plate is positio-

ned on heater base.

Figure 10. Microplate is placed on microplate reader

cradle; optical density is recorded at 650nm for row “A”

(wells in which colour development has occurred).

Figure 11. After microplate is read at 650nm, row “A” is acidiﬁed

with 50 µL of 10% H

;optical density is recorded at 450 nm for

row “A”. The measure at 650 nm is considered low sensitivity while

that at 450 nm is considered high sensitivity. For positive samples,

the optical density at 450 nm should be roughly 2X that at 650 nm.

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 12 Semiautomated sandwich hybridisation

16 Mount 96 well microplate onto processor, make sure to

centre the microplate on the heating plate (Fig. 8).

17 Push the “RUN” button (Fig. 9) and make sure that the

prongs enter the wells without touching the sides of the

wells. The plate will be ready to read in just over an hour.

A digital timer will display time remaining; the counter

will read “0” approximately 90 seconds before the process

is actually complete. The processor will display a mes-

sage indicating that the reaction is complete, and plate is

ready to be read.

Reading the Plate

18 When the processor is ﬁnished, immediately read the

plate(s) (Fig. 10).

19 First read the plate at 650 nm.

20 Next acidify row A with 50 µL of 10 % H

(add and

mix using a 12-channel pipette and avoid introduction of

bubbles) (Fig. 11).

21 After all bubbles have popped (~10-45 seconds) read

plate at 450 nm.

Data Processing

To calculate the concentration of target in a sample, the as-

say requires that you establish an empirically derived dose re-

sponse curve that relates optical density (O.D.) to a known

number of cells per well of lysate (see Greenﬁeld et al. 2008,

Haywood et al. 2007, Ayers et al. 2005 for details in estab-

lishing a dose response curve). Using optical density (from

the assay), the dose response curve, sample volume and lysis

buffer volume (used to lyse the sample) you can estimate the

target abundance in the sample. Below is the equation used to

convert cells per well to cells per mL.

Here is an example of how you use the above equation to

estimate target abundance in a sample. You ﬁlter 500mL (X)

of water onto a ﬁlter and lyse this sample in 2.0 mL of lysis

buffer (Y). Using the SHA system you obtain an O.D. that

translates to 625 cells per well (from the dose response curve).

Then using the formula you estimate a target abundance of

10 cells per mL in your sample.

Controls

Positive and negative controls are available to check system

integrity and performance (e.g. Greenﬁeld et al. 2006).

Sample Collection and Preservation

Samples should be run or archived as soon as possible after

collection. Prior to ﬁltration, samples should be kept cool and

protected from excessive light. If the sample cannot be run

within several hours it should be ﬁltered and stored frozen. A

sample stored in liquid nitrogen can be held for up to 1 year

or longer. Samples can also be stored in a -80

C freezer or on

dry ice for up to 1 week. It may be possible to store preserved

samples at room temperature as well (see Tyrrell et al. 2002).

Discussion and system considerations

The goal of SHA system is to give the research/monitoring

community a method to quickly and conveniently screen

samples for a variety of HAB species. Once samples are col-

lected, processing takes approximately 1.25 hours. No target

ampliﬁcation is required so problems that can affect ampli-

ﬁcation based systems (e.g. extensive sample handling, PCR

inhibition) are avoided. The absolute detection level of the

SHA system is dependant on the designs of the capture and

signal probes. An important feature of the SHA system is that

it is relatively insensitive to biomass, so techniques such as

sieving to collect large volumes of sample can be used with-

out impacting system performance, provided proper control

experiments have been performed to verify assay results from

a given region and wide range of samples. Further increases

in sensitivity can also be achieved by lysing the sample in a

smaller volume of lysis buffer to increase target cell concentra-

tion. The SHA system is not suitable for very rare targets (e.g.

single copy genes); in those cases some kind of ampliﬁcation

technique may be desirable.

All methods relying on molecular probes for detection can

be subject to cross-reaction with non-target species. Therefore

after an initial positive result, an alternative method (micro-

scopy, toxin detection, PCR, etc.) should be used to conﬁrm

results until such time that conﬁdence in the efﬁcacy of probe

is known. Some probes may work well for certain species in

certain regions, but not all probes will work equally well in

different geographic regions. Moreover, species designations

as deﬁned using traditional criteria (morphology, ultrastruc-

ture, pigments, etc.) may not agree with those based on rRNA

sequence identity (e.g. see Scholin et al. 2004, Ayers et al.

2005, Lundholm et al. 2006). Provisions should also be made

to store replicate samples in case of system failure or if results

require further analysis or reconﬁrmation is desired.

Use of the SHA system requires that probes be available for

the target species of interest. Currently, probes are available

for a variety of HAB spp. (see above) as well as other organ-

isms. The creation of probes for this system often requires

iterative probe design (trial and error) which can incur con-

siderable time and expense. Ideally, cultures of the targeted

species are used to create calibration curves and to spot check

the system when reagent batches are changed.

The SHA system uses chemistry that is designed to work at

C. If the ambient temperature exceeds this value, the sys-

tem will not function properly and steps to lower ambient

temperature will need to be taken. Some of the reagents used

in the SHA system are required to be refrigerated and pro-

tected from direct sunlight.

Acknowledgements

























X mL sample

filter

filter Y mL lysate

lysatemL

well

cells

sampleinmLperCells

25.0

























500 mL sample

filter

filter 2 mL lysate

lysate mL

well

625 cells

25 . 0

mL of sample

10 cells

IOC Manuals & Guides no 55

Chapter 12 Semiautomated sandwich hybridisation

Development and application of the SHA for HAB research

have been sponsored in large part by grants from the David

and Lucille Packard Foundation (allocated by the Monterey

Bay Aquarium Research Institute), the National Science

Foundation (9602576 and OCE-031422), and the National

Oceanic Atmospheric Administration Saltonstall-Kennedy

Grant Program (NOAA NA57D009) to C.A.S.

References

Anderson DM, Kulis DM, Keafer BA, Gribble KE, Marin R, Scho-

lin CA (2005) Identiﬁcation and enumeration of Alexandrium

spp. from the Gulf of Maine using molecular probes. Deep-Sea

Res II 52:2467-2490

Ayers K, Rhodes L, Tyrrell J, Gladstone M, Scholin C (2005) In-

ternational accreditation of sandwich hybridisation assay for-

mat DNA probes for micro-algae. N. Z. J. Mar. Freshwater Res.

39:1225–1231

Greenﬁeld D, Marin III R, Doucette GJ, Mikulski C, Jensen S, Ro-

man B, Alvarado N, Scholin CA. (2008) Field applications of the

second-generation Environmental Sample Processor (ESP) for

remote detection of harmful algae: 2006-2007. Limnology and

Oceanography: Methods 6: 667-679.

Greenﬁeld DI, Marin III R, Jensen S, Massio E, Roman B, Feld-

man J, Scholin C (2006) Application of the Environmental Sam-

ple Processor (ESP) methodology for quantifying Pseudo-nitzschia

australis using ribosomal RNA-targeted probes in sandwich and

ﬂuorescent in situ hybridisation. Limnol Oceanogr Methods

4:426-435.

Goffredi SK, Jones W, Scholin CA, Marin R, Hallam S, Vrijenhoek

RC (2006) Molecular detection of marine larvae. Marine Biotech-

nology 8: 149-160.

Haywood AJ, Scholin CA, Marin III R, Steidinger KA, Heil CA,

Ray J (2007) Molecular detection of the brevetoxin-producing

dinoﬂagellate Karenia brevis (Dinophyceae) and closely related

species using ribosomal RNA probes and a semi-automated sand-

wich hybridization assay. J Phycol 43:1271–1286

Jones WJ, Preston C, Marin III R, Scholin C, Vrijenhoek R (2008)

A Robotic Molecular Method for in situ Detection of Marine In-

vertebrate Larvae. Mol Ecol Resour 8:540-550

Lundholm N, Moestrup Ø, Kotaki Y, Scholin C, Miller P (2006)

Inter- and intraspeciﬁc variation of the Pseudo-nitzschia delica-

tissima-complex (Bacillariophyceae) illustrated by rRNA probes,

morphological data and phylogenetic analyses identiﬁcation of P.

decipiens and P. dolorosa spp. Nov. J Phycol 42:464-481

Matweyou JA, Stockwell DA, Scholin CA, Hall S, Trainer VL, Ray

JD, Whitledge TE, Childers AR, Plumley FG (2004) Use of Alex-

andrium rRNA targeted probes to predict PSP events on Kodiak

Island, Alaska. In: Steidinger KA, Landsberg JH, Tomas CR, Var-

go GA (eds) Harmful Algae 2002. Florida Fish and Wildlife Con-

servation Commission, Florida Institute of Oceanography, and

Intergovernmental Oceanographic Commission of UNESCO, St.

Petersburg, Florida, USA., p 267-269

Mikulski CM, Park YT, Jones KL, Lee CK, Lim WA, Lee Y, Scho-

lin CA, Doucette GJ (2008) Development and ﬁeld applica-

tion of rRNA-targeted probes for the detection of Cochlodinium

polykrikoides Margalef in Korean coastal waters using whole cell

and sandwich hybridization formats. Harmful Algae 7:347-359

Preston C., Marin III R, Jenson S, Feldman J, Massion E, De-

Long E, Suzuki M, Wheeler K, Cline D, Alvarado N, Scholin

C. (2009) Near real-time, autonomous detection of marine bac-

terioplankton on a coastal mooring in Monterey Bay, California,

using rRNA-targeted DNA probes. Environmental Microbiology

11:1168-1180

Scholin CA, Buck KR, Britschgi T, Cangelosi J, Chavez FP (1996)

Identiﬁcation of Pseudo-nitzschia australis (Bacillariophyceae) us-

ing rRNA-targeted probes in whole cell and sandwich hybridiza-

tion formats. Phycologia 35:190 – 197

Scholin C, Miller P, Buck K, Chavez F, Harris P, Haydock P, Howard

J, Cangelosi G (1997) Detection and quantiﬁcation of Pseudo-

nitzschia australis in cultured and natural populations using LSU

rRNA-targeted probes. Limnol Oceanogr 42:1265–1272

Scholin CA, Marin III R, Miller PE, Doucette GJ, Powell CL,

Haydock P, Howard J, Ray J (1999) DNA probes and a receptor-

binding assay for detection of Pseudo-nitzschia (Bacillariophyceae)

species and domoic acid activity in cultured and natural samples.

J Phycol 35:1356-1367.

Scholin CA, Vrieling E, Peperzak L, Rhodes L, Rublee P (2004) De-

tection of HAB species using lectin, antibody and DNA probes.

In: Hallegraeff GM, Anderson DM, Cembella AD (eds) Manual

on harmful marine microalgae, Vol 11. IOC, UNESCO Paris, p

131-164

Tyrrell JV, Connell LB, Scholin CA (2002) Monitoring for Heter-

osigma akashiwo using a sandwich hybridisation assay. Harmful

Algae 1:205-214

Tyrrell JV, Scholin CA, Berguist PR, Berguist PL (2001) Detection

and enumeration of Heterosigma akashiwo and Fibrocapsa japonica

(Raphidophyceae) using rRNA-targeted oligonucleotide probes.

Phycologia 40:457-467

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 12 Semiautomated sandwich hybridisation

Appendix

Table 1. Equipment and suppliers. Note that some pieces of equipment, such as the plate reader, sample ﬁltration system, heating block

and refrigerator, are available as other models from a variety of vendors. Prices quote obtained December, 2007.

Equipment Supplier Cat. Number

€

US $

96-well Microplate reader that can

read wavelengths 650 nm and 450 nm

Fisher Scientiﬁc 14-386-27 3821 5510

Optical ﬁlter 650nm Fisher Scientiﬁc 14-386-59 196 283

Vacuum ﬁlter manifold Fisher Scientiﬁc 09-753-39A 540 779

25 mm polysulfone ﬁlter funnel

(250 mL) need 6, costs in total

Pall 4203 504 726

Vacuum pump GAST DOA P704 AA 303 443

Heating block Fisher Scientiﬁc 11-716-68Q 223 322

12-Channel Pipettor 20-300 µL Rainin L12-300 412 595

Single Channel Pipettor 10-1000 µL Rainin PR-1000 184 265

Robotic processor Saigene Inc

. 6000-01 5708 8500

Refrigerator (4-8

C) Maytag MBB1952HE 577 850

Cryogenic Storage vessel Fisher Scientiﬁc 11-676-1C 2350 3393

Sum approx. 14818 21666

IOC Manuals & Guides no 55

Chapter 12 Semiautomated sandwich hybridisation

Table 2. Expendable reagents and supplies required for application of the SHA and relevant suppliers.

Material Supplier Cat. Number

€

US $

Custom Plate One Probe

Set; minimum order of 50

plates

Saigene

call for order 25 38 per plate

Custom Plate Two Probe Set;

minimum order of 50 plates

Saigene

call for order 27 40

Custom Plate Three Probe

Set; minimum order of 50

plates

Saigene

call for order 28 42

Custom Plate Four Probe

Set; minimum order of 50

plates

Saigene

call for order 29 44

Assay Development Kit;

minimum order of 25 plates

Saigene

call for order 24 36

Bulk lysis buffer, 500 mL Saigene

call for order 27 40

Deﬁned Kits (routine

production), incl lysis buffer,

prong, sample ﬁlters, etc.

Call for quote Call for quote

25 mm Durapore ﬁlter (100

count)

Millipore DVPP02500 60 86

13 mm syringe ﬁlter (100

count)

Millipore SLHVT13NL 138 197

Polypropylene 12X75mm

tubes (5000 count)

Fisher Scientiﬁc 14-961-11 318 459

5 cc Syringe (400 count) Fisher Scientiﬁc 14-823-35 47 68

2 mL Cryovial (250 vials) Fisher Scientiﬁc 03-337-7H 127 184

Filling boats (200 count) Fisher Scientiﬁc 07-200-127 76 110

10% H

(dilute stock) Fisher Scientiﬁc A300-500 40 58

Sum approx. 966 1402

Plates made custom to user speciﬁcation. Saigene will ﬁll and seal plates; user supplies capture and signal pro-

bes. Plates are conﬁgured with one to four probe sets. Price includes prongs; lysis buffer sold separately. Discount

available for orders >50 plates.

Plates ﬁlled with all reagents except capture and signal probes (to facilitate assay development). Price includes

prong, buffers for preparing capture and signal probe solutions. Discount available for orders >25 plates.

In addition to lysis buffer, Saigene can provide bulk quantities of other reagents used in the SHA; prices available

on request.

Deﬁned Kits are those prepared entirely by Saigene. They differ from Custom Plate conﬁgurations in that Saigene

provides user-deﬁned probes, or those available through published articles. Deﬁned Kits also include prongs and

lysis buffer (volume based on the intended use of the plates), and can be bundled with sample and lysate ﬁlters if

desired. The most likely application of Deﬁned Kits are for research/monitoring programs where there is a deﬁned

set of target species/probes, sample and lysis volumes are ﬁxed within well speciﬁed range, and users prefer not

to take responsibility for procurement and quality assurance of probe stocks. Depending on the number of plates

ordered, Deﬁned Kits will generally exceed those in cost of the equivalent Custom Plate by 10-20% depending on

probe costs and number of probe sets required per plate.

Saigene Biotech Inc.

ATTN: Thomas Hurford

Box 3048

Monument, CO 80132

USA

Phone Int.+ 1 719-559-1163

[email protected]

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 13 Quantitative PCR

13 Quantitative PCR for detection and enumeration of phyto-

plankton

Luca Galluzzi*

and Antonella Penna

Section for Biotechnology, Department of Biomolecular Sciences, University of Urbino, Via Campanella 1, 61032 Fano (PU), Italy

Section of Environmental Biology, Department of Biomolecular Sciences, University of Urbino, V.le Trieste 296, 61100 Pesaro (PU), Italy

*Author for correspondence e-mail: [email protected]

Introduction

Quantitative Polyemerase Chain Reaction (QPCR) is an ex-

tremely sensitive method which has been applied in recent

years to detect and quantify different phytoplankton spe-

cies in environmental samples (Bowers et al. 2000, Popels et

al. 2003, Galluzzi et al. 2004, Coyne et al. 2005, Park et al.

2007, Touzet et al. 2009). Its application could revolutionise

the study of microalgal population dynamics in marine sys-

tems as it allows the concurrent identiﬁcation, enumeration

and determination of viability of target species (Coyne and

Cary 2005, Kamikawa et al. 2005). QPCR can be used on

both seawater and sediment samples. The method is based

on the ampliﬁcation of speciﬁc DNA sequences. In assays

developed to date for phytoplankton these sequances consist

of ribosomal RNA (rRNA) genes. Many protocols have been

optimised for the QPCR approach. Here, we describe an op-

timised protocol, which is based on the use of SYBR Green (a

dye which speciﬁcally binds to DNA) and relies on a standard

curve constructed with a DNA plasmid containing the cloned

target sequence for quantiﬁcation.

Basic Principles of Quantitative PCR

(QPCR)

Polymerase Chain Reaction is a technique used to amplify in

vitro a target sequence of DNA. The PCR is performed by

heating and cooling an initial reaction mixture in a deﬁned

series of temperature steps. The reaction mix contains DNA

from the sample to be tested, two different primers (small

bits of artiﬁcially synthesised DNA complementary to the tar-

get DNA in which you are interested) nucleotides consisting

of the four different bases required to make DNA, a DNA

polymerase enzyme which will build DNA using the nucle-

otides and buffer containing various salts which are optimal

for the functioning of the DNA polymerase. The different

temperature steps are necessary to separate the two strands

in the double helix DNA (denaturation step), to allow the

binding of the primers to the complementary DNA present

in the sample (annealing step) and to permit DNA synthesis

by the DNA polymerase (elongation step). The speciﬁcity of

the PCR is mainly due to the primer sequences which must

be complementary only to the DNA region targeted for am-

pliﬁcation. The annealing temperature is of particular impor-

tance. If too low an annealing temperature is used, then the

primers may anneal to regions of DNA which are similar but

not identical.

As the PCR progresses, the DNA generated by the reaction

is used as a template for replication leading to the exponen-

tial ampliﬁcation of the target DNA sequence. A typical PCR

ampliﬁcation proﬁle consists of an exponential amplication

of the target sequence, followed by a linear or plateau phase

as reagents become exhausted as seen in Figure 1.

The qualitative analysis of the PCR reactions is performed

at the end point of the reaction. This is usually performed

by agarose gel electrophoresis and ethidium bromide staining.

This method of visualising this PCR DNA product involves

placing the PCR product in a well in a thin block of agar-

ose gel and passing an electric current, negative to positive,

through the gel. DNA is a negatively charged particle and will

migrate with the current through the gel. Because all of the

PCR products are the same size i.e. the size determined by the

distance between where the two primers originally bound to

the opposite strands of the DNA during PCR ampliﬁcation,

all the PCR products will migrate at the same rate forming a

dense band of DNA. The agarose is infused with ethidium

bromide, a stain which binds to DNA. Ethidium bromide

ﬂuoresces under UV light. When the gel is viewed under UV

light the PCR product can be easily seen as a ﬂuorescent band

where the ethidium bromide has concentrated in the DNA.

The brightness of the PCR band is related to the amount of

PCR product present at the end of the PCR reaction (the

plateau phase).

In quantitative real-time PCR (QPCR), reactions are ana-

lysed during the initial exponential phase rather than at the

end point. PCR product formation is monitored after each

cycle in real time by measuring a ﬂuorescence signal which

is proportional to the amount of PCR product generated.

This is performed using a camera incorporated within a real-

time PCR machine. Software converts the data recorded by

the camera and allows the visualization of the ampliﬁcation

curves on a computer screen. An example of this can be seen

in Fig. 2. The ﬂuorescence can be generated by using inter-

calating (binding between the grooves of the double helix

PCR product

Cycle number

A B

Figure 1. General PCR ampliﬁcation proﬁle. The exponential amp-

liﬁcation phase (a) is followed by a linear or plateau phase (b), due

to reactants exhaustion

IOC Manuals & Guides no 55

Chapter 13 Quantitative PCR

Scope

Detection and quantiﬁcation of target phytoplankton species.

Detection range

Detection performance varies with the sample volume, more

precisely: 10 target sequences in a 25 µL reaction tube. The

method is sufﬁciently sensitive to detect one cell.

Advantages

Allows identiﬁcation of target phytoplankton to species level.

Highly sensitive. No taxonomic expertise needed.

Drawbacks

Probes are only available for a limited number of target species.

Speciﬁcity of probes must be established on a regional basis.

Only one species or strain at a time can be analysed in a quanti-

tative manner, unless a multiplex reaction is performed; equip-

ment is still expensive.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of molecu-

lar biology particularly in real-time PCR (RT-PCR). A skilled

molecular biologist should be available to solve any problems

that may arise using this method.

Essential Equipment

Filtration apparatus/centrifuge, hybridisation oven, real-time

PCR instrument.

Equipment cost*

Total set-up cost = €49426 ($69890)

See Appendix, Table 1 for details

Consumables, cost per sample**

€4.10 ($6.00 US) using a Millipore ﬁlter to collect cells or

€3.00 ($4.40 US) using a microcentriguge to collect cells

Processing time per sample before analysis

Approximately 4 hours to process the sample.

Analysis time per sample

Approximately 3 hours to assemble and perform the RT- PCR.

Up to 10 samples can be processed roughly in the same amount

of time. Analysis of the real-time PCR results and calculation of

cell concentration may require 15 minutes.

Sample throughput per person per day

A trained person can process up to 16 samples per day.

No. of samples processed in parallel

Up to 16 samples.

Health and Safety issues

Relevant health and safety procedures must be followed. SYBR

Green is a DNA intercalating dye.

*service contracts not included

**salaries not included

The fundamentals of

The quantitative PCR method

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 13 Quantitative PCR

DNA) ﬂuorescent dyes or a number of alternative real-time

PCR chemistries which use probes (short synthetically made

pieces of DNA) which ﬂuoresce when bound to their com-

plementary target DNA following PCR ampliﬁcation (e.g.

Hydrolysis probes (TaqMan), Hybridisation probes ( FRET).

For each sample, the ﬂuorescence signal of the reporter dye

(i.e. the dye which ﬂuoresces in proportion to the amount of

PCR product produced) (e.g. SYBR) is divided by the ﬂuo-

rescence signal of the passive reference dye (ROX) to obtain

a ratio deﬁned as the normalised reporter signal (Rn). ROX

is a dye present in the reaction mix which gives out a stand-

ard level of ﬂuorescence independent of PCR ampliﬁcation.

It is used to normalise for differences in amount of reaction

mix due to pipetting errors or evaporation. Some QPCR ma-

chines do not require the use of ROX. The higher the start-

ing amount of the target molecule, the earlier a signiﬁcant

increase in ﬂuorescence (Rn value) is observed. The parameter

Ct (threshold cycle) is deﬁned as the fractional cycle number

at which the ﬂuorescence crosses a ﬁxed threshold above the

baseline. The amount of target sequence in an unknown

sample is calculated by plotting the Ct value on the standard

curve. The standard curve is generated by the QPCR instru-

ment software by plotting Ct values versus the log of initial

target concentration, and by performing a linear regression.

The PCR efﬁciency can be calculated from the slope of the

line using the equation:

Efciency = 10

(-1/slope)

–1

If the PCR has 100% efﬁciency, the amount of PCR prod-

uct will double after each cycle and the slope of the standard

curve will be -3.33 (i.e. 3.33 cycles gives a 10 fold increase/

decrease between the 10 fold serial dilutions used to generate

the curves). A PCR efﬁciency of at least 90% (slope = 3.6) is

generally required for reliable quantitative results.

Materials

Laboratory facilities

Laboratory facilities necessary for quantitative analysis of

phytoplankton by PCR are consistent with those found in

a standard molecular biology laboratory equipped for DNA

cloning, puriﬁcation, quantiﬁcation and real-time (quantita-

tive) ampliﬁcation.

Equipment, Chemicals and Consumables

The equipment, chemicals and consumables used in this

method are presented in the Appendix, at the end of this

chapter (Tables 1-2). Suppliers, Catalogue numbers and es-

timated cost in Euros and US Dollars are also listed in Tables

1-2.

Method

Sample processing (culture or seawater samples

ﬁxed with acidiﬁed Lugol’s solution)

Microalgal cultures or seawater samples ﬁxed with acidiﬁed

Lugol’s iodine solution (see chapter 2 for recipe) have to be

collected and lysed appropriately in order to generate lysates

Figure 2. Real-time PCR instrument ABI PRISM 7000 SDS (App-

lied Biosystems).

Figure 3. Filter system (A) with 3 µm Millipore TSTP mem-

brane (B).

Figure 4. Collecting cells from the ﬁlter with 1 mL artiﬁcial sterile

seawater in a 1.5 mL tube .

A B

Figure 5. Cell lysis by sonication.

IOC Manuals & Guides no 55

Chapter 13 Quantitative PCR

(or starting material) suitable for use in QPCR. In many

instances the sample will need to be concentrated prior to

starting the QPCR method. This can be achieved by either

centrifugation or ﬁltration.

Collection of Phytoplankton Cells Using a Centrifuge

1 Spin phytoplankton cells at 3000 rpm for 15 minutes

at 12 ± 1

C and remove the supernatant carefully. This

removes the acidiﬁed Lugol’s ﬁxative. A swinging bucket

centrifuge is required to allow for the generation of a con-

centrated pellet.

2 Resuspend cells in a suitable volume of artiﬁcial sterile

seawater and transfer to 1.5 mL tube.

3 Spin at 3000 rpm for 15 minutes at 12 ±1

C in a swing-

ing bucket rotor and remove the supernatant (leaving ap-

proximately 100 µL).

4 Spin again at 10000 rpm for 10 seconds at 12 ± 1

C and

remove the remaining supernatant leaving the pellet in

the tube.

Collection of Phytoplankton Cells Using a Filter System

1 Filter the appropriate volume of sample to be processed

onto a 3.0 µm Millipore TSTP membrane (Fig. 3).

2 Wash the ﬁlter with 1.0 mL of artiﬁcial sterile seawater

in a 1.5 mL tube (Fig. 4). This collects the cells from the

ﬁlter surface.

3 Spin the cells at 6800 rpm for 5 minutes in a microcentri-

fuge and remove the supernatant (leaving approximately

100 µL).

4 Spin at 10000 rpm for 10 seconds and remove the re-

maining supernatant leaving the pellet dry.

Cell Lysates Preparation

1 Freeze pellet at -80 ± 1

C for 15 minutes.

2 Resuspend the frozen pellet with 400-600 µL lysis buffer

(PCR buffer 1X, NP40 0.5%, Tween 20 0.5%, protein-

ase K 0.1 mg mL

-1

) at a concentration of 1 x 10

– 2 x 10

cells mL

-1

3 Sonicate twice at 50 W for 10 seconds and incubate at 55

± 1

C for 2-4 hours (Figs 5-6), vortexing at intervals of

30 minutes.

4 Boil for 5 minutes and centrifuge at 12000 rpm for 2

minutes. This will eliminate cell debris and impurities.

5 Transfer the supernatant to a clean microcentrifuge tube

and store this sample lysate at -80 ± 1

C for 2-3 days or

process it immediately.

Standard Curve Preparation

Plasmid generation

A PCR product, generated as described previously, which

contains the complementary target DNA sequence to which

the QPCR primers bind (and probe if a probe based chem-

istry is used) is enzymatically joined at both ends (ligated) to

a speciﬁc double stranded DNA sequence called a plasmid.

This forms a circular DNA construct. A plasmid is DNA,

usually in circular form, which is capable of replicating itself

independently of cell replication. A single plasmid containing

the target sequence is introduced into a bacterial cell follow-

ing a chemical or electrical reaction. The bacteria is placed on

a nutritious gel and multiplies to form a visible colony con-

sisting of thousands of bacterial cells. The plasmid is passed

to each new cell following cell division. Within each cell, the

plasmid will also replicate many times, and with it the original

PCR product. The plasmids are puriﬁed from the bacteria re-

sulting in a highly concentrated source of pure PCR product

containing the target sequence for the QPCR assay. There are

many types of plasmids and bacterial cells commercially avail-

able and numerous methods for puriﬁcation, again many of

which are incorporated into commercially available kits.

The standard curve can be constructed with 10-fold serial di-

lutions of a plasmid containing the target sequence. Usually,

the curve range extends from 1.0 × 10

to 1.0 × 10

plasmid

copies but it can be adjusted according to the assay detection

range required. The plasmid copy number is calculated using

the following formula:

Molecules µ L

-1

= (A × 6.022 × 10

)/(660 × B)

where: A = plasmid concentration (g µL

-1

); B = length of

the plasmid containing the cloned sequence; 6.022 × 10

Figure 6. Samples in a thermal block at 55 °C.

Figure 7. Dispersing the reaction mixtures in

the optical plate (25 µL each well).

Reagent Amount per

reaction

Final

concentration

O variable

Commercial mix containing

SYBR Green 2X

12.5 µL 1X

*MgCl

25 mM 1-5 µL 1-5 mM

Forward primer 10 mM 0.25-1.5 µL 100-600 nM

Reverse primer 10 mM 0.25-1.5 µL 100-600 nM

*DNA Polymerase 5U/mL 0.125-0.25 µL 0.025-0.05 U/mL

Diluted cell lysate 1-2 µL -

Final volume 25 µL

Table 1. Master mix preparation for real-time PCR.

*Only if required. Some commercial mixes (i.e. Applied Biosys-

tems) already contain MgCl

and DNA polymerase

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 13 Quantitative PCR

Avogadro’s number; 660 = average molecular weight of one

base pair. The accurate determination of the plasmid con-

centration can be performed using a spectrophotometer or a

ﬂuorimeter. Once quantiﬁed, it is recommended to store the

plasmid at -80

C in small aliquots to avoid repeated freeze/

thaw cycles. Moreover, plasmid dilutions should be freshly

prepared for each PCR run.

Dilutions of Sample Lysates and Plasmid

1 Make scalar dilutions 1:10 of the sample lysates with

PCR-grade H

2 Make scalar dilutions 1:10 of the plasmid with PCR-

grade H

O (from 1.0 × 10

to 1.0 × 10

Quantitative PCR Assay

1 In a separate clean room set up the QPCR master mix

described in the Table 1. Make triplicate PCR tubes for

each lysate and plasmid dilution (see above). This is re-

quired to determine the intra-assay variability of results.

For this purpose, it is practical to aliquot a reaction vol-

ume equivalent to 3 reactions (75 µL) in one standard

PCR tube, add the template, and then disperse the reac-

tions in optical tubes/plates (25 µL per tube/well) (Fig.

7). Quantitative PCR assays are performed in a ﬁnal vol-

ume of 25 µL using the SYBR Green chemistry as pre-

sented in Table 1. To reduce cost, it is possible to set up

QPCR mixtures considering a ﬁnal volume of 12.5 µl

instead of 25 µl. In this case, particular care should be

taken to avoid volume differences between tubes.

2 Perform the QPCR in a real-time PCR instrument under

the following reaction conditions: 95 ºC for 5 minutes;

40 cycles of 15 sec at 95 ºC, 1 min at 60 ºC, with a

ﬁnal dissociation protocol to ensure the absence of non-

speciﬁc PCR products or primer dimers (Fig. 8). These

conditions can be modiﬁed depending on the commer-

cial mix used and the characteristics of each primer.

Due to the sensitivity of the PCR method, it is crucial to

avoid contamination. It is therefore necessary to set up the

reactions in a clean area (i.e. in a PCR cabinet), free of poten-

tial plasmid or PCR product contamination. Always include

one or more negative controls (blank sample with no target

template).

PCR efﬁciency is important for quantiﬁcation purposes: to

maximize efﬁciency, it is advisable to design primers produc-

ing a PCR product no more than 100 bp long. These short

amplicons can also allow the use of partially degraded DNA,

without loss of quantiﬁcation performance. It is also neces-

sary to include the standard curve in each PCR run, due to

the possible variability from one PCR to another.

To avoid variability and low yields in DNA puriﬁcations,

crude cell lysates can be used as the DNA template in the

PCR reactions. These lysates contain components that can

interfere with PCR reactions and it is therefore necessary to

ﬁnd a template amount which can be ampliﬁed without any

inhibitory effect. For this purpose, the QPCR assay is per-

formed, at least during the assay optimisation, with 10-fold

serial diluted lysates until quantiﬁcation results become pro-

portional to sample dilutions. Usually, dilutions from 1:10 to

1:1000 are sufﬁcient to establish the correct conditions. It is

important to note that this approach would be possible when

the target sequence is present in high copy number in the cell

(e.g. rRNA genes), otherwise the sensitivity of the method

will drop signiﬁcantly.

Analysis of Results

The analysis of the results is performed using the speciﬁc

QPCR machine software.

1 Set a suitable baseline and threshold value, if the instru-

ment does not do it automatically. Plasmid ampliﬁcation

plots and a standard curve similar to the one presented in

Fig. 9, should appear.

2 Using experimental results, the number of target DNA

copies per µL of the original sample can be caluclated

Temperature (C)

60 65 70 75 80 85 90 95

0.24

0.20

0.16

0.12

0.08

0.04

0.00

-0.04

Derivative

Figure 8. Example of dissociation protocol results. The graph

shows the derivative of ﬂuorescence emission variation plotted

against the temperature increment. The peak corresponds to a

speciﬁc 173-bp PCR product. No aspeciﬁc products or primer

dimers are visible.

Figure 9. Example of standard curve obtained with a plasmid

containing 28S rDNA sequence of Alexandrium fundyense. (A)

Ampliﬁcation plots with plasmid copy number from 2.0 X 10

to 2.0

X 10

. The cycle number is plotted vs the Delta Rn. The Delta Rn

represents the normalized reporter signal (Rn) minus the baseline

signal established in the early PCR cycles. Three replicates were

performed for each reference DNA sample. T: threshold. (B) Cali-

bration curve plotting log starting copy number vs Ct. Slope: –3.61;

correlation coefﬁcient (R

): 0.9939.

0.1

0.01

Delta Rn

Cycle number

1 10 20 30 40

2 5.5 3 3.5 4 4.5 2.5 5 6 6.5

IOC Manuals & Guides no 55

Chapter 13 Quantitative PCR

100

from the standard curve.

3 In order to estimate the total number of cells in the initial

sample, the number of target DNA copies per µL in the

sample is divided by the number of rDNA copies per cell.

This is then multiplied by the initial lysate volume.

Formulas for Calculating Results:

N = [(A/B) x d] x (V/C)

N: Total number of cells in the initial sample

A: number of target DNA copies per PCR tube (calculated by

instrument software)

B: mL of cell lysate in the PCR tube

C: target DNA copies per cell (to be determined for each tar-

get species using cultured cells)

d: lysate dilution factor

V: initial lysate Volume expressed in mL

Sample Preservation and Storage

Phytoplankton samples can be preserved with acidiﬁed

Lugol’s iodine solution and stored at 4°C in the dark. Experi-

ence has shown that reliable quantitative PCR results can be

obtained from samples stored up to one year.

Discussion

The QPCR has proven to be a powerful method for the quan-

tiﬁcation and detection of phytoplankton species in environ-

mental samples (Galluzzi et al. 2004, Hosoi-Tanabe and Sako

2005, Dyhrman et al. 2006).

The use of commercial PCR master mix containing interca-

lating dyes such as SYBR Green is a simple and inexpensive

approach. The assays based on intercalating dyes are very sen-

sitive, but it is noteworthy that any double stranded non tar-

get DNA can also be detected, leading to misinterpretation of

results. For this reason, the speciﬁcity of the primer is crucial.

An increase in the speciﬁcity of the assay can be obtained us-

ing a TaqMan probe but this may result in a loss of sensitivity.

Theoretically, this technique can be applied to any phy-

toplankton species with DNA sequence data available so

speciﬁc primers or probes can be designed. However, some

drawbacks need to be taken into account when applying

QPCR method to phytoplankton. In particular, when a tar-

get sequence cloned into a plasmid is used as a standard, it

is essential to know the amount of target DNA per cell, in

order to determine the cell number in the ﬁeld sample. This

requires preliminary work with cultured strains to optimise

the method for each target species/strain. Due to the possi-

bility of variations of target gene copy number (particularly

in case of rRNA genes) among different strains, the level of

accuracy of the quantitative real-time PCR assays can be af-

fected, as it has been shown for Mediterranean Alexandrium

species (Galluzzi et al. in press). For this reason, the method

should be tested and optimized with the local phytoplankton

population in the geographical area to be investigated. Primer

or probe speciﬁcity needs to be tested with the local popula-

tion of phytoplankton to ensure absence of non-speciﬁc target

ampliﬁcation.

Compared to classical microscopy techniques, the main ad-

vantages of the QPCR in phytoplankton monitoring include

speciﬁcity, sensitivity and applicability to preserved environ-

mental samples. Sample preservation is often necessary, but

the use of ﬁxatives may cause the morphology distortion of

some phytoplankton species, making it more difﬁcult to dis-

tinguish them from closely related species using a microscope.

The QPCR sample throughput can be up to 27 triplicate

samples per 96-well plate, including the standard curve. This

sample throughput may reduce working time compared to

the microscope-based methods when a high number of sam-

ples need to be analysed. It is also noteworthy that the col-

lection of phytoplankton using a ﬁlter based system is faster

then the centrifugation method. This alternative speeds up

the entire process thus reducing the handling time.

QPCR instrument prices are becoming affordable for small

research groups and are now common in many molecular bi-

ology laboratories. The consumable cost per sample has been

estimated in $4 - $6 depending on the method/master mix/

chemistry used, making the QPCR an affordable method for

monitoring purposes.

In QPCR assays, only one species or strain can be analysed

at a time unless a multiplex reaction is performed. This usu-

ally requires the use of more expensive ﬂuorescent probes

(e.g. TaqMan probes) instead of intercalating dyes. Although

multiplexing and/or multiprobing are powerful tools for mo-

lecular investigations of speciﬁc groups of toxic algae, their

development and validation can be difﬁcult and expensive

(Handy et al. 2006).

This method can be still considered at the developmental

stage. If this method is incorporated into future monitoring

programs for HAB-species, a subset of samples could also be

checked using a more traditional microscopic technique. This

type of quality control would validate the presence of the tar-

get species on a selected subset of samples and highlight any

problems that may arise with the method.

Acknowledgements

EU-Project Seed GOCE-CT-003875-2005.

References

Bowers HA, Tengs T, Glasgow HB, Burkhoelder JMj, Rublee PA,

Oldach DW (2000) Development of real-time PCR assay for rap-

id detection of Pﬁesteria piscicida and related dinoﬂagellates. Appl

Environ Microbiol 66:4641-4648

Coyne KJ, Cary SC (2005) Molecular approaches to the investiga-

tion of viable dinoﬂagellate cysts in natural sediments from estua-

rine environments. J Eukaryot Microbiol 52:90-94

Coyne KJ, Handy SM, Demir E, Whereat EB, Hutchins DA, Por-

tune KJ, Doblin MA, Cary SC (2005) Improved quantitative real-

time PCR assays for enumeration of harmful algal species in ﬁeld

samples using an exogenous DNA reference standard. Limnol

Oceanogr Methods 3:381–391

Dyhrman ST, Erdner D, La Du J, Galac M, Anderson DM (2006)

Molecular quantiﬁcation of toxic Alexandrium fundyense in the

Gulf of Maine using real-time PCR. Harmful Algae 5:242–250

Galluzzi L, Penna A, Bertozzini E, Vila M, Garcés E, Magnani M

101

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(2004) Development of Real-Time PCR assay for rapid detection

and quantiﬁcation of A. minutum (Dinoﬂagellate). Appl Environ

Microbiol 70:1199-1206

Handy, SM, Hutchins, DA, Cary, SC, Coyne, KJ, (2006)

Simultaneous enumeration of multiple raphidophyte species by

quantitative real-time PCR: capabilities and limitations. Limnol.

Oceanogr. Methods 4: 193–204

Hosoi-Tanabe S, Sako Y (2005) Species-speciﬁc detection and quanti-

ﬁcation of toxic marine dinoﬂagellates Alexandrium tamarense and

A. catenella by Real-Time PCR assay. Mar Biotechnol 7:506-514

Kamikawa R, Hosoi-Tanabe S, Nagai S, Itakura S, Sako Y (2005)

Development of a quantiﬁcation assay for the cysts of the toxic

dinoﬂagellate Alexandrium tamarense using real-time polymerase

chain reaction. Fish Sci 71: 987–991

Park TG, de Salas MF, Bolch CJ, Hallegraeff GM (2007) Develop-

ment of a real-time PCR probe for quantiﬁcation of the hetero-

trophic dinoﬂagellate Cryptoperidiniopsis brodyi (Dinophyceae) in

environmental samples. Appl. Environ. Microbiol. 73: 2552–2560

Popels LC, Cary SC, Hutchins DA, Forbes R, Pustizzi F, Gobler

CJ, Coyne KJ (2003) The use of quantitative polymerase chain

reaction for the detection and enumeration of the harmful alga

Aureococcus anophagefferens in environmental samples along the

United States East Coast. Limnol Oceanogr: Methods 1:92–102

Touzet N, Keady E, Raine R, Maher M (2009) Evaluation of taxa-

speciﬁc real-time PCR, whole-cell FISH and morphotaxonomy

analyses for the detection and quantiﬁcation of the toxic

microalgae Alexandrium minutum (Dinophyceae), Global Clade

ribotype. FEMS Microbial Ecol. 67:329-341

IOC Manuals & Guides no 55

Chapter 13 Quantitative PCR

102

Appendix

Table 1. Equipment and suppliers.

Table 2. Chemicals and suppliers.

*Different commercial SYBR Green mixtures can also be used (for example

Hot-Rescue Real Time PCR KIT-SG from Diatheva, Italy).

Equipment Supplier Cat. Number

€

US $

Real-time PCR instrument ABI PRISM 7300 SDS Applied Biosystems 4351101 29840 42099

Ultrasonic Homogeniser Branson S-150D VWR international 33995 2110 2980

Analog Dry Block Heater VWR international 12621-110 344 485

Swing-bucket Centrifuge IEC-CL30 Thermo Scientiﬁc 11210904 5532 7856

Mini-Centrifuge Heraeus ”Biofuge Fresco” Thermo Scientiﬁc 75002420 3600 5110

Spectrophotometer Shimadzu, Japan UV-2401 PC 8000 11360

Sum approx. 49426 69890

Chemicals/consumables Supplier Cat. Number

€

US $

Isopore membrane ﬁlter 3.0 µm TSTP Millipore, USA TSTP02500 129 183

Lugol solution (1L) Sigma-Aldrich Srl, Italy 62650 26 37

*SYBR Green PCR Master Mix, 2 X 200 reactions Applied Biosystems, USA 4364344 664 936

MicroAmp Optical 96-well Reaction Plate, 20/pkg Applied Biosystems, USA 4306737 152 214

Optical Adhesive Covers, 25/pkg Applied Biosystems, USA 4360954 76 107

Tween 20 Sigma-Aldrich Srl, Italy P1379-100ML 26.9 38

NP 40 Sigma-Aldrich Srl, Italy 74385-1L 36 50

Proteinase k Sigma-Aldrich Srl, Italy P6556-25MG 39.1 55

Primers forward and reverse (25 nmol) Invitrogen, Italy - 13 18

Tubes 1.5 mL Sarstedt, Germany 72690 - 500/pack 16 22

Polypropylene conical tubes 50 mL BD Biosciences 352070 - 500/case 99 140

103

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

Introduction

Tyramide Signal Ampliﬁcation (TSA) - Fluorescence in situ

Hybridisation (FISH) provides an enhanced ﬂuorescence sig-

nal from molecular probes The oligonucleotide probe is la-

belled with the enzyme horseradish peroxidase (HRP). After

hybridisation the HRP catalyses the deposition of a phenolic

compound, a FITC (ﬂuorescein isothiocyanate) labelled tyra-

mide. In the presence of hydrogene peroxide, the immobi-

lised tyramide binds to electron rich moieties (mostly tyrosine

residues) of adjacent proteins. This TSA reaction results in a

signal enhancement of up to 30 times more intensity then

traditional labelled ﬂuorescent probes (Fig. 1).

Solid phase cytometry

Solid phase cytometry (SPC) combines the advantages of ﬂow

cytometry with image analysis (Kamentsky 2001), and enables

the fast detection and enumeration of micro-organisms down

to one cell in a sample (Lemarchand et al. 2001). In SPC, the

laser is moved over cells immobilised onto a solid support,

which allows the rapid enumeration of several thousand cells

with a similar accuracy to ﬂow cytometry (Darynkiewicz et al.

2001). The ChemScan™ instrument (Chemunex, France) is

a SPC, which was initially developed for industrial and envi-

ronmental microbiology (Mignon-Godefroy et al. 1997, Rey-

nolds and Fricker 1999). It was adapted for the detection of

toxic microalgae using antibody (West et al. 2006) and oligo-

nucleotides probes (Töbe et al. 2006). The ChemScan™ uses

a 488 nm argon-ion laser and is therefore suited for probes

or tyramides labelled with FITC. Algal cells are collected by

ﬁltration onto a polycarbonate membrane, hybridised, and

subsequently scanned (Fig. 2). Fluorescent events are detected

and a computer applies various criteria to discriminate be-

tween “true” and “false” signals deriving from hybridised cells

using the MatLab-based software (Matworks, Natick, USA)

for comparison with Gaussian curves (Roubin et al. 2002).

The exact positions of positively identiﬁed cells are shown as

14 Tyramide signal ampliﬁcation in combination

with ﬂuorescence in situ hybridisation

Kerstin Töbe

Alfred Wegener Institute for Polar and Marine Research Am Handelshafen 12. D-27570 Bremerhaven, Germany

*Author for correspondence e-mail: Kerstin.T[email protected]

coloured spots on a display of the membrane in a scan map.

The hybridised cells can be visualised by transferring the

membrane with its membrane holder to an epiﬂuorescence

microscope equipped with a computer controlled motorised

stage that is connected to the ChemScan™. This allows each

positive data point to be visually validated by microscopic ex-

amination immediately after scanning as true positives or false

positives (Reynolds and Fricker 1999, Roubin et al. 2002).

This method is very fast, positive hybridised cells are counted

within 3 minutes and no positive hybridised cell can be over-

looked by the operator as in standard FISH applications.

TSA-FISH is required for reliable automated detection of

target cells with the ChemScan™ to increase the peak ﬂuo-

rescence intensity as a discrimination pattern in the compu-

ter software. Since TSA-FISH labelled cells reach very high

ﬂuorescence intensities, this allows the computer software to

develop discrimination patterns between labelled and non-la-

belled cells. This new automated method for counting micro-

algae is, however, only adequate for round and spherical cells

at the moment and not for long colony forming species, like

the diatoms Pseudo-nitzschia. The computer software present-

ly used must be revised in order to count ﬁlamentous colony

forming microalgae. Microscopic veriﬁcation of positive cells

is recommended. This can be performed after conﬁrming that

the FISH labelling has been successfully completed.

Presently, the application of only one single probe label is pos-

sible, because of the single laser of the ChemScan™ and it is

therefore not possible to detect more than one species on a

ﬁlter at a time. The effectiveness of a new probe label is, how-

ever, under development. This new probe label is excited with

the present ChemScan™ laser, like standard FITC-labelled

cells, but it has a different emission wavelength and with the

installation of a suited Photomultiplier, two different probes

could be applied.

Figure 1. Tyramide signal ampliﬁcation.

IOC Manuals & Guides no 55

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

104

Scope

Semi-automated detection and quantiﬁcation of target phyto-

plankton species.

Detection range

Detection of microalgal RNA by FISH is very sensitive. The

number of cells that can be detected depend on the sample vo-

lume. High biomass can obscure the view of target cells.

Advantages

Simple and easy to use. Sample volumes can easily be adjusted.

Simultaneous labeling and detection of multiple species is pos-

sible. Strong yellowish/green labelling of target cells minimises

confusion with non-target cells. Semi-automated analysis.

Drawbacks

Probes are only available for a limited number of target species.

Rigorous optimisation and speciﬁcity testing on local strains is

required before the method can be implemented. Finite storage

time for samples. Complex sample processing procedure may

result in cell loss. At present very high start-up costs. Intensity

of positive reaction may vary with cell conditions. Access to

molecular expertise is essential. Appropriate laboratory facilities

for storage and processing of probes and reagents are necessary.

Type of training needed

Instruction in setting up this technique should come from a

person with an in-depth knowledge and experience of mole-

cular biology. Approximately one week of supervised training

required to properly perform the method.

Essential Equipment

Solid phase cytometer and epiﬂuorescence microscope with

automated stage; ﬁltration unit, vacuum pump, hybridisation

oven.

Equipment cost*

Total set-up cost = €183310, $ 269.795

See Appendix, Table 1 for details

Consumables, cost per sample**

€11 ($16.13), see also Appendix, Table 2

Processing time per sample before analysis

5-30 minutes. Filtering time depends on the amount of phyto-

plankton in the samples.

Analysis time per sample

The TSA-FISH procedure and analysis with SPC requires 4.25

hours of handling time per ﬁlter of 3 minutes for scanning and

2 minutes for veriﬁcation of the results.

Sample throughput per person per day

A trained person can process up to 36 samples per day depen-

ding on the number of target organisms in each sample.

No. of samples processed in parallel

Health and Safety issues

Relevant health and safety procedures must be followed. The

following chemical is particularly hazardous: Formamide.

*service contracts not included

**salaries not included

The fundamentals of

The tyramide signal ampliﬁcation - ﬂuoresence in situ hybridisation in combination with solid

phase cytometry method

105

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

Materials

Laboratory facilities

Molecular biology laboratory

Required Equipment (essential)

The quantitative TSA-FISH in combination with solid phase

cytometry requires the following equipment:

• Solid phase cytometer

• Epiﬂuorescence microscope with motorised stage

• Filter vacuum manifold or glass ﬁlter equipment

• Hybridisation oven

• Vacuum pump

• Pipettes 1-20 µL, 100-100 µL + sterile tips, 1-50 mL pi-

pette + sterile tips

• Autoclaved glassware

• Disposable gloves, tweezers,

• White polycarbonate ﬁlter membranes: 25 mm diameter,

pore size depending on cell size

• Support pads

Chemicals and Consumables

Solutions for Fixation

Saline ethanol (Scholin et al. 1996), prepared freshly for each

experiment because of the formation of precipitates

25 vol. 95 % ethanol

2 vol. Milli-Q water

3 vol. 25X SET

or modiﬁed saline ethanol (Miller and Scholin 2000) stable

at room temperature for several months without precipitate

formation

22 vol. 95 % ethanol

5 vol. Milli-Q water

3 vol. 25X SET buffer

25X SET

3.75 M NaCl

0.5 M Tris/HCl

25 mM EDTA

pH 7.8, ﬁlter sterilised

Solutions for Fluorescence In situ Hybridisation

Hybridisation buffer

0.1 % (v/v) Nonidet-P40 (Sigma N-6507)

x % (v/v) Formamide

2 % blocking reagent (Roche, Mannheim, Germany)

Wash buffer:

1X SET

Solution for quenching endogenous peroxidase activity

3 % Hydrogene peroxide (H

) in ﬁlter sterilised deionised

water

Solutions for ampliﬁcation reaction

TNT-Buffer

0.1 M Tris-HCL, pH 7.5

0.15M NaCl

0.05 % Tween 20

Tyramide substrate solution

One volume of 40 % (wt/vol) dextrane sulfate (Sigma-Aldrich,

Munich, Germany) in sterile deionised water, is mixed with

one volume of ampliﬁcation diluent and a 1:50 dilution of

the ﬂuorescein labelled tyramide (Perkin Elmer, Boston Ma,

USA, diluted in Dimethylsulfoxide) in sterile deionised water

and stored in the dark.

Solution for counterstaining

Citiﬂuor/DAPI (4’,6’-diamidino-2-phenylindoline) mixture

(0.5 mL sterile deionised water, 1 mL Citiﬂuor (Citiﬂuor

Ltd., Cambridge) and 1.5 µL DAPI solution (1 µg µL

-1

, Inv-

itrogen, Germany, in sterile deionised water)).

Probes

Horseradish peroxidase labelled probes purchased from Ther-

mo Scientiﬁc, Germany are delivered lyophilized. The HRP-

label is anchored at the 5’-end of the oligonucleotide. Stock

solution of 1 µg µL

-1

, and working solutions of 500 µL

-1

and

50 ng µL

-1

should be prepared in 1X TE buffer, pH 7.8- 8.0.

Probe stock solution should be stored at -80°C and working

solutions at -20°C. Since, HRP-labelled probes are not light

sensitive it is not necessary to work in the dark. However,

from the tyramide signal ampliﬁcation step on working in

the dark is necessary, because of the incorporated light sensi-

tive FITC-labelled tyramide. More information on the equip-

ment, chemicals and consumables used in this method are

presented in the Appendix at the end of this chapter.

Method

Handling Time

Based on 10 samples processed in parallel

0.5 h Set-up and ﬁltering step

1 h Fixation

0.25 h Wash step and probe addition

1.5 h Hybridisation and tyramide signal ampliﬁcation

0.5 h Analysis of 10 ﬁlter with solid phase cytometer

0.5 h Clean up

TOTAL HOURS 4.25 h

Figure 2. Tyramide Signal Ampliﬁcation FISH with Alexandrium

fundyense cells and HRP-labelled probe ATNA02 (John et al.

2005), photographed at 40x objective lens.

IOC Manuals & Guides no 55

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

106

Sample Preservation and Storage

• Filter 5 mL sample onto a polycarbonate ﬁlter with the

lowest possible vacuum and incubate in the ﬁxative for at

least 1 hour at room temperature or overnight at 4 ºC.

• Remove the ﬁxative by ﬁltrating and incubating for 5 mi-

nutes with hybridisation buffer without a probe.

Filters can be dried and stored for at least one month at room

temperature or hybridised directly afterwards.

Quenching of Natural Occurring Peroxidases

• Quench naturally occurring endogenous peroxidase ac-

tivity by treating the ﬁlters with 100 µL 3 % H

per

ﬁlter at room temperature for 15 to 30 minutes to avoid

unspeciﬁc staining.

• Rinse the ﬁlter in sterile deionised water to remove excess

Hybridisation

• Cover the ﬁlters with 80-100 µL hybridisation buffer

containing the horse radish peroxidase labelled probe

(ﬁnal concentration of probe in hybridisation buffer:

5 ng µL

-1

) and hybridise 1.5-2 hours at 37 ºC.

• Stop the hybridisation by adding pre-warmed (37 ºC) 1X

SET wash buffer and then wash the ﬁlters with 1X SET

for 10 minutes at 37 ºC.

Tyramide Signal Ampliﬁcation

• Equilibrate the ﬁlters for 15 minutes in TNT buffer at

room temperature.

• Remove excess liquid by putting the ﬁlters on blotting

paper, staining should be conducted before they are com-

pletely dry.

• Incubate each ﬁlter with 100 µL Tyramide substrate solu-

tion for approximately 30 minutes at room temperature

in the dark.

• Rinse the ﬁlters in TNT-buffer and wash for 15 minutes

at 55 ºC in TNT-buffer. Then rinse the ﬁlter in sterile

deionised water, air dry and store at -20 ºC pending ana-

lysis by ChemScan™.

Optional Step

Counterstaining

• Counterstain the ﬁlters with a Citiﬂuor/DAPI mixture

for 10 minutes at room temperature. Wash with sterile

deionised water for 1 minute and incubate in 80 % etha-

nol (v/v) for 30 seconds to remove an excess of staining

solution. Air Dry and store at -20 ºC or examine directly

with the ChemScan™. Citiﬂuor is used as an antifade

and the blue DAPI nucleic acid stain effectively stains

double stranded DNA and enables a general overview of

all cells on a hybridised ﬁlter.

Formulas for Calculating Results

The entire ﬁlter is automatically counted by the solid phase

cytometer and afterwards validated randomly by the operator.

Hence the cell number is reﬂective of the amount of original

sample that was processed and preserved as well as a the vol-

ume that was ﬁltered for hybridisation.

To calculate cells L

-1

where N is the number of positive cells on the whole ﬁlter and

V (mL) is the volume of sample used.

Analysis of hybridised ﬁlters with SPC in combi-

nation with epiﬂuorescence microscopy

The ChemScan™ system (Fig. 3) must be calibrated on a

daily basis with a standardised amount of FITC labelled latex

beads, diameter 2-3 µm (Standard C, Chemunex, France), in

100 µL. In order to verify that the laser is working properly,

the number of ﬂuorescence signals recorded by the Chem-

Scan™ must be cross referenced with the number of counted

latex beads in solution.

1. The 100 µL solution with the latex beads is ﬁltered onto

a black polycarbonate membrane (25 mm diameter, 0.2

µm pore size, Chemunex, France). To support the ﬁlter

membrane, a black support pad is mounted by applying

100 µL ChemSol B16 (Chemunex, France) to a mem-

brane holder. Then the ﬁlter membrane with latex beads

is laid onto the support pad. The ﬁlter is scanned with

the application C control.

2. For TSA-FISH ﬁlter also a black support pad is mounted

by applying 100 µL ChemSol B16 (Chemunex, France),

overlaid by the hybridised ﬁlter and the the application

tvcbio1 (tvc: total viable counts) is used. The peak inten-

sity is manually changed from 250 to 2500 to prevent

the enumeration of false positive autoﬂuoresing particles

with lower peak ﬂuorescence intensity.

3. Immediately after the scan, signals are validated using

an epiﬂuorescence microscope (Nikon, Eclipse E 800)

equipped with ﬁlter blocks for FITC (Nikon Filter Block

B-2A) and DAPI (Nikon Filter Block UV-2A) and with

a motorised stage (Prior Scientiﬁc, UK). Images are cap-

tured with a digital camera (CCD-1300CB, Vosskühler,

Germany) and analysed with the Nikon software Lucia

Discussion

Traditional FISH methods have limitations when counting

samples with low target cell densities as well as in the number

of samples that can be analysed per day. The time involved to

count an environmental sample will vary with the diversity

of the sample and the skill of the operator. For a high sample

throughput, FISH has been combined with ﬂow cytometry,

which allows the analysis of different cell parameters.

However, a combination of ﬂow cytometry and microscopy of

single detected ﬂuorescent microalgae is difﬁcult. In addition,

because of the limited sensitivity at lower cell concentrations

this method is not suited for the detection of cells at low

concentrations in environmental samples. The solid phase

−

1000 *

)(

) (













mLsampleofVolume

NfilterwholeoncountcellPositive

Lcells ion concentrat Cell

−

1000 *

)(

) (













mLsampleofVolume

NfilterwholeoncountcellPositive

Lcells ion concentrat Cell

107

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

cytometer has the advantage of a direct combination of

automated counting and epiﬂuorescence microscopy, allowing

the microscopically veriﬁcation of each single cell detected.

The solid phase cytometry in combination with TSA-FISH

enables the efﬁcient detection of a single cell in a ﬁltered

volume in less than 5 h. For a reliable automated detection

of target cells with the ChemScan™, signal ampliﬁcation is

necessary. This method has great potential for application in

analysing ﬁeld material where a rapid and reliable detection

and enumeration of target cells is required. The shape of the

algae may cause problems, e.g., the ChemScan™ cannot

count long chain forming cells like Pseudo-nitzschia cells. The

adaptation of improved software could help to overcome this

problem. Additionally, the actual high price of this machine

and the additional costs of an epiﬂuorescence microscope

equipped with an automatic stage is a limiting factor.

Acknowledgements

The work was funded by the Stiftung Alfred-Wegener-Insti-

tut für Polar und Meeresforschung in the Helmholtz-Ges-

ellschaft, Bremerhaven, Germany, in part by EU DETAL ,

project QLRT-1999-30778 and Chemunex, Ivry, France.

Figure 3. Overlapping scan with the ChemScan.

References

Darynkiewicz Z, Smolewski P, Bedner E (2001) Use of ﬂow

and laser scanning cytometry to study mechanisms regulat-

ing cell cycle and controlling cell death. Clin Chem Lab

Med 21:857-873

John U, Medlin LK, Groben R (2005) Development of spe-

ciﬁc rRNA probes to distinguish between geographic clades

of the Alexandrium tamarense species complex. J Plankton

Res 27:199-204

Kamentsky LA (2001) Laser scanning cytometry. Methods

Mol Cell Biol 63:51-87

Lemarchand K, Parthuisot N, Catala P, Lebaron P (2001)

Comparative assessment of epiﬂuorescence microscopy,

ﬂow cytometry and solid-phase cytometry used in the enu-

meration of speciﬁc bacteria in water. Aquat Microb Ecol

25:301-309

Mignon-Godefroy K, Guillet JC, Butor C (1997) Laser scan-

ning cytometry for the detection of rare events. Cytometry

27:336-344

Miller PE, Scholin CA (2000) On detection of Pseudo-nitzschia

(Bacillariophyceae) species using whole cell hybridization: Sample

ﬁxation and stability. J Phycol 36:238-250

Roubin MR, Pharamond JS, Zanelli F, Poty F, Houdart S,

Laurent F, Drocourt, JL, Van Poucke S (2002) Application

of laser scanning cytometry followed by epiﬂuorescent and

differential interference contrast microscopy for the detec-

tion and enumeration of Cryptosporidium and Giardia in

raw and potable waters. J Appl Microbiol 93:599-607

Reynolds DT, Fricker CR (1999) Application of laser scan-

ning for the rapid and automated detection of bacteria in

water samples. J Appl Microbiol 86:785-795

Scholin CA, Buck KR, BritschigT, Cangelosi G, Chavez FP

(1996) Identiﬁcation of Pseudo-nitzschia australis (Bacillari-

ophyceae) using rRNA-targeted probes in whole cell and

sandwich hybridization formats. Phycologia 35:190-197

Töbe K, Eller G, Medlin LK (2006) Automated detection

and enumeration of Prymnesium parvum (Haptophyta:

Prymnesiophyceae) by solid-phase cytometry. J Plankton

Res 28:643-657

West NJ, Bacchieri R, Hansen G, Tomas C, Lebaron P,

Moreau H (2006) Rapid quantiﬁcation of the toxic alga

Prymnesium parvum in natural samples by use of a speciﬁc

monoclonal antibody and solid-phase cytometry Appl En-

vir Microbiol 72:860-868

IOC Manuals & Guides no 55

Chapter 14 Tyramide signal ampliﬁcation in combination with ﬂuorescence in situ hybridisation

108

Appendix

Table 1. Equipment and suppliers.

Table 2. Chemicals and suppliers.

Equipment Supplier Cat. Number

€

US $

Filter Manifold Millipore, USA XX2702550 400 587

Vacuum pump Omnilab, Germany 9.881 391 574

Incubator ”Shake’n’Stack” VWR, Germany 7996 2310 3387

Epiﬂuorescence microscope, e.g. Nikon

Eclipse E800 with motorized stage

Nikon, Japan MAA600BA 29000 42521

Solid phase cytometer e.g. ChemScan™ Chemunex, France 152000 222870

Chemicals Supplier Cat. Number

€

US $

Ethanol, absolute, 1L Merck 1.009.832.500 73 107

Milli-Q water Most labs have an in-

house supply

Sodium chloride, 1 kg Sigma-Aldrich S9888 23 34

Tris/HCl, 500 g Sigma-Aldrich T3253 96 141

Nonidet-P40, 100 mL Roche 11754599 74 109

EDTA, 500 g Sigma-Aldrich E7889 56 82

Isopore white polycarbonate membrane

ﬁlter, 3 µm pore size, Qty. 100

Millipore TSTP02500 130 191

Hydrogene peroxide Pharmacy - 4 6

HRP-labelled probe, approx. 300 µg Thermo Scientiﬁc - 235 345

Deionized formamide, 100 mL Sigma-Aldrich F 9037 46 67

109

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

Appendix: Acronyms and Notation

Acronym Meaning

ABD Area-Based spherical Diameter

ADPA N-Phenyl-1,4-benzenediamine hydrochloride

ALGADEC Development of a RNA -Biosensor for the Detection of Toxic Algae

approx. Approximately

ARB “arbor ”=tree

BSA Bovine Saline A

CCD Charge Coupled Device

CEN Comité Europeén De Normalisation

ChemScan™ Lazer scanning Process Analyser/solid-phase cytometry (Scan RDI™ in North America)

Chl a Chlorophyll a

CICEET Cooperative Institute for Coastal and Estuarine Technology

CPR Continuous Plankton Recorder

CTD Conductivity, Temperature, Depth

O distilled water

DAPI 4’,6-diamidino-2-phenylindoline

DIC Differential interference contrast

DICANN Dinoﬂagellate Categorisation by Artiﬁcial Neural Network

DSP Diarrhetic Shellﬁsh Poisoning

ECOHAB The Ecology and Oceanography of Harmful Algal Blooms

EPA Environmental Protection Agency

ESD Equivalent Spherical Diameter

ESP Environmental Sample Processor

EU European Union

EU DETAL Rapid and ultra-sensitive ﬂuorescent test for the tracking of toxic algae in the marine environment

FISH Fluorescence In Situ Hybridisation

FIT Fluid Imaging Technologies

FITC Fluorescein isothiocyanate

FlowCAM Flow Cytometer And Microscope

FSW Filtered Sea Water

FTF Filter-Transfer-Freeze

GF/F Glass Fibre Filters

HAB(s) Harmful Algal Bloom(s)

HAB Buoy Harmful Algal Bloom Buoy

HAE(s) Harmful Algal Event(s)

Hybe Hybridisation

ICES International Council for the Exploration of the Sea

IOC Intergovernmental Oceanographic Comission of UNESCO

LED Light-Emitting Diode

LM Light Microscopy

LSU Large SubUnit

MBARI Monterey Bay Aquarium Research Institute

NASA National Aeronautics and Space Administration

NOAA National Oceanic and Atmospheric Administration

NSF National Science Foundation

ONR Ofﬁce of Naval Research

PCR Polymerase Chain Reaction

PFA Paraformaldehyde

PICODIV Picoplankton Diversity

PSP Paralytic Shellﬁsh Poisoning

QC Quality Control

rDNA Ribosomal Deoxyribonucleic Acid

RNases Ribonucleases

rRNA Ribosomal Ribonucleic Acid

RT-PCR Real-Time PCR

Please note that RT-PCR usually refers to reverse transcriptase PCR, In this manual the acronym

refers to Real Time PCR.

SEM Scanning electron Microscopy

SOP Standard Operating Procedure

SSU Small SubUnit

TEM Transmission Electron Microscopy

TSA-FISH Tyramide Signal Ampliﬁcation has been used with FISH

UNESCO United Nations Educational Scientiﬁc and Cultural Organisation

wt/vol weight/volume

IOC Manuals & Guides no 55

110

SI Unit Meaning

Iodine

KI Potassium Iodide

mL millilitre

L Litre

cm Centimetre

hr Hour

ºC Degree Celsius

% Percentage

µm Micrometre

Millimetre squared

Min

-1

Per minute

Hg Mercury

n Number

g Gram(s)

µL Microlitres

v/v Volume to volume

x g Multiplied by gravity

113

Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis

List of previous titles in the series continued:

19 Guide to IGOSS Specialized Oceanographic Centres (SOCs). 1988. 17 pp. (English, French, Spa-

nish, Russian)

20 Guide to Drifting Data Buoys. 1988. 71 pp. (English, French, Spanish, Russian)

21 (Superseded by IOC Manuals and Guides No. 25)

22 GTSPP Real-time Quality Control Manual. 1990. 122 pp. (English)

23 Marine Information Centre Development: An Introductory Manual. 1991. 32 pp. (English, French,

Spanish, Russian)

24 Guide to Satellite Remote Sensing of the Marine Environment. 1992. 178 pp. (English)

25 Standard and Reference Materials for Marine Science. Revised Edition. 1993. 577 pp. (English)

26 Manual of Quality Control Procedures for Validation of Oceanographic Data. 1993. 436 pp. (Eng-

lish)

27 Chlorinated Biphenyls in Open Ocean Waters: Sampling, Extraction, Clean-up and Instrumental

Determination. 1993. 36 pp. (English)

28 Nutrient Analysis in Tropical Marine Waters. 1993. 24 pp. (English)

29 Protocols for the Joint Global Ocean Flux Study (JGOFS) Core Measurements. 1994. 178 pp .

(English)

30 MIM Publication Series:

Vol. 1: Report on Diagnostic Procedures and a Deﬁnition of Minimum Requirements for Providing

Information Services on a National and/or Regional Level. 1994. 6 pp. (English)

Vol. 2: Information Networking: The Development of National or Regional Scientiﬁc Information

Exchange. 1994. 22 pp. (English)

Vol. 3: Standard Directory Record Structure for Organizations, Individuals and their Research

Interests. 1994. 33 pp. (English)

31 HAB Publication Series:

Vol. 1: Amnesic Shellﬁsh Poisoning. 1995. 18 pp. (English)

32 Oceanographic Survey Techniques and Living Resources Assessment Methods. 1996. 34 pp.

(English)

33 Manual on Harmful Marine Microalgae. 1995. (English) [superseded by a sale publication in 2003,

92-3-103871-0. UNESCO Publishing]

34 Environmental Design and Analysis in Marine Environmental Sampling. 1996. 86 pp. (English)

35 IUGG/IOC Time Project. Numerical Method of Tsunami Simulation with the Leap-Frog Scheme.

1997. 122 pp. (English)

36 Methodological Guide to Integrated Coastal Zone Management. 1997. 47 pp. (French, English)

37 Post-Tsunami Survey Field Guide. First Edition. 1998. 61 pp. (English, French, Spanish, Russian)

38 Guidelines for Vulnerability Mapping of Coastal Zones in the Indian Ocean. 2000. 40 pp. (French,

English)

39 Manual on Aquatic Cyanobacteria – A photo guide and a synopsis of their toxicology. 2006. 106

pp. (English)

40 Guidelines for the Study of Shoreline Change in the Western Indian Ocean Region. 2000. 73 pp.

(English)

41 Potentially Harmful Marine Microalgae of the Western Indian Ocean. Microalgues potentiellement

nuisibles de l’océan Indien occidental. 2001. 104 pp. (English/French)

42 Des outils et des hommes pour une gestion intégrée des zones côtières - Guide méthodologique,

vol.II/Steps and Tools Towards Integrated Coastal Area Management – Methodological Guide, Vol.

II. 2001. 64 pp. (French, English; Spanish)

43 Black Sea Data Management Guide (Cancelled)

44 Submarine Groundwater Discharge in Coastal Areas – Management implications, measurements

and effects. 2004. 35 pp. (English)

IOC Manuals & Guides no 55

114

45 A Reference Guide on the Use of Indicators for Integrated Coastal Management. 2003. 127 pp.

(English). ICAM Dossier No. 1

46 A Handbook for Measuring the Progress and Outcomes of Integrated Coastal and Ocean Mana-

gement. 2006. iv + 215 pp. (English). ICAM Dossier No. 2

47 TsunamiTeacher – An information and resource toolkit building capacity to respond to tsunamis

and mitigate their effects. 2006. DVD (English, Bahasa Indonesia, Bangladesh Bangla, French,

Spanish, and Thai)

48 Visions for a Sea Change. Report of the ﬁrst international workshop on marine spatial planning.

2007. 83 pp. (English). ICAM Dossier No. 4

49 Tsunami preparedness. Information guide for disaster planners. 2008. (English, French, Spanish)

50 Hazard Awareness and Risk Mitigation in Integrated Coastal Area Management. 2009. 141 pp.

(English). ICAM Dossier No. 5

51 IOC Strategic Plan for Oceanographic Data and Information Management (2008–2011). 2008. 46

pp. (English)

52 Tsunami risk assessment and mitigation for the Indian Ocean; knowing your tsunami risk – and

what to do about it. 2009. 82 pp. (English)

53 Marine Spatial Planning. A Step-by-step Approach. 2009. 96 pp. (English). ICAM Dossier No. 6

54 Ocean Data Standards Series:

Vol. 1: Recommendation to Adopt ISO 3166-1 and 3166-3 Country Codes as the Standard for

Identifying Countries in Oceanographic Data Exchange. 2010. 13 pp. (English)

55 Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis. 2010. 114 pp. (Eng-

lish)

The Intergovernmental Oceanographic Commission (IOC) of UNESCO celebrates its 50th anniversary in 2010. Since taking the

lead in coordinating the International Indian Ocean Expedition in 1960, the IOC has worked to promote marine research, protection

of the ocean, and international cooperation. Today the Commission is also developing marine services and capacity building, and is

instrumental in monitoring the ocean through the Global Ocean Observing System (GOOS) and developing marine-hazards warning

systems in vulnerable regions. Recognized as the UN focal point and mechanism for global cooperation in the study of the ocean, a

key climate driver, IOC is a key player in the study of climate change. Through promoting international cooperation, the IOC assists

Member States in their decisions towards improved management, sustainable development, and protection of the marine environment.