PLANKTON

Sampling and Cultivation Protocol

Wan Maznah Wan Omar

School of Biological Sciences, Universiti Sains Malaysia, Malaysia
Centre for Marine and Coastal Studies (CEMACS), Universiti Sains Malaysia,
Malaysia
River Engineering and Urban Drainage Centre (REDAC), Universiti Sains
Malaysia, Malaysia

Mohammad Basri Eshak

School of Biological Sciences, Universiti Sains Malaysia, Malaysia

Wong Swe Cheng

Centre of Marine and Coastal Studies (CEMACS), Universiti Sains Malaysia,
Malaysia

Lim Chiew Chin

School of Biological Sciences, Universiti Sains Malaysia, Malaysia
CONTENTS

Preface V
Acknowledgements VII

1. AN INTRODUCTION TO PHYTOPLANKTON 1
1.1. Growth dynamics 2
1.1.1. Lag phase 2
1.1.2. Exponential phase 2
1.1.3. Stationary phase 3
1.1.4. Death phase 3
1.2. Brief classification 3
1.3. Factors affecting phytoplankton growth 5
1.3.1. Culture medium/nutrients 5
1.3.2. Light 5
1.3.3. pH 6
1.3.4. Aeration/mixing 6
1.3.5. Temperature 7
1.3.6. Salinity 7
1.4. Common phytoplankton in tropical waters 8
1.4.1. Freshwater 8
1.4.2. Marine water 10

2. AN INTRODUCTION TO ZOOPLANKTON 15
2.1. Growth stages 16
2.2. Brief classification 16
2.3. Factors affecting zooplankton growth 17
2.4. Common zooplankton in tropical waters 19

3. PHYTOPLANKTON AND ZOOPLANKTON SAMPLES COLLECTION 31

3.1. Pre-sampling 31
 3.1.1. Sampling location and station selection 32
3.1.2. Basic materials 34
3.2. Sampling techniques 36
3.2.1. Bottles/ water samplers 36
3.2.2. Plankton pump 39
3.2.3. Plankton net 40
3.3. Post-sampling 48
3.3.1. Fixation and preservation 48
3.3.2. Storage and labelling 50

4. PLANKTON ENUMERATION AND BIOMASS ESTIMATION 56

4.1. Enumeration 56
4.1.1. Zooplankton enumeration 57
4.1.2. Phytoplankton enumeration 59
4.2. Biomass estimation 61
4.2.1. Dry weight and ash-free dry weight 61
4.2.2. Pigment concentrations 62
4.2.3. Biochemical concentrations 62
4.3. Growth rate measurement 63

5. PHYTOPLANKTON CULTIVATION TECHNIQUES 71

5.1. Sterilization techniques 71
5.1.1. Heat sterilization 72
5.1.2. Filtration sterilization 73
5.1.3. Electromagnetic waves sterilization 74
5.1.4. Chemical sterilization 74
5.2. Preparation of culture media 75
5.2.1. Types of culture media 76
5.2.2. Recipes for freshwater and marine media 78
5.3. Isolation techniques 83
5.3.1. Serial dilution 83
 5.3.2. Agar streaking 85
5.3.3. Micropipette isolation 87
5.4. Purification techniques 89
5.4.1. Mechanical separation 89
5.4.2. Antibiotic treatment 90
5.5. Culture maintenance 91
5.5.1. Short-term maintenance 91
5.5.2. Long-term maintenance 92
5.6. Mass cultivation 97
5.7. Harvesting technique 98
5.7.1. Centrifugation 98
5.7.2. Flocculation 99
5.7.3. Filtration 100

6. ZOOPLANKTON CULTIVATION TECHNIQUES 103

6.1. Zooplankton key attributes and life cycles 104
6.2. Isolation technique 107
6.3. Culture maintenance 111
6.3.1. Rotifer and Daphnia stock culture 111
6.3.2. Copepod stock culture 113
6.4. Daphnia, Rotifer and Copepods cultivation 115
6.4.1. Batch culture for Rotifer 116
6.4.2. Batch culture for Daphnia 117
6.4.3. Batch culture for Copepod (harpacticoid) 117
Preface

The word “plankton” comes from the Greek for “drifter” or “wanderer.”
Plankton are organisms that float or suspend passively in the water column,
without the ability to swim against tides and currents. They dominate the well-
lit surface layers of freshwater or seawater bodies and play a central role in
aquatic food webs. Scientists classify plankton in several ways, but the most
basic categories divide plankton into two functional groups: phytoplankton
(plants) and zooplankton (animals). Plankton play an incredible ecological
importance to the aquatic ecosystem. They possess attributes that can be
further manipulated as part of marine bioresources. Marine phytoplankton (or
planktic microalgae) are unique resources that provide a diverse array of
natural products, which are beneficial for human, directly or indirectly.
Previous bioactivities surveys and nutrient analyses of microalgae have been
reported. The importance of phytoplankton in aquaculture is not surprising as
they are the natural food source of these animals. Phytoplankton has been
cultivated as a feedstock for value added products, which include food, animal
feed, aquaculture, bioremediation, nutraceuticals, pharmaceuticals, biogas and
biofuels. Zooplankton, on the other hand, are important food source for large
vertebrate and invertebrate predators. This aspect puts them at a central
trophic link between autotrophs and tertiary trophic levels in the food chains
leading to fishery production. To meet this purpose as a potential sustainable
source of marine bioresource, plankton cultivation systems are developed.
As cultivation begins with screening for valuable strains that are suitable for
any purposes, plankton have to be collected from wild; thus, sampling protocols
are essential. In this book, Chapter 3 covers techniques for collecting plankton
samples, not only for cultivation but also for ecological studies. Biomass
determination is important in plankton cultivation to ensure of their optimal
growth, whereas cells enumeration is more pronounced in ecological studies.
These growth parameters are outlined in Chapter 4. Biochemical analysis to
determine the protein, carbohydrate, lipid and fatty acid contents are essential
as determinants of plankton nutritional values, related to algal growth. Thus,
this proximate analysis is also outlined in Chapter 4. Although there are various
techniques or systems to cultivate and harvest both phytoplankton and
zooplankton, especially with the advancement of technologies, we did not go
deeper into this aspect, as our aim is to provide a basic guideline to cultivate
and harvest them.

V
 We believe, this e-book will be beneficial for scientists, researchers, and
students who aim to study in more detail of various aspects of plankton, which
must begin with plankton samples collection, isolation, maintenance,
cultivation and harvesting. This knowledge is essential for us to exploit them
for their natural products to generate a wide range of primary and secondary
metabolites of commercial interest. In addition to that, this basic guideline will
also help aquaculture sectors. The potential for the discovery and development
of unique products from plankton cultivation seems unlimited.

Wan Maznah Wan Omar

School of Biological Sciences,
Universiti Sains Malaysia
November, 2021.

VI
Acknowledgement

We would like to thank Dean of Faculty of Fisheries and Marine, Universitas

Airlangga for giving us the opportunity to conduct the workshop, under the
grant awarded by Ministry of Education and Culture of the Republic of
Indonesia. This e-book was constructed and designed to facilitate and guide the
lecturers and students of Aquaculture Study Program of Faculty of Fisheries
and Marine participating in the workshop. Not to forget the staffs of Faculty of
Fisheries and Marine, Mrs Luthfiana and Mr. Ahmad Briwojoyo for facilitating
the workshop and make the production of this e-book a reality. Dean of School
of Biological Sciences, Universiti Sains Malaysia is gratefully acknowledged for
the facilities and technical help from the staffs during the field and laboratory
analysis, from where, the step-by-step procedural guides and photos used in
this book were gathered. Special thanks also go to Mr. Mohd Syaiful Mohammad
who involved direct and indirectly in the making of the videos and photos of
this book.

VII
VIII
 An Introduction to Phytoplankton

1. AN INTRODUCTION TO
PHYTOPLANKTON

OVERVIEW
Phyto + Plankton were coined from the Greek word “phyton”, (plant) and
the word “planktos” (wanderer or drifter). Phytoplankton are mostly tiny
single-celled aquatic organisms that are able to create materials such as
carbohydrates, fats, and proteins from carbon dioxide, water and
inorganic nutrients through photosynthesis. As the primary producer in
the food chain, phytoplankton play a key role as a food source for many
marine species larvae in aquaculture. Despite many methods of feeds
have been developed such as microencapsulated feeds, live microalgae
are still the preferred feed by various larvae.
Moreover, the by-products of microalgae biomass culture have gained
popularity in the market. Certain strains of microalgae undergoing a
specific environmental stress is able to produce a by-product for the food
and feed industry. For example, microalgae cultivation has been used for
the production of polyunsaturated fatty acids (PUFAs). It has also been
known that the main driving force of growing microalgae extensively is
harvesting the metabolite products. Being known for its potential as one
of the promising feedstocks for biodiesel production (Lu et al., 2021), the
microalgae have been massively cultured aiming for high lipid
production. Furthermore, microalgae have been widely research for its
potential as an environmental biotechnological tool. For example, it can
be used as a bioindicator of aquatic ecosystem health (Wan Maznah and
Makhlough 2015) and as a “carrier” in remediation system for
contaminants (Patil et al., 2021).

1|P a g e
 An Introduction to Phytoplankton

1.1 GROWTH DYNAMICS

Within a batch culture, microalgae will exhibit four phases of algal growth.
The four phases are lag, exponential, stationary and lastly, the death phase
(Lavens & Sorgaloos, 1996; Price & Farag, 2013) (Figure 1.1).

Figure 1.1 Four growth phases of microalgae cultures; (1) lag phase; (2)
exponential phase; (3) stationary phase; (4) death phase (modified from
Mohammad Basri & Wan Maznah, 2017)

1.1.1 Lag phase

Lag phase will experience a slow growth with a slow increase in cell density.
The is mostly caused by the adaptation of the cell metabolism towards its
surrounding such as culture media, water pH, temperature, and light.

1.1.2 Exponential phase

In this phase, rapid growth and increased cell division occur. The cell density
will start to increase exponentially. The growth rate of the culture can be
determined from the exponential phase.

2|P a g e
 An Introduction to Phytoplankton

1.1.3 Stationary phase

This phase will start when nutrients in the media depleted. Growth
requirements and microalgal growth rate will be in equilibrium and the cell
density will cease to increase. This is because cell division and cell death are
happening at the same rate.

1.1.4 Death phase

Cells begin to die rapidly due to lack of growth requirement and water quality
declines. Cell death rate will be higher than cell division rate at this phase.

1.2 BRIEF CLASSIFICATION

Understanding the morphology and the classification of microalgae before

culturing is necessary in the work of microalgae culture. Microalgae can
appear in many forms and shapes, due to many factors. For example, to
prevent grazing predator, Scenedesmus sp. will form a colony and the outer
cells of the colony will have an extra spine to prevent grazing (Mayeli et al.,
2004). The extra spines and colony form will be confusing if we are not
familiar with the classification and the microalgae morphology. Descriptions
of few of the common algae phylum within the four evolutionary groups are
shown in Table 1.1.

3|P a g e
 An Introduction to Phytoplankton

Evolutionary Group Description Pigmentation* Example

Group 1 (Prokaryota) Prokaryotic cell organization Blue-green/ phycocyanin Cyanophyta (blue-green algae)

Group 2 (Eukaryota) Chloroplast enveloped by two Red/phycobilliproteins Rhodophyta (red algae)

chloroplast membranes Grass green Chlorophyta (green algae)
envelope
N/A Glaucophyta
Group 3 (Eukaryota) Chloroplasts enveloped by one Grass green Euglenophyta (euglenoids)
membrane of chloroplast N/A Dinophyta (dinoflagellates)
endoplasmic reticulum (ER)

Group 4 (Eukaryota) Chloroplast enveloped by two Phycobiliproteins / Yellow green to Cryptophyta (cryptophytes)
membranes of chloroplast ER golden brown
N/A Eustigmatophyta
Fucoxanthin Chrysophyta (golden-brown algae)
Fucoxanthin Prymnesiophyta (haptophytes)
fucoxanthin Bacillariophyta (diatoms)
N/A Raphidophyta (chloromonads)
N/A Xanthophyta (yellow-green algae)
Olive green to dark brown/ Phaeophyta (brown algae)
fucoxanthin and other xanthophylls

*Chlorophyll pigments are present in all evolutionary group.

*N/A = not available

4|P a g e
 An Introduction to Phytoplankton

1.3 FACTORS AFFECTING PHYTOPLANKTON

GROWTH

The following section will elaborate on the effect of environmental

parameters (light, pH, salinity temperature), nutrients and mixing towards
algal growth (Lavens & Sorgeloos, 1996; Anderson, 2005; Creswell, 2010).

1.3.1 Culture medium/nutrients

In general, algal grown indoor has much higher density than those found in
the natural environment. Algal culture usually has to be cultured in enriched
media. Enriched media is usually consisted of macronutrients (nitrogen and
phosphorus) and micronutrients (trace elements and vitamins) (Anderson et
al., 2005). Algae growth is dependent on the uptake of the most limiting
nutrient in the media or its surrounding environment (Titman, 1976). If
either one of the macronutrients or micronutrients was limited, algal
metabolic pathway will be affected. For example, phosphorus level of
<0.045mg/L, or >1.65mg/L, will inhibit the growth of microalgae (Ren, 2014).
Different algae family will have different requirement for their growth.
For example, silicate must be added into the culture media for the culture of
diatom family and green algae culture media does not require that addition
(Anderson et al., 2005). Due to this reason, nutrient types, nutrient
concentration, and its ratios will be different for different media. Species
found predominantly in freshwater, marine and soil samples has been
successfully cultured based on media recipes which have been improved
throughout the years by the phycological and aquaculture communities
(Anderson et al.,2005).

1.3.2 Light

Most algae are autotrophic, except for a few which are heterotrophic
(Rittmann & McCarty, 2001). Autotrophic algal depends on light source to
photosynthesize. Chlorophyll in the algae helps to convert inorganic
compounds into food required by the algae through the photosynthesis
process in the presence of light (Gatamaneni et al., 2018).

5|P a g e
 An Introduction to Phytoplankton

Light intensity required during algae culture depended on the culture

depth and the density of the algal culture. Stronger light intensity will be
required to penetrate the culture of a larger volume and higher cell densities.
Natural sunlight or fluorescent tubes can be used during algae culture.
During culture, microalgae growth will be affected by three different light
conditions, such as light limitation, light saturation, and light inhibition. If the
lighting condition was limited, increase light intensity will increase algae
growth. However, if the lighting condition was at saturated level, algal cell
photosynthetic activity will be inhibited as the chloroplast in the algal cell
have been saturated with too much light to work efficiently. Light inhibition
will further take place if the light intensity continues to increase without limit.
If this happens, the algal cell chloroplast would experience an irreparable
damage (Chang et al., 2017).

1.3.3 pH

Presence of microalgae have been found in both acidic and alkaline

environment (Ying et al., 2014). However, it is sensitive to pH changes. The
optimum pH for the photosynthesis process of microalgae is between 6 to 10
(Rastogi et al., 2017).
Medium’s pH can influence the photosynthetic process in the microalgae
chloroplast organelle. Too high or too low pH value within a medium have
been known to inhibit the photosynthetic process. There is a co-relation
between the medium’s pH and the carbon dioxide concentration. pH will
increase as carbon dioxide is being used by the microalgae during the
photosynthesis process. So, pre-adjustment of the pH medium before
microalgae inoculation, is vital to ensure optimum growth rate.

1.3.4 Aeration/mixing

Mixing helps to evenly distribute the light and nutrients within a culture
medium to all the algal cells. Mixing can also minimise the algae shading
effect due to high density within a culture.

6|P a g e
 An Introduction to Phytoplankton

1.3.5 Temperature

Temperature plays a role in the growth of algae, and optimal temperature

(20 to 24°C) is required to optimize growth (Raven and Geider, 1988).
Growth optimisation process also have to consider the composition of the
culture medium, algae species and the strain cultured. Once temperature
exceeded the optimal temperature for an algae strain, cell growth will be
hindered by heat stress and denaturation of proteins and inactivation of
photosynthesis enzymes will occur. The temperature range for frequently
cultured microalgae species are between 16 to 27 °C (Lavens and Sorgeloos,
1996).

1.3.6 Salinity

Every strain of microalgae adapted differently to changes in salinity.

However, marine microalgae are more tolerant towards changes in salinity
than freshwater algae (Blinova´ et al., 2015). Microalgae with high tolerance
to salinity is known as halophilic algae, while algae that do not require salt,
but can live in saline medium is known as halotolerant (Rao et al., 2007).
Salinity stress affected the microalgae growth and its lipid production.

7|P a g e
 An Introduction to Phytoplankton

1.4 COMMON PHYTOPLANKTON IN TROPICAL

WATERS

Examples of common phytoplankton found in freshwater and marine water

are shown in Sections 1.4.1 and 1.4.2, respectively.

1.4.1 Freshwater

Oscillatoria sp. Pediastrum sp.

Cymbella sp. Staurastrum sp.

8|P a g e
 An Introduction to Phytoplankton

Dinobryon sp. Aphanizomenon sp.

Nodularia sp. Woronichinia sp.

Navicula sp. Scenedesmus sp.

9|P a g e
 An Introduction to Phytoplankton

1.4.2 Marine water

Ceratium sp. Bacteriastrium sp.

Chaetoceros sp. Corethron sp.

Coscinodiscus sp. Rhizosolenia sp.

10 | P a g e
 An Introduction to Phytoplankton

Noctiluca sp. Dinophysis sp.

Guinardia sp. Skeletonema sp.

11 | P a g e
 An Introduction to Phytoplankton

References
[1] Anderson, R. A. (ed.). (2005). Algal Culture Techniques. Elsevier
Academic Press, Burlington, Massachusetts. Pp. 578.
[2] Baweja, P., & Sahoo D. (2015). Classification of algae. In: Follows
Sahoo D. & Seckbach J.(eds) The Algae world (Cellular Origin, Life in
Extreme Habitats and Astrobiology, vol 26. Springer, Dordrecht
Heidelberg New York London.
[3] Blinova´, L., Bartosˇova´, A., and Gerulova´, K. (2015). Cultivation of
microalgae (Chlorella vulgaris) for biodiesel production. Research
Papers Faculty of Materials Science and Technology. Slovak Univ.
Technol. 23, 87.
[4] Chang, J.S., Show, P.L., Ling, T.C., Chen, C.Y., Ho, S.H., Tan, C.H.,
Nagarajan, D., and Phong, W.N. (2017). 11- Photobioreactors A2 -
Larroche, Christian. In M.A´ . Sanroma´n, G. Du, and A. Pandey, Eds.,
Current Developments in Biotechnology and Bioengineering.
Elsevier, pp. 313–352.
[5] Creswell, L. (2010). Phytoplankton Culture for Aquaculture Feed.
Southern Regional Aquaculture Center. 5004: 1-55.
[6] Gatamaneni, B.L., Orsat, V., Lefsrud, M. (2018). Factors Affecting
Growth of Various Microalgal Species. Environmental Engineering
Science. 1-12.
[7] Lavens, P. & Sorgeloos, P. (1996). “Manual on the Production and
Use of Live food for Aquaculture: 2.3. Algal Production." FAO
Fisheries Technical Paper.
http://www.fao.org/docrep/003/W3732E/w3732e06.htm
[8] Lee, R.E., (2008). “Phycology”, 4th Ed. Cambridge University Press,
pg. 561.
[9] Lim, H. C. Teng, S. T., Leaw, C. P. Iwataki, M., Lim, P. T. (2014).
Phytoplankton assemblage of the Merambong Shoal, Tebrau Straits
with note on potentially harmful species. Malayan Nature Journal,
66(1&2), 198-211.
[10] Lu, J., Zhang, Z., Fan, G., Zhang, L., Wu, Y., Yang, M. (2021).
Enhancement of microalgae bio-oil quality via hydrothermal
liquefaction using functionalized carbon nanotubes. J. Clean Prod.
28:1–8.
[11] Mayeli, S.M., Nandini, S., Sarma, S.S.S. (2004). The efficacy of
Scenedesmus morphology as a defense mechanism against grazing

12 | P a g e
 An Introduction to Phytoplankton

by selected species of rotifers and cladocerans. Aquatic Ecology, 38,

515-524.
[12] Mohammad Basri, E., Wan Maznah, W. O. (2017). Isochrysis
maritima Billard and Gayral Isolated from Penang National Park
Coastal Waters as a Potential Microalgae for Aquaculture. Tropical
Life Sciences Research, 28(2), 163–177.
[13] Patil R. R, Kambhar S. V., Giriyappanavar B. S., Chakraborty S. (2021).
Algae as environmental biotechnological tool for monitoring health
of aquatic ecosystem, chap 2. In: Maddela N. R. et al. (eds) Advances
in the domain of environmental biotechnology, environmental and
microbial biotechnology. Springer, Singapore.
[14] Price & Farag, I. H. (2013). “. -Resources conservation in Microalgae
Biodiesel Production”. International Journal of Engineering and
Technical Research (IJETR), 1(8), 49-56.
[15] Rao, A. R., Dayananda, C., Sarada, R., Shamala, T. R., Ravishankar, G.
A. (2007). Effect of salinity on growth of green alga Botryococcus
braunii and its constituents. Bioresource Technology, 98, 560-564.
[16] Rastogi, R.P.,Madamwar, D., and Pandey, A. (2017). Algal Green
Chemistry: Recent Progress in Biotechnology. Netherlands: Elsevier
[17] Raven, J.A., and Geider, R.J. (1988). Temperature and algal growth.
New Phytologist, 110, 441.
[18] Ren, T. (2014). Primary Factors Affecting Growth of Microalgae
Optimal Light Exposure Duration and Frequency. Thesis. Iowa State
University.
[19] Richardson, K., (1997). Harmful or Exceptional Phytoplankton
Blooms in the Marine Ecosystem. Advances in Marine Biology, 31,
301-385.
[20] Rittmann, B. E., McCarty, (2001). “Environmental Biotechnology:
Principles and Applications”, McGraw-Hill Higher Companies, 1st
Ed., pg 26. – 31, 26. – 31,551, 535—545: 599—600
[21] Rocha, J. M. S., Garcia, J. E. C., Henriques, M. H. F. (2003). Growth
aspects of the marine microalga Nannochloropsis gaditana.
Biomolecular Engineering, 2, 237–242.
[22] Titman, D. (1976). Ecological competition between algae:
Experimental confirmation of resource-based competition theory.
Science, 192, 463.
[23] Wan Maznah, W.O., Makhlough, A. (2015). Water quality of a
tropical reservoir based on spatio-temporal variation of
13 | P a g e
 An Introduction to Phytoplankton

phytoplankton composition and physico-chemical analysis. Int J

Environ Sci. Technol, 12(7),2221–2232.
[24] Yingying, S., Changhai, W. (2009). The optimal growth conditions for
the biomass production of Isochrysis galbana and the effects that
phosphorus, Zn2+, CO2, and light intensity have on the biochemical
composition of Isochrysis galbana and the activity of extracellular
CA. Biotechnology and Bioprocess Engineering, 14, 225–231.

14 | P a g e
 An Introduction to Zooplankton

2. AN INTRODUCTION TO
ZOOPLANKTON

OVERVIEW
Zooplankton are planktic animals that are non-motile and drifting along
with the currents in the freshwater or seawater bodies. Zooplankton play
an extremely important role in food webs of aquatic ecosystem. Unlike
phytoplankton, zooplankton are heterotrophs which are unable to
synthesize their own food. Most of zooplankton are filter feeders. They
consume phytoplankton and strains of bacteria, as well as other fine
particles in water columns. Carnivorous zooplankton also grazes on
herbivorous and small-sized zooplankton (Farrell & Hodgson, 2012).
Then, planktivorous fish feed on the larger zooplankton. Zooplankton are
an essential food source for large vertebrate predators such as
planktivorous and larval fish, and invertebrate predators such as insects.
They are the central trophic link between autotrophs and tertiary trophic
levels in the food chains leading to fishery production (Sardet, 2015;
Yağci, 2016).
Zooplankton have several adaptation strategies against their
predators particularly fish to survive and maximize their populations
size. These adaptations include cyclomorphosis (Chang & Hanazato,
2003), transparent anatomy (McClintock et al., 2001; Hirst, 2017) and
diel vertical migration (Meester, 2010; Mendonça et al., 2015; Picapedra
et al., 2015). Despite such mechanisms that are actually for predation
avoidance purpose, many studies showed that feeding and mating
behaviors of zooplankton also influence their predation risk and
subsequently impact their community structure (Derry & Arnott, 2007;
Ekhande et al., 2013; Almeda et al., 2017).
 An Introduction to Zooplankton

2.1 GROWTH STAGES

According to McClintock et al. (2001), zooplankton are constituted of

holoplankton (permanent members of plankton) and meroplankton
(temporary members of plankton). Holoplankton spend their entire life cycle
as plankton whereas meroplankton comprises larval and young stages of
animals that will develop different lifestyle once they reach their maturity
stage (McClintock et al., 2001). Zooplankton undergo diapausing stage like
some other organisms to overcome adverse environmental conditions in
order to survive (Ślusarczyk, 2004; Lass et al., 2005). Most of zooplankton
species establish short generation period which usually took a day or weeks
(Jaiswal et. al., 2014).

2.2 BRIEF CLASSIFICATION

Most animal phyla including Protista, Porifera, Coelenterata, Gastrotricha,

Hydracarina, Ostracods, Rotifera, Copepoda, Cladocera, and larval members
of insects and fishes are the constituents of zooplankton in freshwater lakes
and streams (Osborne, 2002). In freshwater systems, Rotifera, Cladocera and
Copepoda are the predominant groups of zooplankton (Osborne, 2002; Moss,
2010; Yağci, 2014) whereas Copepoda is the predominantly found in marine
waters (Chen & Liu, 2015; Fernandes & Ramaiah, 2019; Ismail et. al., 2021).
Rotifers are a group of tiny or microscopic zooplankton (50-2000 µm in
size) that constitutes the phylum Rotifera. They are mainly constituted from
Monogononta and Bdelloidea (Segers, 2008). Most of them occur in
freshwater bodies. Corona, a ciliary organ around the mouth at the anterior
end, is an important structure for their locomotion and feeding mechanism.
They are named rotifer because of the rapid movements of the coronal cilia
that resemble a rotating wheel. Most of the rotifers feed on particles with the
size range 1-20 µm shared with filter-feeding cladocerans (Moss, 2010).
Among the plankton, rotifers exhibit a very short lifespan (Supratim et al.,
2015). The lifespan of the rotifers are different from one species to another
which ranges from several days to a few months (Ricci, 2001).
Cladocerans are one of the crustacean zooplankton that are widely
distributed throughout the world. They are commonly present in various
16 | P a g e
 An Introduction to Zooplankton

freshwater habitats such as lakes, ponds and wetlands (Boehler et al., 2012).
They occur in various size that range from < 250 µm (representative genus is
Alonella) to 4-6 mm (e.g. Daphnia and Simocephalus) (Shiel, 1995).
Cladocerans are covered with carapace which gives them a smooth body
outline and propel through the waterbody by rowing their two large second
antennae. Female cladocerans are mostly predominant in the populations
and reproduce by parthenogenesis. Eggs are resistant to unfavourable
conditions like desiccation. Cladocerans (especially Daphnia) are important
model organisms in ecological and evolutionary studies due to their easy
culturing, short generation time, and clonal reproduction (Forro et. al., 2008).
Some cladocerans are herbivorous such as Daphnia and Bosmina but some
are carnivores (Leptodora and Polyphemus) that feed on smaller zooplankton
(Moss, 2010). They are important food source for fish as reported by
Medeiros & Arthington (2014) and Gogoi et al. (2016).
Copepoda is the largest constituent of the class Maxillopoda, and some of
them are freshwater species (Suárez-Morales, 2015). Calanoida, Cyclopoida
and Harpaticoida are the three main groups of copepods that are free-living
in freshwater habitats (Shiel, 1995).
Copepods are relatively active and excellent swimmers. For instance,
cyclopoids escape from their predators with high velocity which may
accelerate up to 350 mm/s with continuous and vigorous hops (Suárez-
Morales, 2015). Smaller zooplankton, larger colonies of phytoplankton and
detritus are the food sources of copepods and the food particles size ranges
from 5-100 µm (Moss, 2010)

2.3 FACTORS AFFECTING ZOOPLANKTON GROWTH

Many studies demonstrated that zooplankton respond sensitively to the

environmental changes such as variations in dissolved oxygen (DO), nutrient,
temperature, pollution, food availability, light intensity, predation and pH by
changing their behaviours, abundance and community structure (Park et al.,
2001; Sousa et al., 2008; Imoobe & Adeyinka, 2010; Paul & Liu, 2012; Jong-
Yun et al., 2014; Almeda et al., 2017). pH below 5.0 would affect zooplankton
population (Zhuang, 1995). Azma Hanim et al. (2019) reported that
zooplankton abundance was influenced by the combined effects of water
17 | P a g e
 An Introduction to Zooplankton

quality variables. Water quality changes, food quality, food limitation and fish
predation are the factors affecting cladocerans’ body size and abundance
(Hart & Bychek, 2011; Jong-Yun et al., 2014; Straile, 2015). Food, predators
and light are the factors changing the swimming behavior of copepods
(Suárez-Morales, 2015). Besides changing their swimming behavior,
different reproductive and feeding strategies are demonstrated by small
copepods against predators in order to maximize their population size and
compensate heavy losses (Turner, 2004). Positive correlation between
copepods and orthophosphate might indicate that they required food with
higher phosphorus contents which are useful in promoting the growth rate
of copepods and the formation of RNA and DNA (Carrillo et al., 2001).
Hong-Wu et al. (2003) revealed that temperature affected calanoid
copepod Pseudocalanus newwani through temperature-dependent growth
and reproduction, high-temperature inhibition, and temperature-controlled
sex ratio under condition with sufficient food supply. Higher temperatures
increase the growth rate of rotifers which have the ability to tolerate with a
wide range of temperature (Sulehria & Malik, 2012). Ger et al. (2016) pointed
out that an environment with lower food supply and higher temperature is
dominated by small-bodied cladocerans species. The species composition
and species richness of cladocerans can be influenced by food quantity and
quality (Ghidini et al., 2009).
Park et al. (2001) and Paul & Liu (2012) stated that the growth of rotifers
are very dependent on DO. When DO was < 2 mg/L, the density of cultured
rotifers declined. Rotifera are sensitive on variations of food type and density
caused by changing in nutrient concentrations (Duggan et al., 2001). Ji et al.
(2013) highlighted that temperature was the factor affected species
composition and total abundance of Rotifera in subtropical lakes. Negreiros
et al. (2010) reported that variation in chlorophyll a was one of biotic factors
associated with rotifers densities and diversity. Other than that, rotifers can
be suppressed by the presence of cladocerans through competition
(MacIsaac & Gilbert, 1991) and regulated by invertebrate predators (Ulloa,
2004; Negreiros et al., 2010).

18 | P a g e
 An Introduction to Zooplankton

2.4 COMMON ZOOPLANKTON IN TROPICAL

WATERS

Species stated in Table 2.1 are some common zooplankton in tropical waters
(Wan Maznah et al., 2017; Azma Hanim et al., 2019)

Table 2.1 Common zooplankton in tropical waters

Group Rotifera Cladocera Copepoda
Taxa • Anuraeopsis fissa • Alona karua • Cyclopoida
• Anuraeopsis navicula • Bosminopsis deitersi • Harpacticoida
• Anuraeopsis sp. • Bosminopsis sp. • Calanoida
• Ceriodaphnia cornuta • Mesocyclops
• Ascomorpha sp.
• Chydorus barroisi leukarti
• Asplanchna brightweli • Microcyclops
• Diaphanosoma excisum
• Asplanchna sp. • Diaphanosoma sarsi varicans
• Bdelloidea • Diaphanosoma sp. • Thermocyclops
• Brachionus calyciflorus • Moina micrura crassus
• Brachionus caudatus • Tropocyclops
prasinus
• Brachionus falcatus
• Brachionus forficula
• Brachionus patulus
• Collotheca sp.
• Conochilus sp.
• Euchlanis dilatate
• Filinia opoliensis
• Keratella cochlearis
• Keratella tecta
• Keratella tropica
• Keratella sp.
• Lecane (Monostilla)
bulla
• Lecane hamata
• Lecane luna
• Lecane lunaris
• Lecane papuana
• Notommata sp.
• Polyarthra vulgaris
• Polyarthra sp.
• Pompholyx complanata
• Ptygura sp.

19 | P a g e
 An Introduction to Zooplankton

• Testudinella patina
• Trichocerca pusilla
• Trichocerca similis
• Trichocerca sp.

The following photos show some of the common zooplankton in tropical

waters:

Conochilus sp. Polyarthra sp.

Ptygura sp. Trichocerca sp.

20 | P a g e
 An Introduction to Zooplankton

Bdelloidea Cyclopoida

Anuraeopsis sp. Diaphanosoma sarsi

Lecane lunaris Notommata sp.

21 | P a g e
 An Introduction to Zooplankton

Keratella tecta Collotheca sp.

Harpacticoida Lecane hamata

Keratella cochlearis Trichocerca similis

22 | P a g e
 An Introduction to Zooplankton

Brachionus calyciflorus Ascomorpha sp.

Asplanchna sp. Brachionus forficula

Bosminopsis deitersi Diaphanosoma excisum

23 | P a g e
 An Introduction to Zooplankton

Lecane papuana Trichocerca pusilla

Anuraeopsis navicula

24 | P a g e
 An Introduction to Zooplankton

References
[1] Almeda, R., van Someren Gréve, H., & Kiørboe, T. (2017). Behavior is
a major determinant of predation risk in zooplankton. Ecosphere,
8(2), 1-20.
[2] Azma Hanim, I., Lim, C. C., & Wan Maznah, W. O. (2019). Evaluation of
spatial and temporal variations in zooplankton community structure
with reference to water quality in Teluk Bahang Reservoir,
Malaysia. Tropical Ecology, 60(2), 186-198.
[3] Boehler, J. A., Keller, T. S., & Krieger, K. A. (2012). Taxanomic atlas of
the water fleas, “Cladocera” (Class Crustacea) recorded at the Old
Woman Creek National Estuarine Research Reserve and State Nature
Preserve, Ohio. Final report to Ohio Department of Natural Resources,
Division of Wildlife, Columbus.
[4] Carrillo, P., Villar-Argaiz, M., & Medina-Sánchez, J. M. (2001).
Relationship between N: P ratio and growth rate during the life cycle
of calanoid copepods: An in-situ measurement. J. Plankton Res., 23(5),
537-547.
[5] Chang, K. H., & Hanazato, T. (2003). Seasonal and reciprocal
succession and cyclomorphosis of two Bosmina species (Cladocera,
Crustacea) co-existing in a lake: Their relationship with invertebrate
predators. J. Plankton Res., 25(2), 141-150.
[6] Chen, H., & Liu, G. (2015). Zooplankton community structure in the
Yellow Sea and East China Sea in autumn. Brazilian Journal of
Oceanography, 63, 455-468.
[7] Derry, A. M., & Arnott, S. E. (2007). Zooplankton community response
to experimental acidification in boreal shield lakes with different
ecological histories. Can. J. Fish. Aquat. Sci., 64, 887-898.
[8] Duggan, I., Green, J., & Thomasson, K. (2001). Do rotifers have
potential as bioindicators of lake trophic state? Verh. Internat. Verein.
Limnol., 27, 3497-3502.
[9] Ekhande, A. P., Patil, J. P., Patil, R. D., & Padate, G. S. (2013). Water
quality monitoring - study of seasonal variation of rotifer and their
correlation with physicochemical parameters of Yashwant lake,
Toranmal (M.S.) India. Arch. Appl. Sci. Res., 5(1), 177-181.
25 | P a g e
 An Introduction to Zooplankton

[10] Farrell, A. M., & Hodgson, J. R. (2012). Zooplankton diel vertical

migrations in lakes of contrasting food webs. BIOS, 83(1), 12-16.
[11] Fernandes, V., & Ramaiah, N. (2019). Spatial structuring of
zooplankton communities through partitioning of habitat and
resources in the Bay of Bengal during spring intermonsoon. Turkish
Journal of Zoology, 43(1), 68-93.
[12] Forro, I., Korovchinsky, M. M., Kotov, A. A., Petrusek, A. (2008).
Global diversity of cladocerans (Cladocera; Crustacea) in freshwater.
Hydrobiologia, 595, 177-184.
[13] Ger, K. A., Urrutia-Cordero, P., Frost, P. C., Hansson, L.-A., Sarnelle, O.,
Wilson, A. E., & Lürling, M. (2016). The interaction between
cyanobacteria and zooplankton in a more eutrophic world. Harmful
Algae, 54, 128-144.
[14] Ghidini, A. R., Serafim-Júnior, M., Perbiche-Neves, G., & Brito, L.
(2009). Distribution of planktonic cladocerans (Crustacea:
Branchiopoda) of a shallow eutrophic reservoir (Paraná State, Brazil).
Pan-Am. J. Aquat. Sci., 4(3), 294-305.
[15] Gogoi, B., Safi, V., & Das, D. N. (2016). The cladoceran as live feed in
fish culture: A brief review. Res. J. Animal, Veterinary and Fishery Sci.,
4(3), 7-12.
[16] Hart, R. C., & Bychek, E. A. (2011). Body size in freshwater
planktonic crustaceans: An overview of extrinsic determinants and
modifying influences of biotic interactions. Hydrobiologia, 668(1),
61-108.
[17] Hirst, A. G. (2017). Chapter 4: Zooplankton productivity. In C.
Castellani & M. Edwards (Eds.), Marine plankton: A practical guide to
ecology, methodology, and taxonomy (pp. 34-41). UK: Oxford
University Press.
[18] Hong-Wu, L., Syuhei, B., Tsutomu, I., & Takashi, M. (2003). Effect of
temperature on development, growth and reproduction in the
marine copepod Pseudocalanus newmani at satiating food condition J.
Plankton Res., 25(3), 261-271.

26 | P a g e
 An Introduction to Zooplankton

[19] Imoobe, T. O. T., & Adeyinka, M. L. (2010). Zooplankton-based

assessment of the trophic state of a tropical forest river. Int. J. Fish.
Aquac., 2(2), 64-70.
[20] Ismail, J., Mustafa Kamal, A. H., Idris, M. H., Amin, S. M. N., Hamli, H.,
Sien, L. S., Al-Asif, A., Abualreesh, M. H. (2021). Zooplankton species
composition and diversity in the seagrass habitat of Lawas, Sarawak,
Malaysia. Biodiversity Data Journal, 9, e67449.
[21] Jaiswal, D. P., Ahirrao, K. D., Shejule, K. B. (2014). Study of
Zooplankton Population in A Freshwater, Rangavali Dam, Navapur,
Dist- Nandurbar (MS) India. Scholarly Research Journal for Inter
Disciplinary Studies, 2(12),1355-1365.
[22] Ji, G., Wang, X., & Wang, L. (2013). Planktonic rotifers in a
subtropical shallow lake: Succession, relationship to environmental
factors, and use as bioindicators. Sci. World J., 2013, 1-14.
[23] Jong-Yun, C., Seong-Ki, K., Kwang-Hyeon, C., Myoung-Chul, K.,
Geung-Hwan, L., Gea-Jae, J., & Kwang-Seuk, J. (2014). Population
growth of the cladoceran, Daphnia magna: A quantitative analysis of
the effects of different algal food. PLOS ONE, 9(4), e95591.
[24] Lass, S., Vos, M., Wolinska, J., & Spaak, P. (2005). Hatching with the
enemy: Daphnia diapausing eggs hatch in the presence of fish
kairomones. Chemoecology, 15(1), 7-12.
[25] MacIsaac, H. J., & Gilbert, J. J. (1991). Discrimination between
exploitative and interference competition between Cladocera and
Keratella cochlearis. Ecology, 72(3), 924-937.
[26] McClintock, J. B., Baker, B., & Steinberg, D. (2001). Chapter 5: The
chemical ecology of invertebrate meroplankton and holoplankton. In
J. B. McClintock & B. J. Baker (Eds.), Marine chemical ecology (pp. 195-
225). New York: CRC Press. Retrieved from
https://books.google.com.my/books?id=XQtM4j5qkUoC&printsec=f
rontcover#v=onepage&q&f=false.
[27] Medeiros, E. S. F., & Arthington, A. H. (2014). Fish diet composition
in floodplain lagoons of an Australian dryland river in relation to an
extended dry period following flooding. Environ. Biol. Fishes, 97(7),
797-812.

27 | P a g e
 An Introduction to Zooplankton

[28] Meester, L. D. (2010). Zooplankton: Diel vertical migration. In G. E.

Likens (Ed.), Plankton of inland waters (pp. 239-246). USA: Academic
Press. Retrieved from
https://books.google.com.my/books?id=nx46BanT_V4C&printsec=f
rontcover#v=onepage&q&f=false.
[29] Mendonça, M. M., Picapedra, P. H. d. S., Ferronato, M. C., & Sanches,
P. V. (2015). Diel vertical migration of predators (planktivorous fish
larvae) and prey (zooplankton) in a tropical lagoon. Iheringia. Série
Zoologia, 105, 174-183.
[30] Moss, B. (2010). Ecology of freshwaters: A review for the twenty-first
century (4th ed.). UK: John Wiley & Sons. pp 60-451.
[31] Negreiros, N. F., Santos-Wisniewski, M. J. d., Santos, R. M. d., & Rocha,
O. (2010). The influence of environmental factors on the seasonal
dynamics and composition of Rotifera in the Sapucaí River arm of
Furnas Reservoir, MG, Brazil. Biota Neotrop., 10(4), 173-182.
[32] Osborne, J. A. (2002). Zooplankton populations dynamics in Lakes
Jesup and Washington Final report January-December 2001: St. Johns
River Water Management District, Palatka, Florida. pp 1-165.
[33] Park, H. G., Lee, K. W., Cho, S. H., Kim, H. S., Jung, M.-M., & Kim, H.-S.
(2001). High density culture of the freshwater rotifer, Brachionus
calyciflorus. Hydrobiologia, 446(1), 369-374.
[34] Paul, E., & Liu, Y. (2012). Biological sludge minimization and
biomaterials/bioenergy recovery technologies: John Wiley & Sons. pp
1-20
[35] Picapedra, P., Lansac-Tôha, F., & Bialetzki, A. (2015). Diel vertical
migration and spatial overlap between fish larvae and zooplankton in
two tropical lakes, Brazil. Braz. J. Biol., 75(2), 352-361.
[36] Ricci, C. (2001). Dormancy patterns in rotifers. Hydrobiologia,
446(1), 1-11.
[37] Sardet, C. (2015). Plankton: Wonders of the Drifting World:
University of Chicago Press. pp 1-250
[38] Segers, H. (2008). Global diversity of rotifers (Rotifera) in
freshwater. Hydrobiologia, 595, 49-59.

28 | P a g e
 An Introduction to Zooplankton

[39] Shiel, R. J. (1995). A guide to identification of rotifers, cladocerans

and copepods from Australian inland waters. Albury: Co-operative
Research Centre for Freshwater Ecology Canberra.
[40] Ślusarczyk, M. (2004). Environmental plasticity of fish avoidance
diapause response in Daphnia magna. J. Limnol., 63(Suppl. 1), 70-74.
[41] Sousa, W., Attayde, J. L., Rocha, E. D. S., & Eskinazi-Sant'Anna, E. M.
(2008). The response of zooplankton assemblages to variations in the
water quality of four man-made lakes in semi-arid northeastern
Brazil. J. Plankton Res., 30(6), 699-708.
[42] Straile, D. (2015). Zooplankton biomass dynamics in oligotrophic
versus eutrophic conditions: A test of the PEG model. Freshwater Biol.,
60(1), 174-183.
[43] Suárez-Morales, E. (2015). Class Maxillopoda. In J. H. Thorp & D. C.
Rogers (Eds.), Ecology and general biology: Thorp and Covich's
Freshwater Invertebrates (4th ed., Vol. 1, pp. 709-755). Boston:
Academic Press.
[44] Sulehria, A. Q. K., & Malik, M. A. (2012). Population dynamics of
planktonic rotifers in Balloki Headworks. Pakistan J. Zool., 44(3), 663-
669.
[45] Supratim, P., Amal Kumar, P., & Kaushik, C. (2015). Prospect of
Brachionus calyciflorus, a holoplankton, for its potential bio-indicator
property: A review. Int. J. Recent Sci. Res., 6(11), 7603-7608.
[46] Turner, J. T. (2004). The importance of small planktonic copepods
and their roles in pelagic marine food webs. Zool. Stud., 43(2), 255-
266.
[47] Ulloa, V. (2004). Density and biomass of planktonic rotifers in
different habitats in upper Parana River (PR, Brazil). Acta Limnol.
Bras., 16(3), 281-292.
[48] Wan Maznah, W. O., Intan, S., Sharifah, R., & Lim, C. C. (2017). Lentic
and lotic assemblages of zooplankton in a tropical reservoir, and their
association with water quality conditions. Int. J. Environ. Sci. Technol.,
15(3), 533-542.

29 | P a g e
 An Introduction to Zooplankton

[49] Yağci, M. A. (2014). Seasonal variations in zooplankton species of

Lake Gölhisar, a shallow lake in Burdur, Turkey. Pakistan J. Zool.,
46(4), 927-932.
[50] Yağci, M. A. (2016). Variations in the zooplankton species structure
of eutrophic lakes in Turkey. In M. N. Rashed (Ed.), Lake sciences and
climate change: InTech. Retrieved from
https://www.intechopen.com/books/lake-sciences-and-climate-
change/variations-in-the-zooplankton-species-structure-of-
eutrophic-lakes-in-turkey.
[51] Zhuang, D. (1995). Effects of low pH on zooplankton in some
suburban waterbodies of Chongqing City. J. Environ. Sci., 7(1), 31-35.

30 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3. PHYTOPLANKTON AND
ZOOPLANKTON SAMPLES
COLLECTION

OVERVIEW
As previously noted in the preceding chapter, phytoplankton and
zooplankton have a wide range of uses in both ecological and industrial
fields of study. However, before plankton can be used for any purpose,
we must first learn how to gather samples in the proper manner. Sample
collection for cultivation may not be as complex as collecting for
ecological research, which necessitates extensive preparation and
planning. The sampling method used, and the field of study have an
impact on the way plankton data is analysed from an ecological
standpoint. Sampling should be designed to include spatial and temporal
variations, for example, collecting samples at several stations or localities
(spatial variation) and repeatedly done for several months in a year
(temporal variation). It is recommended that spatial and temporal
sampling events to be done in the same manner to reduce irregular
horizontal/spatial and temporal spread. Sample collection for
cultivation, on the other hand, is considerably simpler because we may
choose any acceptable region and sample collection can be carried out at
any time. In recent years, there has been a major advancement in the
processes for collecting plankton samples, thus, this section will focus on
the most regularly used plankton sampling methods that are applicable
to both freshwater and marine habitats.

3.1 PRE-SAMPLING

Before plankton samples collection begin, a few tasks must be completed.

This section will address briefly how to choose an appropriate site and
station, as well as the basic items required for collecting plankton samples.
 Phytoplankton and Zooplankton Samples Collection

3.1.1 Sampling location and station selection

a) Sampling location selection

The sampling site is highly dependent on our aim, the study's timeliness,
and financial viability. Plankton analysis is typically used in conjunction
with environmental monitoring in areas of major interest to authorities,
such as water catchment areas, tourism, breeding, and construction areas.
Assume that a research's objective is to describe the phytoplankton and
zooplankton of a certain geographic region (baseline study), which
necessitates sampling of several lakes in the case of a freshwater
environment. In that situation, sampling from a single point inside each
lake or core basin of bigger, more complex lakes may be viable. In
comparison, if extensive environmental monitoring is required, numerous
stations should be sampled. Additionally, the sample collection period
should be extended to at least six months and maybe longer, as plankton
biomass can fluctuate significantly between months.

b) Sampling station selection

In elongated reservoirs, samples are often taken at many locations along

the longitudinal axis (Figure 3.1), since these lakes contain diverse zones
of water chemistry, phytoplankton, and zooplankton as one goes from the
often-turbid upstream end to the algal-dominated downstream end
(Jacobs & Grant, 1978). In the event of an open ocean or lake, sample sites
can be identified more easily by dividing the region into a gridded pattern
or transects (Figure 3.2). After reviewing data and information, gaining a
deeper understanding of events, and establishing trends, (e.g., no
significant different in all parameters between different sites) it is
possible to begin scaled-down monitoring. If it is possible to minimise the
number of stations and samples without compromising the quality of the
data, this should be done.
Essentially, monitoring programmes should be designed to deliver
the most amount of information with the least amount of time and effort.
When monitoring pollution-related issues, it is better to take a series of
samples from impacted to unaffected locations, with the latter samples
serving as controls. If this is not practicable, control data can be obtained

32 | P a g e
 Phytoplankton and Zooplankton Samples Collection

from surveys done in the same region prior to the introduction of a

pollutant, or from surveys conducted in adjacent unpolluted areas with
similar hydrographic and environmental characteristics. When possible,
sampling sites should be close enough to one other to detect gradient
effects when they occur. Monitoring should be carried out until the
recovery process is complete. Studies that monitor the impacts of direct
environmental additions can be conducted by placing stations along
several transect lines that radiate out from the source site of the
environmental addition.

Figure 3.1 Sampling stations at Temengor Reservoir, Perak, Malaysia was

chosen along the longitudinal axis due to diverse water zonation.

33 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Figure 3.2 Sampling stations at Kuala Kedah coastal area, Kedah, Malaysia
was chosen according to gridded pattern.

3.1.2 Basic Materials

a) Sample collection tools

Different sampling tools are required based on the type of sample
technique being utilised, and these techniques will be explained in each
part of the following sections.

b) Handheld GPS navigation

This is necessary to record the precise coordinates of the sample point
and will be useful in the case that you got lost in the middle of the ocean.

c) Life jacket
This is required, especially for open water sampling, and is not optional.
First and foremost, safety!

d) Data sheet
The data sheet should be prepared at least a day before the sample is
collected, and it should be placed on a clipboard with a piece of plastic

34 | P a g e
 Phytoplankton and Zooplankton Samples Collection

over it to avoid ripping from water splashes. Appendix 3.1 provides a

great example of a field data sheet.

e) Pen and permanent marker pen

A pen is clearly necessary for recording data, but a permanent marker
pen is essential to label the sample bottles or any apparatus on a wet
surface.

f) Camera/smartphone
It is important to take a photograph or video of the sampling site for your
own records and reference. A photograph or video will assist you in
remembering the condition of the sampling ecosystem for the sake of
analysis, as well as assisting others in understanding your results. All you
need is a waterproof camera case or plastic to protect your smartphone
or digital camera. Water and cameras/smartphones don't get along very
well.

g) Duct tape & cable tie

It is advisable that you include duct tape and cable tie in your sampling
list material. Both are aversatile material that can be used to repair
almost anything that has been damaged, for example, we applied duct
tape to patch holes in our plankton nets that were ripped during sampling
in the middle of the ocean.

h) Box / Ice cooler box

The box should be of appropriate size to accommodate all the sample
equipment and instruments. This will make it easier to carry all the
equipment and instruments throughout the landscape and to avoid water
splashes. Additionally, a different box to store the collected samples is
required, specifically for fresh samples.

i) Spare battery
All the equipment, including GPS, will not function unless they are
powered by batteries, so it is preferable to have a few extras.

j) Distilled water and tissue

All equipment must be cleaned with distilled water and tissue before and
after use.
35 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3.2 SAMPLING TECHNIQUES

Plankton can be collected using a variety of ways, such as bottles or water

samplers, pumps, and nets, among others. Each of them has their own
advantages and disadvantages. In this part, we will go through three
approaches, which are applicable to both phytoplankton and zooplankton.

3.2.1 Bottles/ water samplers

Plankton collection using bottles or water samplers is the most basic

approach. It is also the most effective at capturing all sizes of phytoplankton
and zooplankton. Using no filter to catch plankton implies that it captures
anything that floats on the surface of water body, but bigger zooplankton
with moderate locomotion may avoid the capture (Jacobs & Grant, 1978). As
a result, this approach is the most accurate way to determine the
phytoplankton and microzooplankton diversity in the study region, and it has
become the primary reason most researchers, particularly ecologists, favour
this method above any other method. In fact, there are some editors and
reviewers who would reject any article that covers phytoplankton diversity
that employed a filter to collect their plankton sample, such as a net.
The primary drawbacks of this technique, in our opinion, would be the
large number of water samples required to ensure that all species present at
a specific site are included. If the research involves several sites, it will be
difficult to get all of the samples back to the laboratory for additional
examination and interpretation. In the shallow environment or on the
surface of water, we can collect plankton samples using simple bottles with a
cap. However, if it involves several depths, there are a few other samplers
that could be employed (Pal & Choudhury, 2014):

I. Meyer’s Water Sampler (Weighting bottle)

⟡ Meyer’s water sampler is one of the oldest sampling gears used in

plankton sample collection. This bottle is made of glass and has a
capacity of 2L. It is secured with a metal band. This device is
weighted with a lead weight below, and there are two strong nylon
graded ropes: one attached to the neck of the bottle and the other
to the cork (Figure 3.3).
36 | P a g e
 Phytoplankton and Zooplankton Samples Collection

⟡ Sampling procedure:

1. Close the bottles with the stopper and let the bottles to drop
to the desired depth using gravity.
2. The weight at the bottom of the bottle will assist it in sinking
smoothly. If the current is too strong, add extra weight.
3. Pull the cork rope and let the water flow inside the bottle
once it has reached the desired depth.
4. Next, using the neck rope, carefully lift the bottle out of the
water and place it in another container.
5. Repeat the process as replicates for at least three times.

⟡ Depth recommendation: surface to 20 metres.

Figure 3.3 Meyer’s Water Sampler (pic from World Bank & Government of
The Netherlands funded, 1999)

I. Niskin Water Sampler & Van Dorn Water Sampler

⟡ The Niskin Water Sampler and the Van Dorn Water Sampler are
both covered in the same subchapter owing to the similarities in
how the two devices are used and operated. A more complex
37 | P a g e
 Phytoplankton and Zooplankton Samples Collection

apparatus compared to Meyer's Water Sampler, Niskin and Van

Dorn Water Samplers are becoming increasingly popular among
researchers and the most used water sampler in recent years. They
can be used for small-scale studies or for large-scale sample
collection in river systems and seas, depending on the situation. It
is used to collect water samples for plankton enumeration at
various depths, from subterranean levels to the surface of the ocean.
The sizes range from 1L all the way up to 8L. Both samplers were
made up from non-metallic materials and both ends are attached
with two strong cables which need to be hooked at the trip
mechanism and will be activated by a messenger (Figure 3.4).

⟡ Sampling procedure:

1. Attach both cables to the trip mechanism (Figure 3.5 a)

2. Lower down the water sampler to the aimed depth and
subsequently release the messenger weight (Figure 3.5 b)
3. The messenger weight will fall along the attached rope, hit
the water sampler's release mechanism, and close on both
ends.
4. Carefully lift the bottle out of the water (Figure 3.5 c) and
place it in another container (Figure 3.5 d)
5. Repeat the process at least three times as replicates.

⟡ Depth recommendation: surface to 100 metres

(a) (b)

Figure 3.4 (a) Niskin Water Sampler; (b) Van Dorn Water Sampler

38 | P a g e
 Phytoplankton and Zooplankton Samples Collection

(a) (b)

(c) (d)

Figure 3.5 Water sampler sampling procedure

3.2.2 Plankton pump

A plankton pump (Figure 3.6) is an equipment that pumps up saltwater

through a net with a variable mesh size depending on the study aim. The
mesh size of the net is determined by the plankton pump. Continuous
filtering will allow the phytoplankton or zooplankton to be rapidly
concentrated after being pumped to the surface using this equipment.
Due to the continuous collection capability of the pumps as the tube is
lowered down the water column, samples are integrated from the surface
to the required depth. Within a section of the pump, there is a flow metre
built in. As a result, the precise volume of filtered water will be measured
automatically.
The equipment can be deployed from the sampling vessel/boat or
from a dock. This approach is not recommended for phytoplankton and
big size zooplankton research since the frictional resistance of the
sampled water in the hose can produce turbulence, which can result in
the disintegration of colonies, huge setae, and even the cell itself in the
process.
39 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Figure 3.6 Plankton Pump (source: http://www.vliz.be/en/planktonpump)

3.2.3 Plankton Net

Plankton nets are the most frequently used sampling equipment for
collecting plankton samples. It is not unexpected given the fact that
plankton net is simple to operate, dependable, and inexpensive in cost.
The use of plankton nets allows us to filter a huge volume of water, which
allows a significant number of species to be contained within the net.
However, one of the major drawbacks of utilising nets is that a substantial
percentage of phytoplankton is tiny enough to slip through even the
smallest mesh net, making it impossible to establish the actual quantity
and variety of species in a given region.
It is important to note that the size of the plankton net varies from
large to small depending on how and where it will be utilised. A larger
plankton net may be required when employing a towing technique or
sampling in the wide ocean to minimise damage to the net, whereas a
smaller plankton net may be required while sampling in a small river or
on the pier due to the ease with which it may be handled in these
situations. The mesh size of the plankton net varies as well. For
40 | P a g e
 Phytoplankton and Zooplankton Samples Collection

phytoplankton collection, we propose adopting a plankton net with a

mesh size of 5 to 40 µm for phytoplankton, 20 to 200 µm for small
zooplankton, and 200 to 1000 µm for large zooplankton including larvae.
Table 3.1 gives information on net mesh sizes and plankton species to be
collected according to Pal & Choudhury (2014).
Many types of nets have been developed; the most common of which
is the basic net with a conical or truncated neck shape (Figure 3.7). It
comprises of a gauze bag, formed like

Figure 3.7 Plankton net

a cone, with a metal or plastic ring at the far side and a removable
plankton – a collecting vessel, closed at the narrow end. The mouth ring
of the net is connected to the towing line by three rope bridles that are
fastened to the net mouth. A weight is added to the end of the towing wire
to prevent it from being towed away. The net's front and tail sections are
strengthened with nonporous textile cuffs to prevent it from tearing. The
gauzes used in nets are composed of a variety of materials, such bolting
silk, polyester, nylon, and other synthetic fibres.

Table 3.1 Sizes of the net mesh and the plankton group to be collected
(Pal and Choudhury, 2014)
Net sizes (µm) Plankton group
1024 Largest zooplankton and ichthyoplankton
752 Larger zooplankton and ichthyoplankton
569 Large zooplankton and ichthyoplankton

41 | P a g e
 Phytoplankton and Zooplankton Samples Collection

366 Large microcrustacea

239 Zooplankton – microcrustacea
158 Zooplankton – microcrustacea and most rotifers
76
64 Phytoplankton – macroplankton, microplankton
53
2 Nanophytoplankton

There are two methods for collecting samples using plankton net. It
can be combined with the use of a water sampler or with the towing
technique. Both approaches are discussed in the following sections:

I. Plankton net + water sampler

⟡ This technique is commonly employed in eutrophic conditions since

it eliminates the need to filter a large amount of water in order to
get a concentrated sample. Furthermore, too many cells in the
sample container will make further analysis difficult. When using
this approach for ecological research, a water sampler is required
since the precise amount of filtered water must be documented.
However, if the exact volume of filtered water is not required (for
example, plankton collected for culture), any water container, even
a bucket, would suffice. The benefit of this approach is that it can be
used anywhere, including on a sample vessel, a pier, a small river,
and any shallow condition, including a mangrove habitat.

⟡ Sampling procedure:

1. Collect water using a water sampler/container (Figure 3.8

a) and filter it with a plankton net (Figure 3.8 b).
2. Repeat the process until the amount of water filtered
reaches the desired level (Figure 3.8 c). It is suggested to
filter at least 40L of water at each station.

42 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3. Transfer the filtered sample to another container for

storage (Figure 3.8 d).
4. Repeat the process at least three times as replicates.

⟡ Depth recommendation: surface to 30 metres

(a) (b)

(c) (d)

Figure 3.8 Plankton net + water sampler sampling procedure.

II. Towing technique

⟡ Towing can be done vertically or horizontally. A vertical pull is more

suited for collecting samples from the entire water column, whereas
a horizontal haul is only suitable for collecting surface water
samples. The quality and number of phytoplankton and
zooplankton gathered will be determined by the mesh size, type,
length, mouth area of the net, towing speed, time of collection, and
haul type (Goswami, 2004).

43 | P a g e
 Phytoplankton and Zooplankton Samples Collection

⟡ Vertical haul:

• The vertical haul is performed to sample plankton for the whole

transect of water body. As zooplankton migrate vertically in
response to light conditions, their occurrence is poor in upper
layers during daytime. To reduce the possibilities of missing
species for quantitative and qualitative analysis, the plankton
net needs to be towed from the lowest point of light visibility to
the surface. A flowmeter should be attached to the net to
calculate the amount of filtered water.

• Sampling procedure:

1. Make certain that the plankton net is securely fastened to

the towing line or rope.
2. Ascertain that the plankton net is weighted (vertical type of
plankton net has a built-in weight at the bottom area of the
net). If not, place some weight on it to ensure that it settles
properly.
3. Attach the flowmeter to the mouth of the net if the volume
of filtered water is required (Figure 3.9 a).
4. Slowly lower the net to the desired depth (Figure 3.9 b).
5. Slowly raise the net after the rope is in a straight line (Figure
3.9 c)
6. Record the flowmeter reading and collect the sample and
place it into a different container for storage (Figure 3.9 d).
7. Repeat the process at least three times as replicates.

44 | P a g e
 Phytoplankton and Zooplankton Samples Collection

(a) (b)

(c) (d)

Figure 3.9 Vertical tow sampling procedure

⟡ Horizontal haul:

• Horizontal towing is often used for the surface and subsurface

layers. Even with the use of a depressor, there is difficult to
ensure that the plankton net remains at the same depth during
the towing process. The net must be dragged at a modest speed
for 5 to 10 minutes. A suitable towing speed should be
maintained so that the greatest quantity of water can enter
through its mouth for improved filtration, while ensuring the
gear employed can bear the strain. The suggested net towing
speed for horizontal samples is 1.5 to 2.0 knots. When the
towing speed is increased, a static cone of water forms, directing
water outside the net and lowering effective filtering and the net
might potentially be harmed.

• Sampling procedure:

45 | P a g e
 Phytoplankton and Zooplankton Samples Collection

1. Make sure the plankton net is securely attached to the

towing line or rope.
2. Attach the flowmeter to the mouth of the net if the volume
of filtered water is required (Figure 3.10 a).
3. Allow the net to float away and submerge (Figure 3.10 b).
4. Slowly manoeuvre the boat or sample vessel until the cable
or rope linked to it is completely stretched out.
5. Begin the towing at 1.5 to 2.0 knots for 5 to 10 minutes. Take
careful note of the time.
6. Pull the net back into the boat at the end of the tow (Figure
3.10 c). Maintain the net's mouth up and the collection
vessel's end down. Record the end time of the tow and the
volume of filtered water on your datasheet.
7. Wash the outside of the net gently with seawater. This
process removes any plankton trapped in the nets and
deposits it in the sample bottle.
8. Collect the plankton sample and place it in a different
container for storage (Figure 3.10 d).
9. Repeat the process at least three times as replicates.

(a) (b)

(c) (d)

Figure 3.10 Horizontal tow sampling procedure

46 | P a g e
 Phytoplankton and Zooplankton Samples Collection

⟡ Calculation for the volume of filtered water

1. Using flow meter:

𝑽 = 𝝅𝒓𝟐 × 𝑭
V = Volume (m3)
π = 3.142
r2 = radius of the net opening (m)
F = flow meter reading (m3)

2. Without flow meter:

𝑽 = 𝝅𝒓𝟐 × 𝑳
V = Volume (m3)
π = 3.142
r2 = radius of the net opening (m)
L = Length (distance net was towed) (m)

3. Net tow by boat/vessel:

The “L” (length of tow in metres) after a boat or any
vessel tow is computed by knowing the vessel's
speed in metres per hour and the duration. The
following formula is used to compute length:
𝑳𝒆𝒏𝒈𝒕𝒉 (𝒎) = 𝑽𝒆𝒔𝒔𝒆𝒍 𝒔𝒑𝒆𝒆𝒅 × 𝑻𝒊𝒎𝒆
Because most vessels' speeds are recorded in knots
(nautical miles per hour), we must multiply the
speed in knots by 1852 metres per nautical mile to
get the speed in metres per hour. If the time
(duration) was measured in minutes, we must
divide it by 60 minutes per hour to convert it to
hours.

47 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3.3 POST-SAMPLING

Several procedures must be completed following the acquisition of plankton

samples. The following section will go over sample preservation as well as a
few other tasks that must be achieved after plankton samples are collected.

3.3.1 Fixation and preservation

Samples for ecological research and cultivation must be handled differently.

Both activities will be addressed briefly in this section.

a) Plankton collection for ecological study

Plankton collection for ecological research often involves

quantitative and qualitative analysis, which entails determining the
density and abundance of plankton in each location. In order to make
this precise calculation, samples must be preserved and stored.
Please keep in mind that plankton counting, and plankton
identification are not the same thing. It is always preferable to
identify plankton from fresh samples. If possible, identify the
plankton in your sample before preserving it, or collect one more
replicate and keep it alive for identification.
The importance of effective plankton fixing, and preservation
cannot be overstated. The samples will be difficult to be analysed if
they are not properly treated and stored. In the improperly fixed
samples, white precipitate and ruptured exoskeletons may be visible.
Fixation of samples should be performed as soon as feasible after
collection, preferably within 5 minutes, to avoid bacterial action and
autolysis damage to animal tissue (Goswami, 2004). A good fixative
should be inexpensive and kill the creatures rapidly, plus it should
not be corrosive or harmful.
In general, 2–5% formalin is employed to preserve and fix
zooplankton samples by diluting the formaldehyde obtained
commercially to the necessary concentration. Eighty percent pure
ethyl alcohol is also utilised as an excellent preservative, although it
frequently caused shrinkage and discoloration. Lugol's iodine
solution is a common preservative for phytoplankton of various sizes.
48 | P a g e
 Phytoplankton and Zooplankton Samples Collection

The iodine component stabilises, preserves, and colours the

plankton, whilst the acetic acid component stabilises and preserves
the flagella and cilia, acting as a wonderful preservative when
maintained in the dark (Pal and Choudhury, 2014). Appendix 3.2
provides steps on how to use formalin, ethyl alcohol and iodine
solution to preserve the plankton.

b) Plankton collection for cultivation

Plankton samples collected for cultivation must be kept alive.

Whatever method we employ to collect plankton samples, we can't
prevent catching both phytoplankton and zooplankton at the same
time, which will be a major issue if our goal is to isolate
phytoplankton because phytoplankton is a natural diet for
zooplankton. Once these creatures are concentrated, phytoplankton
may fast be eaten or otherwise killed. The use of any relevant filters
or nets can remove large creatures (i.e. copepodes, rotifers, ciliates)
and undesirable colonial algae (Andersen and Kawachi, 2005).
Generally, prefiltering samples immediately upon collection is
efficient in removing these undesirable species. Similarly, if the
sample has a high concentration of undesirable microscopic
creatures, such as other algae and bacteria, filtering technique can be
used to capture the bigger, desired species while allowing the smaller
organisms to pass through the filter. Nonetheless, extreme caution
must be used to prevent harm or desiccation to the target species,
and this approach is often employed only when the smaller
organisms represent a threat.
Phytoplankton and zooplankton isolation require careful
consideration of time. Some species die very quickly, sometimes
within an hour or a few hours of being collected. If our sampling site
is close to our laboratory, this will not be a problem. However, if it
will take more than one hour for our sample to reach our laboratory,
it will be important to place it on ice or in a cool container with ice
packs. The plankton are then normally capable of surviving for at
least another 24 hours, if not longer (Andersen and Kawachi, 2005).

49 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3.3.2 Storage and labelling

Clear glass containers with a wide mouth (Figure 3.11) are ideal for storing
plankton samples. Glass containers will not react to any preservative
chemicals, and clear glass will make further analysis easier later. Although a
clear glass can expose the sample and preservative chemicals to light, this
issue is easily avoided by placing the samples in a closed box. However, the
big issue will arise when we have a lot of sampling stations and replicates,
and our sampling site is a long distance away from our laboratory.
Furthermore, glass bottles are heavy and fragile, leaving them vulnerable to
damage during the sampling process. Unexpected events, such as a rough sea
and poor road conditions, will exacerbate the situation. In this regard,
polyethylene jars with polypropylene lids are the best choice for plankton
fixation and storage. It is much lighter than a glass bottle, and the chances of
the container breaking during the sampling process due to unexpected
events are almost nil. It may react to some preservative chemicals, but the
reaction has no effect on the chemicals' ability to perform their function.
Preserved samples should be kept in the dark and chilled to 4°C
(maximum 10°C) (Bellinger and Sigee, 2015). They can only be kept at room
temperature and in the dark if the sample is to be analysed within a week. It
is needed to reduce the pace of physical and chemical reactions that result in
a loss in sample quality. Storage in the dark is always required to prevent
photo-oxidation and to maintain the autofluorescence of pigments in
formaldehyde samples. The maximum storage duration for Lugol's preserved
samples in the dark and below 5°C if oxidation is avoided is 6 months. Only
with the addition of formaldehyde it is feasible to preserve and store for long
periods of time. Living samples should be stored in the dark at temperatures
ranging from 4 to 10 °C. They can only be stored unpreserved for up to 24
hours. Depletion of oxygen should be avoided in samples with a high density
of organisms by diluting the sample, allowing a significant amount of air in
the container, or exposing it to a little quantity of light.
Labelling sampling bottles is essential to avoid confusion during analysis.
The labels should include the following information:
1. Station’s number
2. Date
3. Time

50 | P a g e
 Phytoplankton and Zooplankton Samples Collection

4. Depth
5. Reading from the flow metre (if applicable)
6. Filtered volume (if applicable)
7. The collector's name

The label should be directly applied to the container. It is strictly

forbidden to put the label on the lid or cover of your sampling bottles because
it could change accidentally during analysis.

Figure 3.11 (a) Clear glass containers with a wide mouth; (b) Polyethylene
jars with lid

51 | P a g e
 Phytoplankton and Zooplankton Samples Collection

References
[1] Andersen, R.A. and Kawachi, M. (2005). Traditional microalgae
isolation techniques. In (Anderson, R.A. eds.) Algae Culturing
Techniques. Elsevier Academic Press, USA. pp. 83-100.
[2] Bellinger, G.E. and Sigee, C.D. (2015). Freshwater Algae:
Identification, Enumeration and Use as Bioindicators. (2nd Ed.),
Wiley-Blackwell, West Sussex, UK.
[3] Eaton, D. R., Brown, J., Addison, J. T., Milligan, S. P., & Fernand, L. J.
(2003). Edible crab (Cancer pagurus) larvae surveys off the east coast
of England; implications for stock structure. Fisheries Research, 65,
191–199.
[4] Goswami, S.C. (2004) Zooplankton Methodology, Collection and
Identification – A Field Manual. Natural Institute of Oceanography
Dona Paula, Goa- 403004. pp. 684.
[5] Jacobs, F. and Grant, G.C. (1978). Guidelines for zooplankton sampling
in quantitative baseline and monitoring programs. Virginia Institute
of Marine Science. U.S. Environmental Protection Agency,
Washington, D.C., pp. 52.
[6] Pal, R. and Choudhury, A.K. (2014). An Introduction to
Phytoplanktons: Diversity and Ecology, Springer, India.
[7] Retrieve from http://www.vliz.be/en/planktonpump on 18/9/2021
[8] World Bank & Government of The Netherlands funded (1999).
Training module # WQ - 13. How to sample surface waters for water
quality analysis. New Delhi.

52 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Appendix 3.1: Field data sheet

53 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Appendix 3.2: Fixatives and preservatives commonly used in the

preservation of plankton

1) Formaldehyde (Formalin)

Typically, the manufacturer provides a 40% solution. After dilution, the term
"formalin" is frequently employed. A solution of 2% to 4% is frequently used
for preservation. To make a 4% formaldehyde solution (10% Formalin), nine
parts waters are added to one part 40% (aqueous) formaldehyde; that is, 100
ml of 40% formaldehyde is dissolved in 900 ml saltwater or distilled water.
In general, it serves as a preservative for all types of phytoplankton. The
fixing agent must be freshly made a few days before sample collection, stored
at 5-6 °C, and stored in dark glass bottles. Two mL of neutralised
formaldehyde is enough to fix 100 mL of water sample. For long-term
storage, water sample should be stored in the dark.

IMPORTANT NOTE

Formaldehyde is carcinogen and should be handled with

care. Proper safety equipment includes gloves and plastic
goggles.

2) Ethanol

Ethanol is a common fixative used in molecular studies for zooplankton

samples. The final alcohol concentration in the sample must be carefully
monitored. A concentration that is too high may cause the zooplankton to dry
and become brittle, making them extremely difficult to detect. If the alcohol
content is too low, the samples will not be sufficiently preserved, resulting in
the decomposition of your zooplankton in their containers. For long-term
storage, we recommend a 70-80% ethanol solution. We propose using a 95%
ethanol solution in the field since the water entrained in the sample dilutes
the alcohol significantly. A 1:1 preservative/sample material ratio is a solid
thumb rule.

54 | P a g e
 Phytoplankton and Zooplankton Samples Collection

3) Lugol’s solution

Calcareous flagellates (coccolithophorids) can make up a large percentage of

the algal biomass in saltwater, thus neutral or alkaline Lugol's solution is
preferred. Diatoms with silicate cell walls retain their viability well in acidic
Lugol’s. Fungal infections may occur in samples fixed with alkaline Lugol's
solution, and such samples should be post-fixed by adding a few drops of
concentrated formalin solution for long-term storage. One advantage of
Lugol's solution is that it enters the cell faster than formaldehyde, which
preserves shock-sensitive organisms better in the sample. The following are
the steps for making acidic, neutral, and alkaline Lugol's solutions:

a) Acidic Lugol’s solution: Mix 100 g of Potassium iodide (KI),

1000 mL of distilled water, 50 g of Iodine (I2), and 100 mL of
Acetic acid glacial (100 % CH3COOH).

b) Neutral Lugol’s solution: Mix 100 g of Potassium iodide (KI),

1000 mL of distilled water, 50 g of Iodine (I2).

c) Alkaline (basic) Lugol’s solution: Mix 100 g of Potassium iodide

(KI), 1000 mL of distilled water, 50 g of Iodine (I2), 100g of
Sodium acetate (CH3COONa).

In each 100 ml sample, add 1 mL of Lugol's solution. To avoid photooxidation,

keep preserved phytoplankton samples in the dark at room temperature.
Lugols-fixed samples kept in a dark and cold environment (1 to 5 °C) have a
maximum storage period of 12 months. Throughout this time period,
samples should be examined every two months and topped up with Lugol's
solution as needed.

55 | P a g e
 Plankton enumeration and biomass estimation

4. PLANKTON ENUMERATION AND

BIOMASS ESTIMATION

OVERVIEW
Density of phytoplankton and zooplankton may be assessed by two
primary parameters: enumeration and biomass. Enumeration is the
process of counting plankton samples using counting chambers in order
to determine the species composition and relative abundance of the
various species. The enumeration approach is always favoured by
ecologists since it provides details down to the genus level or even to the
species level, even though it is laborious and time consuming. All this
information is necessary to ascertain the sample site's condition since
knowing the plankton genus or species provides a wealth of information
about the status of a given environment. Meanwhile, biomass
determination is typically chosen in plankton cultivation due to its
simplicity and expediency. Biomass determination includes the wet
weight, dry weight, ash-free dry weight, pigments concentration,
volumetric method (displacement volume and settling volume) and
chemical method (determination of carbon, nitrogen, protein,
carbohydrate and lipid contents). Both enumeration and biomass
techniques will be discussed in further detail in the subsequent sections.
Today, owing to technology developments, plankton density
measurements are done using genetic barcoding (RNA, DNA), automated
measuring equipment (FerryBox system), and remote sensing
techniques.

4.1 ENUMERATION

Prior to enumeration in a counting chamber, preserved samples in formalin,

ethanol, or Lugol's iodine solutions need to be concentrated particularly
when the water sampler approach was employed to obtain the samples
 Plankton enumeration and biomass estimation

(section 3.2.1). This may be accomplished using the sedimentation technique

(Bellinger and Sigee, 2015).
Sedimentation technique can be accomplished by placing the sample
into a measuring cylinder and leaving it on a vibration-free surface for 24–72
hours to allow all the algae to settle. Sedimentation should be performed in
complete darkness and away from any heat sources. The top 80% of
supernatant can then be gently syphoned out with a pipette fitted with a ‘U'
tip, causing the least amount of disruption possible. The remaining 20% can
be resuspended in the bottom of the cylinder using agitation and then kept at
a constant low temperature (4–10 °C) in the dark until enumeration step.

4.1.1 Zooplankton enumeration

Zooplankton enumeration and identification is conducted by observing

the samples under a compound microscope. Recommended model of
compound microscope for this task is Olympus BX41-CCD equipped with a
camera. Firstly, disperse 1 mL of sample onto Sedgwick-Rafter counting
cell using an adjustable volume pipette (e.g.: Rainin Pipet-Lite SL5000).
Sedgwick-Rafter counting cell of glass type is recommended if the samples
are preserved with ethanol as ethanol can be corrosive to plastic/acrylic type
of Sedgwick-Rafter counting cell. Then, place a cover slip across the top of
Sedgwick-Rafter counting cell and slide it in place, assuring no air bubbles
are trapped in the slide (Figure 4.1). If there are air bubbles, we can remove
them by lightly manoeuvring the cover slip from side-to-side. We may need
to reload the slide with a new sample if this does not work. The purpose of
using cover slip is to prevent evaporation of ethanol-preserved samples.
After that, the counting cell is let to stand for approximately 15 minutes to
allow the plankton to settle at the bottom into a single layer prior to the
analysis. Observe zooplankton samples at 10X, 20X, 40X and 100X
magnifications. Species identification is based on the taxonomic keys,
drawings and descriptions in Idris (1983), Korovchinsky (1992) and Shiel
(1995).
Zooplankton abundance is expressed as the number of individuals per
litre (ind./L) based on the following formula (Hauser, 1981):

AC
N=
V

57 | P a g e
 Plankton enumeration and biomass estimation

where N = Number of individuals per litre (ind/L)

A = Mean number of individual (per mL)
C = Volume of concentrated sample (mL)
V = Volume of filtered water (L)

Pipette with sample

Cover slip Sedgewick-Rafter counting chamber

Figure 4.1 Filling method of Sedgewick-Rafter counting cell

Other than Sedgwick-Rafter counting cell, Bogorov counting chamber

(Figure 4.2) is also used. It is a low-cost apparatus designed for enumerating
large quantities of zooplankton, ideally for enumeration of planktonic micro-
crustacea. Bogorov counting chamber is suitable to hold a large volume of
sample ranging from 6 mL to 39 mL for observation under a dissecting
microscope. The shape and configuration of the shallow, etched grooves help
locate specific areas on the chamber. Straight sides prevent focusing up and
down and the round edges prevent specimens from catching. Its compact size
helps to slip easily under a dissecting microscope. Since this chamber is not
covered with cover slip, thus it is not suitable for enumerating samples
preserved with ethanol.

58 | P a g e
 Plankton enumeration and biomass estimation

Figure 4.2 Bogorov counting chamber

4.1.2 Phytoplankton enumeration

Enumeration of phytoplankton is fairly similar to enumeration of

zooplankton. Phytoplankton can be counted using the Sedgwick-Rafter and
Bogorov counting chambers as mentioned in zooplankton enumeration
section (Section 4.1.1). Counting phytoplankton, on the other hand, may be
more difficult since certain species live as a single cell, filament of cells, or
globular colony (morphological types). Species counts are typically
performed in relation to the 'living unit,' which can take into the form of any
of those three morphological types. While there are no hard and fast rules
regarding whether colonies and filaments should be treated as single units
or constituent cells, consistency within and between samples is essential
(Bellinger and Sigee, 2015). Generally, we treat one cell as a single unit
regardless of whether it appears as filament cells or as a collection of
colonies. However, for filamentous algae, measuring filament length is a more
precise method. This is because the filaments of these algae vary in length
(both within and between samples), rendering the concept of individuals
useless. Additionally, filaments may stretch across more than one side of the
counting square, complicating enumeration.
Both Sedgwick-Rafter and Bogorov counting chambers are ideal for
measuring phytoplankton diversity. If a statistical analysis of species
diversity is necessary, we have to ensure that the sample contains as many
species as feasible. At least 400 units (cells or colonies) of each species should

59 | P a g e
 Plankton enumeration and biomass estimation

be counted if feasible to keep the counting error to less than 10% (Lund et al.
1958).
However, for counting single cell species from cultivation, we prefer to use
Haemocytometer counting chamber (Figure 4.3). It is a modified
microscope slide, containing two identical wells (chambers) into which a
small volume of cell suspension is pipetted. To count, load both chambers by
pipetting the suspension under the cover slip. Then, place the
haemocytometer under the microscope. Each chamber is divided into grid
pattern, consisting of 9 large squares. Each square has the same dimensions
and contains 10-4 mL of suspension. In the centre squares, there are 25
smaller square with the size of 1 mm each. The rules for counting cells
sometimes differ in different laboratory. In our lab, we count five squares in
the centres. For the highest degree of accuracy, it is best to count all the cells
in both sets of grids and average the two counts. The calculation is as follows:

𝑋
Cell number/mL = x 1000
0.02
where,
x = cell number (average cells from both wells),
0.02 represents 5 per 25 squares of 1 mm2, multiply with 0.1 mm depth of
haemocytometer (5/25 mm2 x 0.1 mm = 0.02 mm3),
1000 represents (10 mm x 10 mm x 10 mm)/cm3 = 1000 mm3/cm3

Figure 4.3 Haemocytometer counting chamber

60 | P a g e
 Plankton enumeration and biomass estimation

4.2 BIOMASS ESTIMATION

The determination of zooplankton and phytoplankton biomass is essential

because it gives information on the aquatic system's primary productivity as
well as the quantity of organic material available for ingestion by
zooplankton, fish, and the rest of the food chain. Biomass determination is
equally crucial in the aquaculture industry, particularly in feed production.
The assessment of biomass can provide a good picture of the quality of
phytoplankton and zooplankton utilised as feed for aquaculture species.
There are several methods for calculating plankton biomass, and we will go
through several of them in the next sections.

4.2.1 Dry weight and ash-free dry weight

One of the most direct methods to measure plankton biomass is to filter a

volume of plankton samples through a net or a membrane filter paper (glass
fibre or Millipore fibre). The biomass that can be measured are wet weight
(inorganic and organic biomass plus water), dry weight (inorganic and
organic biomass), or ash-free dry weight (organic biomass only). Wet weight
is seldom calculated because of the variability in removing free water from
the sample (Bellinger and Sigee, 2015).
The dry weight of a sample can be determined by drying it to a constant
weight in a thermostatically controlled oven at 105°C for 24 h. The sample is
subsequently heated in a thermostatically controlled muffle furnace for 1
hour (or to constant weight) at 300–500 °C to produce ash-free dry weight,
and the resulting ash-weight is subtracted from the dry weight to obtain the
ash-free value. All results should be divided by the filtered volume, and the
final unit should be in the form of weight/volume (e.g., g/L, mg/L). This
method is applicable to both zooplankton and phytoplankton. To ensure that
the data obtained is reliable, the dry weight or ash-free dry weight of water
samples is calculated from at least three replicate water samples. Dry wight
and ash-free dry weight calculations can be referred in Appendix 4.1.

61 | P a g e
 Plankton enumeration and biomass estimation

4.2.2 Pigment concentrations

Another technique for assessing phytoplankton biomass is to measure the

concentration of a component associated with biomass, such as pigment (e.g.,
chlorophyll-a). However, this approach is relevant only to phytoplankton, as
pigment is found exclusively in phytoplankton. The concentration of
phytoplankton can be observed directly using chlorophyll-a or translated
into estimated biomass using a suitable conversion factor (Bellinger and
Sigee, 2015). Chlorophyll-a concentrations can be determined using a variety
of procedures, including on-site measurements or subsequent laboratory
analysis of samples.
Fluorimeter probes are one of the tools used on-site to determine
chlorophyll content. Fluorimeters determine the intensity and wavelength
distribution of light produced as fluorescence by excited molecules. They can
be used to determine the concentrations of chlorophyll-a and other algal
pigments in the water column by using different excitation wavelengths and
collecting light at varied wavelength emissions.
Pigments such as chlorophyll-a can be determined manually in the
laboratory. It is a straightforward method that consists of three stages:
filtering, pigment extraction, and spectrophotometry analysis. Filtration is
performed using a cellulose acetate filter membrane (0.45 µm) or a glass
fibre filter (Whatman GF/C, 1.2 µm). To minimise pigment degradation,
samples should not be exposed to intense light or heat prior to filtration.
Once filtering is completed, the filter should be removed immediately, and
pigment extraction should take place promptly. If extraction is going to be
delayed, fold the filters, and store them at - 20 °C until needed. The whole
technique is detailed in Appendix 4.2.

4.2.3 Biochemical concentrations

Another way to determine the biomass of zooplankton and phytoplankton

is to estimate the quantity of organic carbon stored within the cells. Protein,
carbohydrates, and lipids are the three major organic components found
within the cell, and they serve as the cell's structural and functional building
blocks (Postel et al. 2000). Occasionally, biochemical values of a specific
taxon or species are determined to assess food energy transferred at higher

62 | P a g e
 Plankton enumeration and biomass estimation

trophic levels. The calorific content of plankton may then be used to estimate
the biomass of zooplankton and phytoplankton. This approach necessitated
the freeze-drying of plankton samples prior to examination. Appendix 4.3
contains the technique for quantifying protein, carbohydrate, and lipid.

4.3 GROWTH RATE MEASUREMENT

The growth rate is a parameter which is used to determine the increase in

biomass over time during the exponential stage (Figure 4.4) of the culture. It
is a critical means of describing a species' or strain's relative ecological
effectiveness in adapting to its native habitat or the imposed experimental
environment. Knowing our culture's growth rate is critical, particularly for
aquaculture feed, to ensure that everything is optimum in terms of time and
cost. Michelle Wood et al. (2005) provided a method for calculating the
specific growth rate and doubling time as mentioned below:

Specific growth rate (µ):

(l n 𝑋2 − ln 𝑋1 )
𝜇=
(𝑡2 − 𝑡1 )

where X2 and X1 are the cell biomass at time t2 and t1, respectively (Figure
4.4).

Doubling time (Dt) is the time required for cells to double in size and can
be calculated from the specific growth rate (µ):

0.6931
Dt (day-1) =
𝜇

63 | P a g e
 Plankton enumeration and biomass estimation

Figure 4.4 Growth curve of Isochrysis maritima (Mohammad Basri and Wan
Maznah, 2017). X1 and X2 indicate that the cell biomass (in this case, cell
density) at early and late exponential, respectively.

64 | P a g e
 Plankton enumeration and biomass estimation

References
[1] Bellinger, G.E. and Sigee, C.D. (2015). Freshwater Algae:
Identification, Enumeration and Use as Bioindicators. (2nd Ed.),
Wiley-Blackwell, West Sussex, UK.
[2] Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction
and purification. Can. J. Biochem. Physiol. 8, 911-917.
[3] Chu, W.L., Phang, S.M.& Goh, S.H. (1996). Environmental effects on
growth and biochemical composition of Nitzschia inconspicua
Grunow. J Appl Phycol, 8, 389–396
[4] DuBois, M., Gilles, K. a., Hamilton, J. K., Rebers, P. a., & Smith, F. (1956).
Colorimetric Method for Determination of Sugars and Related
Substances. Analytical Chemistry, 28(3), 350–356.
[5] Hauser, W. J. (1981). Manual for estuarine environmental and
zooplankton studies. Alaska: Alaska Department of Fish and Game,
Division of Fisheries Rehabilitation Enhancement and Development.
[6] Hulatt, C.J., Lakaniemi, A.M., Puhakka, J.A., & Thomas, D. N. (2012).
Energy Demands of Nitrogen Supply in Mass Cultivation of Two
Commercially Important Microalgal Species, Chlorella vulgaris and
Dunaliella tertiolecta. Bioenergy Research, 5, 669–684.
[7] Idris, B. A. G. (1983). Freshwater zooplankton of Malaysia (Crustacea:
Cladocera). UPM, Selangor.
[8] Korovchinsky, N. M. (1992). Sididae and Holopedidae: Guides to the
identification of microinvertebrates of the continental waters of the
world. The Netherlands: SPB Academic Publishing.
[9] López, C.V.G., García, M.D.C.C., Fernández, F.G.A., Bustos, C.S., Chisti, Y.,
Sevilla, J. M.F. (2010). Protein measurements of microalgal and
cyanobacterial biomass. Bioresource technology, 101(19), 7587–
7591.
[10] Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R. J. (1951). Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–
275.

65 | P a g e
 Plankton enumeration and biomass estimation

[11] Lund, J.W., Kipling, G., Le Cren, E.D. (1958). The inverted microscope
method of estimating algae numbers and the statistical basis of
estimation by counting. Hydrobiologia 11, 143–170.
[12] Michelle Wood, A., Everroad, R.C. and Wingard, L.M. (2005).
Measuring growth rates in microalgal cultures. In (Anderson, R.A.
eds.) Algae Culturing Techniques. Elsevier Academic Press, USA. pp.
269-285.
[13] Mohammad Basri, E. and Wan Maznah, W.O. (2017). Isochrysis
maritima Billard and Gayral isolated from Penang National Park
coastal waters as a potential microalgae for aquaculture. Tropical Life
Sciences Research, 28(2):163–177.
[14] Postel, L., Fock, H. and Hagen, W. (2000). Biomass and Abundance.
In (Harris et al. eds.) ICES Zooplankton Methodology Manual.
Academic Press, UK. Pp. 83 – 174.
[15] Rausch, T. (1981). The estimation of micro-algal protein content
and its meaning to the evaluation of algal biomass I. Comparison of
methods for extracting protein. Hydrobiologia, 78, 237–251.
[16] Ryckebosch, E., Muylaert, K., Foubert, I., 2012. Optimization of an
analytical procedure for the extraction of lipids from microalgae. J.
Am. Oil Chem. Soc., 89, 189-198.
[17] Shiel, R. J. (1995). A guide to identification of rotifers, cladocerans
and copepods from Australian inland waters. Albury: Co-operative
Research Centre for Freshwater Ecology Canberra.

66 | P a g e
 Plankton enumeration and biomass estimation

Appendix 4.1: Dry weight and ash-free dry weight analysis

Dry weight analysis begins with vacuum filtering of a 10 mL suspension of

plankton on a dry 0.45 m Whatman GF/C filter paper with a diameter of 47
mm that had been pre-dried in an oven at 105°C the day before. The filter
paper with the plankton suspension was then dried in the oven at 105°C for
an additional 24 hours. The initial weight of dry filter paper will be recorded,
as well as the weight of filter paper containing plankton suspension. To
eliminate salt from plankton (for marine species), wash the sample with 0.5M
ammonium formate (Hulatt et al., 2012). Calculate the dry weight as follows:

Dry weight, Wt (g/L) = (Wf – Wi)/V

Where;
Wf = dry filter weight after filtration (g)
Wi = dry filter weight before filtration (g)
V = volume filtered (L)

The technique was then continued to determine the amount of ash in the
dry plankton. Place the sample in a muffle furnace set to 540°C for 1 hour.
The sample is cooled in a desiccator and weighed to get the ash and ash-free
dry weight values (AFDW). The following is the calculation:

Total ash (%) = (Wa / Wt) x 100

AFDW (g/L) = (Wt – Wa) / V
Where;
Wt = actual dry weight (g/L)
Wa = ash weight after incineration (g)
V = volume filtered (L)
 Plankton enumeration and biomass estimation

Appendix 4.2: Chlorophyll-a analysis

Filter 250 mL of sample through a cellulose acetate filter membrane (0.45m).

To prevent pigment deterioration, samples should be kept out of direct
sunlight. In a grinder tube, place the filter paper. Add 2 to 3 mL of 90%
acetone and let the solution overnight at 4°C in the dark. Grind the filter paper
after 24 hours to remove the chlorophyll pigment from the filter paper.
Transfer the mixture to a centrifuge tube and dilute with 90 % acetone to
make the total volume 5.0 mL. Wrap the tube with tinfoil to keep it out of
direct light source. Centrifuge the mixture at 1000 g for 10 minutes. Transfer
the supernatant with care into a clean (non-acidic) spectrophotometer
cuvette. Using a spectrophotometer, determine the optical density of the
sample at 664 nm, 647 nm, and 630 nm. Calculate the chlorophyll-a
concentration as follows:

[(11.85 × 𝐴664 ) – (1.54 × 𝐴647 ) – (0.08 × 𝐴630 )] × 𝑉𝑎

Chlorophyll-a (mg/L) =
𝑉𝑠

Where;

Va = volume of acetone 90%

Vs = volume of sample
A664 = optical density value at 664 nm
A647 = optical density value at 647 nm
A630 = optical density value at 630 nm

68 | P a g e
 Plankton enumeration and biomass estimation

Appendix 4.3: Biochemical analysis

Total Protein

The protein is extracted using the Rausch technique (1981). Place a dried
sample (freeze-dried) of 50 mg in a 12 mL test tube. Following that, add 3 mL
of 0.5 N NaOH to the sample. Next, heat the mixture in a water bath at 80°C
for 10 minutes, stirring it occasionally. After the mixture has been heated,
cool it to ambient temperature and centrifuge for 5 minutes at 3000 g.
Transfer the supernatant into a 10 mL test tube. Repeat the second extraction
method using the same samples to ensure that most of the protein was
extracted. Combine the supernatant from the second extraction with the
supernatant from the first extraction and use it for protein quantification.
The Lowry assay (Lowry et al., 1951) is used to determine the protein
content using Bovine Serum Albumin (BSA) as a standard. The Lowry assay
utilises an interaction between cupric ions and protein to form a complex,
which then reduces the Folin-Ciocalteu reagent to produce a blue colour. The
protein concentration is calculated using the Lopez et al. (2010) formula,
which is detailed below.

𝐶𝑉𝐷
Protein (w/w %) = × 100
𝑚
Where;
C = Protein concentration (mg/L)
V = Volume of buffer used to resuspend the biomass (L)
D = dilution factor
m = amount of biomass (mg)

Total Carbohydrate

The carbohydrate content is extracted using the technique described by Chu

et al. (1996). Weigh 100 mg of dried sample and place it in a boiling tube.
After mixing the sample with 5 mL of 2.5N HCl, hydrolyze it for 3 hours in a
heater block at 100 °C. After three hours, the mixture is cooled to room
temperature and then neutralised with sodium carbonate until no longer
69 | P a g e
 Plankton enumeration and biomass estimation

effervescent. The volume is increased to 100 mL using distilled water. Finally,

centrifuge the mixture at 3000 g for 5 minutes and use the supernatant to
determine the total carbohydrate content of the sample.
The carbohydrate content is determined using the DuBois et al. (1956)
technique, which employs a phenol-sulphuric assay. In this analysis, glucose
is employed as a standard. The carbohydrate concentration is calculated
using the same procedure as for the protein content.

Total Lipid

Total lipid is determined using a modified version of Bligh and Dyer (1959)
technique by Ryckebosch et al. (2012). In this procedure, 100 mg of
lyophilized microalgae is added to a falcon tube followed by 9 mL of a mixture
of chloroform and methanol (1:2, v/v). Vortex the solution for 30 seconds.
This mixture, together with an additional 3 mL solvent mixture and 3 mL
water, are placed in an ultrasonic bath set to 25°C for 10 minutes, followed
by further vortexing of the test tubes. Centrifuge the test tubes for 10 minutes
at 3000 g to create a two-phase layer. Using a Pasteur pipette, remove the top
phase (water + methanol). The lower chloroform phase is transferred to a
transparent test tube containing the isolated lipids. The solid material left at
the bottom of the extraction tube is extracted twice more using the 6 mL
solvent mixture. Using Whatman no. 1 filter paper in a funnel, the mixed
solvent layers are passed through a layer of anhydrous sodium sulphate to
make sure all water is completely removed from the mixture. The filtrate is
then concentrated at 40°C in a rotary evaporator and dried using nitrogen
gas. The total lipid concentration was determined gravimetrically as follows:

𝐴−𝐵
Total crude lipid (%) = × 100
𝑊

Where;
A = Weight of tube with lipid extract (mg)
B = Weight of empty tube (mg)
W = Weight of sample (mg)

70 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5. PHYTOPLANKTON CULTIVATION
TECHNIQUES

OVERVIEW
The cultivation of phytoplankton was initially pioneered in the late 1800s
and has subsequently evolved. Cultivating phytoplankton is a lengthy and
laborious process that begins with sample collection (Section 3.0),
sterilization, medium preparation, isolation, culture maintenance, and
finally harvesting for application. Each of these procedures employs a
unique approach, which varies according to a variety of parameters,
including the species cultivated, the species habitat origin, and others.
This section will describe each of the procedures. With the progress of
technology, several new methods including a high-tech equipment have
been created, such as robotic automated flow cytometers that are able to
isolate hundreds of cells per minute, whereas old isolation methods
required days or months. However, this chapter will not go into greater
detail on the high-tech equipment because purchasing it would cost at
least $100,000 USD, which is a bit excessive for a scientific study or
commercial application.

5.1 STERILIZATION TECHNIQUES

Sterilization is the process of producing an aseptic environment, which

entails the elimination or destruction of all contaminants (Kawachi and Noel,
2005). Sterilization is critical in phycological research, particularly when live
organisms are maintained as isolated strains in culture. There are several
sterilizing procedures employed in phytoplankton cultivation; the majority
of which are determined by the size of the culture and the application of the
culture itself. The purpose of this section is to discuss several sterilizing
procedures widely employed in phycological research.
 Phytoplankton and Zooplankton Samples Collection

5.1.1 Heat sterilization

Heat sterilisation is the most often used sterilisation procedure, and it

generally necessitates high temperatures (100°C and above), suggesting that
the materials to be sterilised can withstand high temperatures (e.g.,
glassware, metallic instruments, heat resistance plastic, and aluminium foil).
This technique includes:

a) Flame
⟡ Method: Expose the items immediately to the flame (Bunsen
burner, alcohol burner) for a few seconds (Figure 5.1).
⟡ Application: Surface sterilization (glassware openings, transfer
loops).
⟡ Limitation: Can’t be use for non-heat resistant materials, some
spores can still survive.

Figure. 5.1 Flame technique

b) Autoclaving
⟡ Method: Set the autoclave machine (Figure 5.2) to 2 atm (steam
pressure) and 121°C for 10 to 20 minutes.
⟡ Application: Liquids, agar, glassware, heat-resistance plastic.
⟡ Limitation: Cannot be used for non-heat resistant materials, pH
can sometimes change.

72 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Figure. 5.2 Autoclave machine

c) Dry heat
⟡ Method: Heat the items in the oven at 150 - 250°C for 3 to 5 hours.
⟡ Application: Glassware.
⟡ Limitation: Cannot be used with heat-sensitive goods or liquids.

d) Pasteurization
⟡ Method: Heat the materials to a temperature of 66–80°C for at
least 30 minutes, followed by rapid cooling (4–10°C).
⟡ Application: Liquid containing heat-labile constituents.
⟡ Limitation: Certain contaminants may survive.

5.1.2 Filtration sterilization

Filtration sterilisation (Figure 5.3) is often used for heat-labile components

such as vitamins and antibiotics, as well as volatile liquid components such
as organic solvents. When only a small amount of liquid needs to be sterilised,
this technique is also utilised for ease and speed. A number of commercial
filters are available, but the pore size should be smaller than 0.2 mm.
Autoclavable membrane filters are commonly used to sterilise culture media.
This method is only appropriate for small volume liquids; and tiny
microorganisms, such as viruses, may pass through the filter.

73 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Figure 5.3 Filtration method

5.1.3 Electromagnetic waves sterilization

Electromagnetic waves are usually employed in place of heat-sensitive

materials. This procedure is generally faster than any other technique. The
microwave and UV radiation are the two most commonly used methods for
this procedure.

a) Microwave
⟡ Method: Heat the items in microwave for 10 to 20 minutes
⟡ Application: Small volume of media (liquid and agar), glassware,
⟡ Limitation: Cannot be used for non-microwave resistant
materials, only applicable for small volume

b) UV radiation
⟡ Method: Expose the items to UV light for 5 to 10 minutes
⟡ Application: Surface of materials and working bench
⟡ Limitation: Bad for skin and eyes, only work on surface

5.1.4 Chemical sterilization

There are several types of chemical sterilization methods available, and most
of them are extremely hazardous for laboratory usage. As a result, we will
only describe two of them in this section: bleach (sodium hypochlorite) and
ethanol.

74 | P a g e
 Phytoplankton and Zooplankton Samples Collection

a) Bleach (sodium hypochlorite)

⟡ Method: Add 1 – 5 mL of sodium hypochlorite to 1 L of water and
wait for several hours; leave overnight for large volume (500 L
and above). After the bleach treatment is finished, the solution is
neutralized with sodium thiosulfate (if 250 g of sodium
thiosulfate are dissolved in 1 L of water, then 1 mL of the sodium
thiosulfate solution is added for each 4 mL of bleach used)
⟡ Application: Mostly used for large volumes of water in
aquaculture industry
⟡ Limitation: Not applicable to non-heat resistant materials; some
spores can still survive

b) Ethanol (50 – 70% solution)

⟡ Method: Use as general disinfection. Put in water sprayer and
spray any surface that needs disinfection.
⟡ Application: Surface of materials and working bench
⟡ Limitation: Some resistant microorganism may survive

5.2 PREPARATION OF CULTURE MEDIA

Choosing and preparing culture media is essential for optimizing

phytoplankton growth. Different phytoplankton, particularly those
belonging to different species groups, need specific media because certain
species require chemical compositions that are compatible with their
morphology and physiology. For instance, silicate is required for diatom
growth to produce their external shell (Lavens and Sorgeloos, 1996).
Additionally, nutritional value of phytoplankton can be increased by
changing the culture media compositions, particularly their nutrient content
(e.g., nitrate, phosphate, and silicate). In general, media are made of three
components: macronutrients, trace elements, and vitamins; all these three
components are frequently produced as stock solutions (Watanabe, 2005).
Stock solutions ranging in sizes of 100 mL to 1 L are generally produced at
100–1,000 times the needed nutrient concentration. To use, a portion (e.g., 1
mL) is aseptically extracted and utilised. Stock solutions are advantageous
for a variety of reasons. Individual weighing of chemicals is time intensive
and may result in weighing mistakes. The stock solution is prepared on a
75 | P a g e
 Phytoplankton and Zooplankton Samples Collection

sporadic basis, but once prepared, it provides a convenient and constant

source. In this section, we will discuss a variety of culture medium that are
widely used to cultivate phytoplankton species from both freshwater and
marine environments.

5.2.1 Types of culture media

Culture media can be either in the form of liquid or solid (agar). Either way,
both forms of media use a similar concentration of media stock solution
(Section 5.2.2); and the solid form can be achieved by adding 1 – 2 % of agar
powder in it. Both types of culture media are generally prepared as follows:

a) Liquid media (freshwater)

I. Fill a beaker with 80-90 percent of the needed amount of
distilled or deionized water.
II. Add the necessary amount of nutrients from the prepared
stock solutions (Section 5.2.2) one at a time, stirring
continuously.
III. Dilute to final volume with distilled water.
IV. If required, adjust the pH with 0.1 N HCl (hydrochloric acid)
or 0.1 N NaOH (sodium hydroxide).
V. Pour the medium into culture containers and sterilize with
any suitable technique (Section 5.1).
VI. If autoclaved, cool the medium and let it stand for 24 hours
after autoclaving to allow for re-equilibration of inorganic
carbon species (Watanabe, 2005).

b) Liquid media (marine)

I. Collect high-quality natural seawater with a salinity more
than 30 ppt.
II. Filter the seawater as quickly as possible and keep it in the
dark and cold (4 °C)
III. If using artificial seawater, please follow the manufacturer's
preparation instructions, since various types of artificial
seawater may require a different method of preparation.
IV. Sterilize using an appropriate method (Section 5.1).
V. Follow the procedures outlined in Section 5.2.1 (a).

76 | P a g e
 Phytoplankton and Zooplankton Samples Collection

c) Agar media (freshwater and marine)

I. Prepare the liquid media (Section 5.2.1 (a) or Section 5.2.1
(b))
II. Heat a 2 L Erlenmeyer flask containing 1 L of culture media
on a hotplate.
III. Slowly add 1-2 % agar (if using 1L culture medium, use 10–
20 g of agar powder) while stirring constantly to ensure that
all agars are spread equally.
IV. To prepare agar plates in petri dishes, autoclave the nutrified
agar. Pour the mixture onto sterile petri dishes once the
temperature reaches 50°C–60°C. Condensation will occur if
the temperature of the mixture in the petri dishes is too high.
To get around this, stack the petri dishes and place a flask
filled with hot tap water on top of the stacking plates. It will
reduce the condensation process. Flip the plates (agar up)
and store in airtight containers (e.g., plastic bags, covered
containers) at 4°C after the agar has completely gelled.
V. To produce agar slants in test tubes (Figure 5.4), pour the
nutrified agar into test tubes and autoclave. Remove the test
tubes from the autoclave and allow them to cool and shape at
the appropriate angle (e.g., 45°).

Figure 5.4 Agar slant

77 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.2.2 Recipes for freshwater and marine media

a) Bold’s Basal Media (BBM) (Bold, 1949; Bischoff and Bold, 1963)
This media was developed from a modified version of Bristol's solution
(Bold, 1949) and is a commonly used medium for any freshwater
phytoplankton. To prepare, add 10 mL of each of the preceding six stock
solutions to 900 mL of distilled/deionized water. Add 1 mL of each of the
following solutions: alkaline EDTA, acidified iron, boron, and trace
metals. Make up to 1 L with distilled water. Autoclave and the final pH
should be 6.6.

No. Component Stock solution Quantity used

(g/L dH2O)

Macronutrients
1. NaNO3 25.00 10 mL
2. CaCl2 · 2H2O 2.50 10 mL
3. MgSO4 · 7H2O 7.50 10 mL
4. K2HPO4 7.50 10 mL
5. KH2PO4 17.50 10 mL
6. NaCl 2.50 10 mL

7. Alkaline EDTA Solution 1 mL

EDTA 50.00
KOH 31.00

8. Acidified Iron Solution 1 mL

FeSO4 · 7H2O 4.98
H2SO4 1 mL

9. Boron Solution 1 mL
H3BO3 11.42

10. Trace Metals Solution 1 mL

ZnSO4 · 7H2O 8.82
MnCl2 · 4H2O 1.44
MoO3 0.71
CuSO4 · 5H2O 1.57
78 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Co(NO3)2 · 6H2O 0.49

b) BG-11 Media (Stanier et al., 1971)

This medium was developed for the use of both freshwater and marine
blue-green phytoplankton. To prepare, add 10 mL of each of the
preceding eight stock solutions to 900 mL of distilled water and
subsequently add 1 mL of trace metal solution. Make up to 1 L with
distilled water. Adjust pH to 7.1 with 0.1N NaOH or HCl and autoclave.
For marine species, replace distilled water with seawater and add 1.0 mg
vitamin B12 and 10.0 g NaCl per liter of medium. Make sure to add sterile
vitamin solution aseptically at last after sterilization.

No. Component Stock solution Quantity used

(g/L dH2O)

Macronutrients
1. NaNO3 150.00 10 mL
2. CaCl2 · 2H2O 3.60 10 mL
3. MgSO4 · 7H2O 7.50 10 mL
4. K2HPO4 4.00 10 mL
5. Citric acid 0.60 10 mL
6. Ferric ammonium citrate 0.60 10 mL
7. EDTANa2 0.10 10 mL
8. Na2CO3 2.00 10 mL

9. Trace Metals Solution 1 mL

ZnSO4 · 7H2O 0.22
MnCl2 · 4H2O 1.81
H3BO3 2.86
CuSO4 · 5H2O 0.08
Co(NO3)2 · 6H2O 0.05
Na2MoO4 · 2H2O 0.39

79 | P a g e
 Phytoplankton and Zooplankton Samples Collection

c) Diatom Media (https://www.ccap.ac.uk/index.php/media-recipes)

This media was created for the use of freshwater diatom phytoplankton.
To prepare, add 1 mL of each of the eight stock solutions to 900 mL of
distilled water and subsequently make up to 1 L with distilled water.
Adjust pH to 6.9 with 0.1N NaOH or HCl and autoclave.

No. Component Stock solution Quantity used

(g/L dH2O)

Macronutrients
1. Ca(NO3)2.4H2O 20.00 1 mL
2. KH2PO4 12.40 1 mL
3. MgSO4 · 7H2O 25.00 1 mL
4. NaHCO3 15.90 1 mL

5. Alkaline EDTA Solution 1 mL

EDTANa2 2.25
EDTAFeNa 2.25

6. Trace Metals Solution 1 mL

H3BO3 2.48
MnCl2 · 4H2O 1.40
(NH4)6Mo7O24.4H2O 1.00

7. Vitamin solution 1 mL
Cyanocobalamin 0.04
Thiamine HCl 0.04
Biotin 0.04

8. Na2SiO3.9H20 57.00 1 mL

80 | P a g e
 Phytoplankton and Zooplankton Samples Collection

d) Walne’s Media (Walne, 1970)

This media was developed for the use of marine phytoplankton especially
for mass cultivation. To prepare, add 1 mL of nutrient solutions to 900
mL of filtered seawater and subsequently add 100 µL of vitamins
solution. Sodium metasilicate (40 mg/L) should be added for the growth
of diatoms. Bring the final volume to 1 L and autoclave.

No. Component Stock solution Quantity used

(g/L dH2O)

1. Nutrient solution 1 mL
NaNO3 100.00
H3B03 33.6
EDTANa2 45.00
NaH2PO4. H2O 20.00
FeCl3. 6H2O 1.30
MnCl2. 4H2O 0.36
Trace Metals Solution * 1mL

2. Vitamin solution 100 µL

Cyanocobalamin 0.05
Thiamine HCl 1.00

* Trace Metals Solution

ZnCl2 21.00
CoCl2. 6H2O 20.00
(NH4)6Mo7O24.4H2O 9.00
CuS04. 5H20 20.00

81 | P a g e
 Phytoplankton and Zooplankton Samples Collection

e) F/2 Media (Guillard, 1975)

This is a common and widely used general enriched seawater medium
designed for growing coastal marine algae, especially diatoms. To
prepare, add 1 mL of each of the four stock solutions to 900 mL of filtered
seawater and subsequently add 0.5 mL of vitamins solution. Bring the
final volume to 1 L and autoclave. If silicate is not required, omit to reduce
precipitation.

No. Component Stock solution Quantity used

(g/L dH2O)

1. NaNO3 75.00 1 mL
2. NaH2PO4. H2O 5.00 1 mL
3. Na2SiO3.9H20 30.00 1 mL
4. Trace Metals Solution * - 1 mL
5. Vitamin solution * - 0.5 mL

* Trace Metals Solution

FeCl3. 6H2O - 3.15 g
Na2EDTA · 2H2O - 4.36 g
CoCl2. 6H2O 10.00 1 mL
CuS04. 5H20 9.80 1 mL
MnCl2. 4H2O 180.00 1 mL
ZnSO4 · 7H2O 22.00 1 mL
Na2MoO4 · 2H2O 6.30 1 mL

* Vitamin solution
Cyanocobalamin 1.00 1 mL
Thiamine HCl - 200 mg
Biotin 1.00 1 mL

82 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.3 ISOLATION TECHNIQUES

The goal of isolation is to eradicate foreign organisms such as alien algae,

protozoans, bacteria, fungi, and zooplankton. According to Andersen and
Kawachi (2005), an effective isolation procedure may be broken down into
three phases. The first stage is to comprehend and imitate naturally existing
environmental conditions. Temperature and salinity are critical for coastal
marine algae, whereas water quality and metal toxicity are additional issues
for open ocean phytoplankton. Freshwater algae on the other hand, are
usually less temperature sensitive and have a noticeable effect on pH. The
second stage toward effective isolation is the removal of contaminants,
particularly those that are capable of outcompeting the target species. The
most often used techniques include serial dilution, single-cell isolation using
a micropipette, and agar streaking. All of these techniques will be discussed
further in this section. The last stage needs ongoing development after
subculturing, and as the culture continues to grow healthily, it is a positive
indicator that the growth conditions, including culture medium and culture
parameters, are in satisfactory quality.

5.3.1 Serial dilution

For many years, the dilution approach has been employed; it is useful for
organisms that are relatively abundant in the sample but not for rare species.
The purpose of the dilution approach is to deposit only one cell into a test
tube, flask, or multiwell plate, resulting in the establishment of a single cell
isolate at least at the last of the used containers. The procedures for this
approach are as follows:
a) Prepare 5-10 sterilized 15 mL test tubes (can be replaced with other
container, e.g., flask, multiwell plate)
b) Using a burner, heat the neck and mouth of the test tubes.
c) Next, fill one test tube with 9 mL liquid media and the other with 10
mL liquid media.
d) Transfer 1 mL of sample (or less if the sample is dense) to a test t ube
containing 10 mL medium (Figure 5.5a). Shake the test tube
vigorously.

83 | P a g e
 Phytoplankton and Zooplankton Samples Collection

e) Transfer 1 mL of the preceding test tube's mixture to the second test

tube (containing 10 mL media). Repeat with additional test tubes
(containing 10 mL media).
f) Then, using the last test tube (which contains 10 mL medium),
transfer 1 mL of the combination to the 9 mL test tube. Shake
vigorously (Figure 5.5b).
g) Incubate the culture for a period of 2-6 weeks under suitable culture
conditions (e.g., temperature: 25 ± 1°C; light intensity: 50 mol photons
m2 s1; 12:12-h light/dark cycle) (Figure 5.5c).
h) Next, examine the algae grown in the final 2-4 test tubes (Figure 5.5d).
It's because the chances of finding a single species in the last few test
tubes are higher than earlier tubes.

(a) (b)

(c) (d)

Figure 5.5 Serial dilution method

84 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.3.2 Agar streaking

Streaking approach for isolating coccoid phytoplankton is favoured since it is

simple to apply and extremely efficient. To isolate phytoplankton
successfully using this approach, targeted strains must be able to grow on
agar. Certain flagellates (e.g., Pelagomonas, and Peridinium) do not grow well
on agar, as contrast to others (e.g., Chlamydomonas, Pavlova, and
Tetraselmis). Coccoid cells and most of diatoms may also thrive on agar.
Bear in mind that agar is also an excellent medium for the growth of fungal
and bacterial cells. Field samples contaminated with fungus can be difficult
since fungus develops fast, generating sporangia and spores that
compromise efforts to separate the phytoplankton. To eliminate filamentous
fungus, filters and organic substrates might be employed. On the other hand,
bacteria often generate tiny, isolated colonies, and unialgal cultures can be
obtained if the plate is streaked appropriately. Isolation on agar can be
performed by streaking the collected sample across the agar surface in the
same manner that bacteria are isolated. The procedures for this approach are
as follows:
a) Prepare the agar plate and the obtained sample.
b) Flame the bacterial loop until it becomes red (Figure 5.6a), then allow
it to cool for a few seconds. After that, load a tiny amount of sample
into the bacterial loop (Figure 5.6b) and distribute it across the agar
(Figure 5.6c) with one of the streaking patterns (Figure 5.7). Single
cells begin to detach as the distance from the streak's beginning point
expands.
c) Following streaking, the agar plate is incubated under suitable culture
conditions (e.g., temperature: 25 ± 1°C; light intensity: 50 mol photons
m2s1; 12:12-h light/dark cycle) until cell colonies form. The incubation
time varies from a few days for soil and freshwater algae up to several
months for oceanic species.
d) Once the phytoplankton have formed a colony, move it to liquid
medium or streak it to another agar plate using a bacterial loop
(Figure 5.6d).
e) Allow the phytoplankton to grow for at least two weeks before
confirming the culture under a microscope.

85 | P a g e
 Phytoplankton and Zooplankton Samples Collection

(a) (b)

(c) (d)

Figure 5.6 Agar streaking technique

Figure 5.7 Agar streaking patterns

86 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.3.3 Micropipette isolation

Single-cell separation with a micropipette is a very rapid approach for

phytoplankton isolation, possibly the quickest of the techniques discussed in
this chapter. Nonetheless, this technique requires a significant amount of
effort for beginners before achieving dependable output. The purpose of
micropipette isolation is to pick up a cell from a sample, deposit it into a
sterile droplet without damaging it, then pick up the cell again and transfer it
to a second sterile droplet. This procedure is continued until a single algal
cell is securely placed into growth media, free of all other contaminants. An
Inverted Microscope is required for viewing and isolating cells using this
technique. The target species sample can be put on a glass or plastic dish, a
multiwell plate, a microscope slide, or similar containers. Before isolation
begins, sterile droplets (a tiny quantity of culture medium) should be
prepared. The following procedure demonstrates how to make a
micropipette from a Pasteur pipette:
a) The Pasteur pipette is held in the flame's hottest zone, with a hand
on the left and forceps on the right (Figure 5.8a). While the glass is
being heated to a soft, flexible state, the pipette should be turned.
b) While the glass is still soft, the pipette is gently pulled away from the
flame to form a narrow tube (Figure 5.8b).
c) The forceps are then repositioned in the proper location on the thin
tube.
d) Using the forceps, carefully bend the thin region until it fractures
(Figure 5.8c), creating a micropipette (Figure 5.8d).

e) Insert a cotton swab into the end of the micropipette and sterilize
using the appropriate method.

After preparing the micropipette, the following steps for phytoplankton

isolation can be followed (Parvin et al. 2007):
a) Discard the cotton and replace it with a soft rubber tube.
b) On a glass slide (or other containers), place one drop of culture
medium (droplet). Repeat the procedure for the remaining nine glass
slides (or other containers).

87 | P a g e
 Phytoplankton and Zooplankton Samples Collection

c) Following that, a drop of phytoplankton sample will be introduced and

observed under an inverted microscope.
d) Once the target phytoplankton species has been verified, one drop of
the sample will be transferred to the subsequent droplet.
e) Repeat the procedures until the target species is free of
contamination.
f) Transfer the droplet containing the target phytoplankton to a sterile
test tube containing culture media or to an agar plate and cultivate
under appropriate culture conditions.
g) Allow at least two weeks for the phytoplankton to grow before
verifying the culture under a microscope.

(a) (b)

(c) (d)

Figure 5.8 Preparation of a micropipette from a Pasteur pipette

88 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.4 PURIFICATION TECHNIQUES

There are a few instances where fully pure phytoplankton is necessary (e.g.,
for molecular studies, medicine, and cosmetic usage), and therefore the
isolated phytoplankton must undergo purification. Purification techniques
are designed to produce a viable culture of a particular species that is free of
contaminants. If there were no identifiable impurities in cultures, they were
referred to be pure cultures or axenic cultures. There are several techniques
for producing an axenic culture. We infer from all of the techniques used,
there are two main processes involved: mechanical separation and antibiotic
purification.

5.4.1 Mechanical separation

Mechanical separation is a critical step in increasing the likelihood of

antibiotic treatment effectiveness. Our preferred method of mechanical
separation is a mix of vortexing and centrifugation. Vortexing is used to
separate impurities adhering to isolated cells, while centrifugation aids in the
concentration of contaminants and separated cells into distinct groups by
gravity separation. The contaminants in the supernatant can then be
removed and replaced with new sterile culture medium. Both techniques
must be done two or three times to ensure that most of the contaminants are
eliminated. We apply the following approach for mechanical separation:
a) Vortex the isolated cells for 30 seconds at 2000 × g to detach the
contaminants from the cells.
b) Centrifuge the sample for 5 minutes at 6000 × g to separate the
contaminants and sample by gravity.
c) The produced supernatant is discarded and replaced with new sterile
media.
d) Repeat steps (a) through (c) three times to verify that most of the
contaminants are eliminated.
e) Proceed with the antibiotic treatment.

89 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.4.2 Antibiotic treatment

Antibiotic treatment is theoretically effective in eliminating bacteria without

harming phytoplankton cells, but in practise, it is not as simple as it seems. A
good understanding of which antibiotics to use, at what concentrations, and
for how long the treatment is performed is required for a successful
antibiotic treatment. Furthermore, if one antibiotic combination fails to
eliminate all contaminated bacteria, a subsequent series of antibiotic mixture
or concentration may be necessary. The following are the antibiotic
treatment procedures we use to effectively purify our marine species:
a) After mechanical separation, grow the isolated cells in sterile media
with the mixture of penicillin G., dihydrostreptomycinsulphate and
gentamycin sulphate with a ratio of 4:1:1.
b) Incubate the culture for 18 to 72 hours.
c) Streak the culture on agar plate to observe any bacterial growth (agar
culture is the easiest way to determine the presence of bacteria or
other contaminants).
d) The treatment is considered successful if no bacteria or other
contaminants are discovered after one week of culture.
e) If bacteria and other contaminants persist, try a different antibiotic
combination.

Figure 5.9 Axenic culture of Isochrysis maritima

90 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.5 CULTURE MAINTENANCE

Culture maintenance is classified into two approaches: short-term and long-

term. Short-term maintenance is often used when the culture is required for
immediate use, such as aquaculture feed, cosmetics, or pharmaceuticals,
whereas long-term maintenance is typically used for culture collection. Both
approaches will be discussed further in the next section.

5.5.1 Short-term maintenance

The most frequent method of maintaining phytoplankton cultures in the

short term is perpetual maintenance under controlled conditions. Serial
subculturing is one of the common procedures that includes moving an
inoculum from a late stationary phase culture to new, sterilized media,
resulting in metabolically active cultures that may be utilized immediately.
The goal of this method is to maintain a population that is healthy,
physiologically, morphologically, and genetically representative (Lorenz et
al. 2005). It is important to consider that various ages of subcultures may
contain distinct phases of life cycle, making it critical to have a firm grasp on
the phytoplankton growth stages. The procedure of subculturing is as follow
(Figure 5.10):
a) Make sure the workplace is aseptically clean.
b) Prepare a sterilized 250 mL flask with 100 mL of sterilized media in it
(can be replaced with other bigger container with bigger volume of
media). Make sure the flask is covered with cotton wool or aluminium
foil (Figure 5.10a).
c) Using a burner, heat the neck and mouth of the mother culture (the
original culture which need to be maintained) (Figure 5.10b).
d) Next, transfer 10 mL of phytoplankton sample from the mother
culture to the 250 mL flask with 100 mL of sterilized media (the final
concentration should be 10% of the inoculum; if using 1L sterilized
media, transfer 100 mL of phytoplankton sample) (Figure 5.10c).
e) Shake well and label the flask accordingly.

91 | P a g e
 Phytoplankton and Zooplankton Samples Collection

f) Incubate the culture under suitable culture conditions (e.g.,

temperature: 25 ± 1°C; light intensity: 50 mol photons m2 s1; 12:12-h
light/dark cycle) (Figure 5.10d).
g) Repeat this procedure weekly, biweekly, or monthly depending on
demand and growth.

(a) (b)

(c) (d)

Figure 5.10 Subculturing procedures

5.5.2 Long-term maintenance

Since we have our own algal culture collection, long-term maintenance is

crucial in order to maintain all of our cultures alive without the need to
subculture every week. We always utilise three techniques: streaking on agar
slant, alginate beads, and liquid nitrogen cryopreservation. Each of these
methods will be explored in further detail in the following section.

a) Streaking on agar slant

Generally, streaking phytoplankton on agar slant can sustain it for a longer

period of time than streaking on agar plate. The agar is distributed vertically

92 | P a g e
 Phytoplankton and Zooplankton Samples Collection

inside the plate, creating additional surface area for phytoplankton to grow
on. However, because the agar is distributed vertically, the thickness of the
agar containing all important nutrients is much thinner, and the growing
phytoplankton uses up all nutrients in a short period of time. Agar slant, on
the other hand, is usually prepared in a long glass tube (Figure 5.4), with the
agar media distributed horizontally, requiring longer time for phytoplankton
to consume all the nutrients. As a result, the phytoplankton will survive
longer than if they were grown in an agar plate. According to our experience,
phytoplankton can survive for up to six to nine months after streaking on
agar slant. Prepare agar slant according to the techniques outlined in Section
5.2.1 (c) whereas streaking can be applied in a manner similar to that
described in Section 5.3.2.

b) Alginate beads
Most researchers believe that phytoplankton preserved in alginate beads
may persist more than one year (Faafeng et al. 1994; Chen, 2001). Our
experience has shown that it varies according on the species, but on average
it can last between five and eight months. Preparing alginate beads is a
straightforward process that requires only three ingredients: sodium
alginate, calcium chloride, and glycerol. The steps are as follows:
I. Prepare the following materials accordingly.

Materials Volume used (mL)

4 % of sodium alginate solution 5
2 % of calcium chloride solution 200
Phytoplankton sample (same volume as sodium
5
alginate solution)
Pour until all beads
10% of glycerol
suspended

II. The concentration of the sodium alginate solution may be varied from
2 – 5% depending on your objective & algae species.
III. Wash the phytoplankton culture to remove salts (which may
interfere with the gelatinization process of the alginate). Suspend in
sterile water.

93 | P a g e
 Phytoplankton and Zooplankton Samples Collection

IV. Mix the microalgae suspension with an equal volume of sodium

alginate solution, hence, in this case, the final alginate concentration
is 2% after mixing.
V. Mix well & drop the mixture slowly using a micropipette or syringe
into a solution of calcium chloride 2%. Control the drop rate to obtain
the desired bead shape and size.
VI. Collect the beads and store in a glass bottle or glass plate. Fill the
bottle or plate with 10% glycerol until all beads are suspended It is
preferable to include some glass beads to facilitate easy removal of
the alginate beads.
VII. Store in a freezer.
VIII. Remove the beads and immerse them in sterile culture medium for a
few days to recover them. The beads will disintegrate, but the
phytoplankton will continue to grow.

(a) (b)

Figure 5.11 (a) Phytoplankton in alginate beads; (b) Beads suspended

in glycerol solution

c) Cryopreservation
Cryopreservation is the storing of a live organism at an ultralow
temperature (usually colder than -130°C) such that it can be thawed and
survive. Most algae do not survive cryopreservation unless the
circumstances under which they are frozen and thawed are controlled.
Cryopreservation methods are meant to prevent intracellular ice
formation as well as the consequences of excessive osmotically induced
changes in cell volume, since both can cause permanent physical and/or
94 | P a g e
 Phytoplankton and Zooplankton Samples Collection

chemical damage (Steponkus et al. 1992). Only when the rate of

temperature change is controlled, and an adequate CPA (cryoprotective
agents) is given to the algal suspension prior to freezing can algae survive
freezing and thawing. CPA are water-soluble chemicals that, when given
to an algal suspension before freezing, improve its survivability after
thawing (Day and Brand, 2005). The following is the simple method to
preserve our phytoplankton using cryopreservation technique. Please
adhere to the correct procedures for handling liquid nitrogen to avoid
personal injury.
I. Refrigerate a freezing container (used to store 2mL cryogenic
vials) overnight to equilibrate at about 4°C.
II. Dilute MeOH (methanol) to 20% (v/v) in the culture media used
to grow the phytoplankton. If cryopreservation of axenic strains
is required, sterilize the 20% methanol in culture medium by
passing it through a 0.45mm filter.
III. Fill a prelabelled 2mL cryogenic vial with the previously
produced 20% MeOH solution (0.45 mL for vials referenced
above).
IV. Transfer the culture of algae (phytoplankton) to the cryogenic
vial (3 volumes of culture to 1 volume of 20 percent MeOH; e.g.,
1.35 mL of culture to 0.45 mL of MeOH).
V. Immediately secure the vial cover and gently invert the cryogenic
vial several times to evenly distribute the MeOH. Keep the vial
away from light.
VI. Transfer the cryogenic vial to a precooled freezing container and
place the container immediately in a -80°C freezer. Allow 1.5
hours for the container to cool to a temperature of less than -50°C.
VII. Remove the freezing container from the -80°C freezer and the
liquid nitrogen storage container from the permanent storage
box. Transfer the cryogenic vial to the storage box as quickly as
possible and place the storage box in the liquid nitrogen storage.
The cryogenic vial may then be stored in the box forever without
being disturbed.

95 | P a g e
 Phytoplankton and Zooplankton Samples Collection

(a) (b)

Figure 5.12 (a) Cryogenic vials; (b) Liquid nitrogen storage

For thawing and revival of the sample:

I. Preheat a water bath at 35°C and place a floating vial-holder into the
water bath.
II. Remove the storage box from the liquid nitrogen storage and quickly
transfer the frozen cryogenic vial from the box to the floating holder
in the water bath. Agitate the cryogenic vial by rotating the holder
irregularly to enhance heat transmission.
III. Remove the cryogenic vial from the water bath once all ice in the vial
has melted but before the algal culture hits a temperature of 20°C
(typically less than 3 minutes). The culture may be left undisturbed
in darkness at ambient temperature for a brief period (no more than
1 hour) to provide time for algae that settle fast to settle at the bottom
of the vial. Remove the cryogenic vial's cover gently to prevent
disturbing the settled algae, then carefully remove and discard the
liquid from above the settled algae. Transfer the algae to a new media
for culture. The amount of new medium should be sufficient to
decrease the MeOH content to less than 0.2 percent (v/v). If the
culture does not settle fast, the cryogenic vial may be gently
centrifuged to pellet the culture. Remove the supernatant and
resuspend the pellet in fresh culture media.
IV. The culture should be left in the dark for several hours, ideally
overnight, or in dim light (normal room light is typically okay, but not
close proximity to a source of artificial illumination or an exposed
window to bright outside light). After that, return the culture to
regular culture conditions and anticipate viable cell development to
resume in 1 to 2 days.

96 | P a g e
 Phytoplankton and Zooplankton Samples Collection

5.6 MASS CULTIVATION

Smaller culture containers, in general, provide more control over the culture
environment and algal yields. Upscaling from tiny flasks to big tanks or even
outdoor ponds is impossible without modifying the culture, sacrificing
control, compromising nutritional balance, and reducing yield per unit
volume. Algal densities are often lower on a mass scale, but what matters
most is the number of algal cells per unit volume. Since smaller vessels are
easier to handle, using five 100 L tanks rather than one 500 L tank is typically
preferable.
Batch culture system is the most frequently used culture system owing to
its simplicity and low cost (Barsanti and Gualtieri, 2006). A basic batch
culture system entails the addition of a small volume of complete culture
media and algal inoculum to a culture vessel and incubation at specific
culture conditions for growth. Under optimum parameters, the algal culture
will grow rapidly until the rate of cell division begins to slow, marking the
end of the exponential phase and the beginning of the stationary phase. At
that time, the culture needs to be harvested entirely (Lavens and Sorgeloos,
1996), and the cleaned container need to be replaced with sterilized and
enriched media and subsequently inoculated to start a new culture. Culture
containers can be as basic as a conical flask or as sophisticated as an
environment-controlled fermenter (Richmond 2004). The following is a
simple way for mass producing phytoplankton:
I. Grow the phytoplankton in a 250 mL culture vessel using the
procedure described in Section 5.5.1.
II. When the culture has reached the late exponential stage, transfer it
to a 2 L culture vessel using 10% of the inoculum rule (if using 1L
sterilized media, transfer 100 mL of phytoplankton sample).
III. Repeat the procedure in a 20 L culture vessel before moving on to a
100L culture vessel. The procedure will be repeated until the desired
number of cells was obtained. However, keep in mind that the larger
the culture vessel, the more difficult it is to manage the culture state
which may lead to sudden crash.
IV. Once the desired number of cells is obtained, harvest them at an early
stationary stage using any technique mentioned in the next section.

97 | P a g e
 Phytoplankton and Zooplankton Samples Collection

Figure 5.13 Cultivation of I. maritima through batch culture system; (A)

100 mL (B) 2 L (C) 18 L (D) 1000 L

5.7 HARVESTING TECHNIQUES

In algal cultivation, the harvesting procedure refers to the separation of algal

cells from the culture media prior to further processing of the cells for any
purpose. There are several ways the cells can be harvested; but we employ
just three: centrifugation, flocculation, and filtration. These techniques will
be described in further detail in the next section.

5.7.1 Centrifugation

Centrifugation is a technique that utilizes centrifugal force to aid in the

separation of cells and liquid media. This technique of harvesting is
frequently utilized in the beverage, food, and pharmaceutical sectors, where
chemical addition is prohibited. While this is a simple approach, its efficiency
is relatively poor in algal cultivation. The efficiency rate can be increased by
subjecting the culture to higher gravity or treating it for a longer period of

98 | P a g e
 Phytoplankton and Zooplankton Samples Collection

time; however, these treatments may result in cell death. We usually employ
centrifugation speed of 3000 to 4000 g for 5 to 10 minutes.

(a) (b)

(c) (d)

Figure 5.14 Centrifugation technique; a) Place algal sample in centrifuge

tubes; b) Place the tubes inside the container and make sure all containers
are filled with tubes; c) Set up the speed and gravity; d) Discard the
supernatant and collect the paste.

5.7.2 Flocculation

The flocculation technique is the most promising for large-scale application.

This technique operates by concentrating the cells (coagulation) and settling
them to the bottom of the cultivating equipment owing to the increased
density of the concentrate. To flocculate the algal cells, cationic, anionic, and
non-ionic polyelectrolytes are often employed. The flocculation process
using these chemicals operates at low pH levels to produce cationic
hydrolysis products (Al Hattab et al. 2015). Numerous chemicals have been
used for flocculation purposes, including ferric sulphate, ferric chloride,
aluminium chloride, and aluminium sulphate, with the most successful being
aluminium sulphate (Oh et al. 2001). The concentration of flocculant

99 | P a g e
 Phytoplankton and Zooplankton Samples Collection

employed has a significant effect on the effectiveness of algal recovery, and

the optimal amount of aluminium sulphate is 50 mg/L.

(a) (b)

Figure 5.15 Flocculation technique; a) Add the aluminium sulphate into the
culture vessel at a concentration of 50 mg/L; b) Turn off the aerator and let
the algae to suspend naturally. Remove the supernatant. Note: if the algal
suspension remains too liquid, proceed with the centrifugation method.

5.7.3 Filtration

The filtration process employs a permeable material capable of retaining the

algal biomass while allowing the liquid to pass through. This method requires
a differential pressure across the filter, which can be generated by vacuum,
pressure, or gravity. The harvesting effectiveness of filtering techniques
varies between 20% and 90%. While the procedures vary depending on the
filtration medium used, one thing that all methods have in common is that
the algal culture must pass through the filtration medium.

Figure 5.16 Filtration using algal harvester

100 | P a g e
 Phytoplankton and Zooplankton Samples Collection

References
[1] Al Hattab, M., Ghaly, A. and Hammouda, A. (2015). Microalgae
Harvesting Methods for Industrial Production of Biodiesel: Critical
Review and Comparative Analysis. J Fundam Renewable Energy Appl,
5, 154.
[2] Andersen, R.A. and Kawachi, M. (2005). Traditional Microalgae
Isolation Techniques. In (Anderson, R.A. eds.) Algae Culturing
Techniques. Elsevier Academic Press, USA. pp. 83 – 100.
[3] Barsanti, L. & Gualtieri, P. (2006). Algae: anatomy, biochemistry and
biotechnology. Taylor and Francis Group. LLC, 301 pp.
[4] Bischoff, H.W. and Bold, H.C. (1963). Phycological Studies IV. Some
Soil Algae from Enchanted Rock and Related Algal Species. University
of Texas, Austin, 6318, 1–95.
[5] Bold, H.C. (1949). The morphology of Chlamydomonas chlamydogama
sp. nov. Bull. Torrey Bot. Club., 76,101–108.
[6] Chen, Y.C. (2001). Immobilized microalga Scenedesmus quadricauda
(Chlorophyta, Chlorococcales) for long-term storage and for
application for water quality control in fish culture. Aquaculture,
195(1-2), 71-80.
[7] Day, J.G. and Brand, J.J. (2005). Cryopreservation methods for
maintaining microalgal cultures. In (Anderson, R.A. eds.) Algae
Culturing Techniques. Elsevier Academic Press, USA. pp. 165-187.
[8] Faafeng, B.A, von Donk, E., and Källqvist, S.T. (1994). In situ
measurement of algal growth potential in aquatic ecosystems by
immobilized algae. J. Appl. Phycol., 6, 301-308.
[9] Guillard, R.R.L. (1975). Culture of phytoplankton for feeding marine
invertebrates. In: Smith, W. L., and Chanley, M. H., eds. Culture of
Marine Invertebrate Animals. Plenum Press, New York, pp. 26–60.
[10] Kawachi, M. and Noel, M.H. (2005). Sterilization and Sterile
Techniques. In (Anderson, R.A. eds.) Algae Culturing Techniques.
Elsevier Academic Press, USA. pp. 65-82.

101 | P a g e
 Phytoplankton and Zooplankton Samples Collection

[11] Lavens, P. and Sorgeloos, P. (1996). Manual on the Production and

Use of Live Food for Aquaculture. FAO Fisheries Technical Paper. No.
361. Rome, 295p.
[12] Lorenz, M., Friedl, T. and Day, J.G. (2005). Perpetual maintenance of
actively metabolizing microalgal cultures. In (Anderson, R.A. eds.)
Algae Culturing Techniques. Elsevier Academic Press, USA. pp. 145-
156.
[13] Oh, H.M., Lee, S.J., Park, MH. et al. (2001). Harvesting of Chlorella
vulgaris using a bioflocculant from Paenibacillus sp. AM49.
Biotechnology Letters, 23, 1229–1234.
[14] Parvin, M., Zannat, M.N. and Habib, M.A.B. (2007). Two important
techniques for isolation of microalgae. Asian Fisheries Science,
20,117-124.
[15] Retrieved from https://www.ccap.ac.uk/index.php/media-
recipes/ on 10/10/2021.
[16] Richmond, A. (2004). Handbook of Microalgal Culture:
Biotechnology and Applied Phycology. Blackwell Science Ltd.pp. 34.
[17] Stanier, R.Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971).
Purification and properties of unicellular blue-green algae (Order
Chroococcales). Bacteriol. Rev., 35, 171-205.
[18] Steponkus, P.L., Langis, R., and Fujikawa, S. (1992).
Cryopreservation of plant tissues by vitrification. In: Steponkus, P. L.,
ed. Advances in Low-Temperature Biology. Jai Press, London, pp. 1–
61.
[19] Walne, P.R. (1970). Studies on food value of nineteen genera of algae
to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria and
Mytilus. Fish. Invest. Lond. Ser. 2. 26(5), 1–62.
[20] Watanabe, M.M. (2005). Freshwater Culture Media. In (Anderson,
R.A. eds.) Algae Culturing Techniques. Elsevier Academic Press, USA.
pp. 13 – 20.

102 | P a g e
 Zooplankton cultivation techniques

6. ZOOPLANKTON CULTIVATION
TECHNIQUES

OVERVIEW
Despite the advancement of zooplankton culture as live feed throughout
the years, zooplankton culture only revolves around the common species
(daphnia, rotifer, and copepod). Many potential species have yet to be
explored and widely used in the aquaculture industry. One of the many
reasons is, zooplankton culture tends to be contaminated easily and it is
not as hardy as the commonly used species. Thus, this incurs more
production costs. One example will be the Trocophora larvae, larvae of
the Bivalvia class that have a short planktonic stage in its life cycle. Till
today it is a challenge to culture Trocophora larvae due to their pristine
living requirement. Furthermore, making zooplankton production
economical while at the same time packing nutritional values into the
guts of the zooplankton for larviculture remains a bottleneck for many
aquaculturists till today. Below is the schematic diagram on the chapter
summary (Figure 1).

Figure 6.1 Schematic diagram of Chapter 6 summary

 Zooplankton cultivation techniques

6.1 ZOOPLANKTON KEY ATTRIBUTES AND LIFE

CYCLES

Generally, there are a few criteria to be met before a zooplankton species is

considered for its potential to be cultured. Firstly, will be their nutritional
values. Most zooplankton used as aquaculture feed lacks the essential fatty
acids profile required for larvae development, but copepod is an exception
here. Copepods are famous for being known as a “lipid pump”(Tarrant et al.
2019), as they contain high levels of highly unsaturated fatty acids (Dhont et
al., 2013). To solve this bottleneck, aquaculturists will enrich the zooplankton
(rotifer, daphnia and artemia), few hours before the zooplankton is fed to the
fish or crustacean larvae (Cheban et al. 2017; Dhont et al., 2013; Evjemo et al.,
2003). Table 6.1 summarises the nutritional values found in common
zooplankton used, as feed in the aquaculture sector.
The second and third criteria will be the ease of culture and the growth
rate of zooplankton culture. Commonly cultured zooplankton such as
daphnia, rotifer and certain species of copepods (harpacticoid) met such
criteria. They have high reproduction potential, a short turnover time and
fast population growth. Another important factor that contributes to the
simplicity of culture is the zooplankton tolerance to a wide range of
environmental factors; especially in terms of temperature and salinity range
(Dhont et al., 2013). Furthermore, different developmental stages during the
zooplankton growth, as can be seen in Figure 6.2-6.4; provided a broad
spectrum of prey size suitable for different developmental growth stages of
fish or crustacean larvae (Dhont et al., 2013; Mile et al., 2001).
To start a zooplankton culture, it is always important to understand the
life cycle and requirements of the cultured species. As can be seen in Figure
6.2 and Figure 6.3, both rotifer and daphnia can reproduce asexually under
favourable conditions. Both species reproduce mostly parthenogenetic
daughters, except for during unfavourable conditions (changes in salinity
and lacking food). These two species met the criteria of a short turnover time
and fast population growth for aquaculture purposes. Despite reproducing
sexually (Figure 6.4), certain species of copepods, such as those in the order
of Harpacticoida; have been proven to have high fecundity and short
generation time. Harpacticoid has the potential to produce high biomass
densities if provided with enough food. They are able to increase rapidly from
104 | P a g e
 Zooplankton cultivation techniques

0.05 individual/mL to 9.5 individual/mL in 12 days (Lavens & Sorgeloos,

1996b).

Table 6.1 Nutritional values of common zooplankton species and their target
group of organisms in the aquaculture sector.

Group Nutritional value Target group of

organisms
Rotifer 37-51% protein level Freshwater & marine
1.5-13% lipid level fish larvae
Crustacean larvae
*Inadequate fatty acid profiles (commonly used in mud
*Needs to be enriched before crab culture)
feeding
Copepod 44-52% protein level Freshwater & marine
(Harpacticoid) 7-14% lipid level fish larval
Crustacean larvae
*Contains essential fatty acid.
*Enriching prior to feeding is
not a priority, but it’s a common
practice.

Daphnia 45-70% protein level Freshwater fish larvae

(Mostly found in 11-27% lipid level
freshwater, small
number in *Depends on the quality of the
brackish water) feed substrate used during
culture.
*Best to be enriched prior to
feeding

Artemia 50.6% protein level Freshwater & marine

14.2% lipid level fish larvae
Crustacean larvae
*Inadequate fatty acid profile
*Needs to be enriched prior to
feeding

105 | P a g e
 Zooplankton cultivation techniques

Figure 6.2 Schematic diagram of Daphnia life cycle (modified from Hite et al.,
2017).

Figure 6.3 Schematic diagram of Rotifer life cycle (modified from Hoff &
Snell, 1987)

106 | P a g e
 Zooplankton cultivation techniques

Figure 6.4 Schematic diagram of Copepod (modified from Støttrup,

2003)

6.2 ISOLATION TECHNIQUE

This method is applicable for rotifer, copepod and daphnia. Natural pre-
filtered seawater or freshwater is suitable for zooplankton culture work.
Wild samples that have been collected from the wild (marine or
freshwater environment) with a plankton net, will be brought back to the
laboratory and rinsed with a filter net (mesh size between 64 - 120 μm). The
filter net used depends on the size of the zooplankton that will be cultured.
For example, the L-type rotifer of the genus Brachionus sp. is around the size
of 130-340 μm. Filtering with a mesh net of varying sizes can remove smaller
particles and other unwanted organisms (smaller zooplankton, protozoa,
bacteria, viruses and fungus). This step will increase the overall success rate
for the isolation process. In this case, the targeted zooplankton here is a
rotifer species, Brachionus sp.
Two mesh nets with the size of 250 μm and 120 μm, will be used here. 250
μm mesh net will be stacked on top of the 120 μm mesh net. 250 μm mesh
net is for filtering big unwanted particles, while the 120 μm mesh net is for
trapping adult size rotifer. The wild plankton water will be poured slowly
over the two stacked up sieves. It is always advisable to submerge the 120
μm sieve into the water while sieving the rotifers or any zooplankton. This is
to prevent damaging the rotifer or zooplankton unnecessarily during the
filtering process.
107 | P a g e
 Zooplankton cultivation techniques

Zooplankton isolation (modified from Parvin et al. 2007). Procedures

are as follows:

a) Prepare a few petri dishes, 500mL of culture water in a beaker and a few
disposable plastic pipettes (2mL). All these will be needed during the
isolation work.

b) 1mL of zooplankton sample will be siphoned up by using the plastic

pipette and distributed dropwise on a petri dish, about as many small
droplets as possible, until the 1mL sample has been spread out evenly,
onto the petri dish. This method will distribute the many zooplankton into
different droplets of water, and it will ease the task of individually
targeting them for isolation work (Figure 6.5a).

c) All the small droplets will be observed under dissecting microscope (20x
magnification) (Figure 6.5a).

d) You have to work under a microscope for the rest of these procedures.
Once a targeted zooplankton species has been identified in a water droplet,
transfer it to a petri dish. A clean drop of culture water will be added for
rinsing purposes. Then, this targeted zooplankton species will be picked
up again by the plastic pipette, transferred to another corner of the same
petri dish, and rinsed again with another drop of clean culture water.
(During each "picking" process, try to siphon up the targeted zooplankton
species with as minimal surrounding water as possible while viewing
through the dissecting microscope) (Figure 6.5b).

e) Repeat this rinsing procedure until the targeted species is free of

contamination (Figure 6.5b & 6.5c).

f) Transfer the droplet containing the targeted zooplankton to a clean 100mL

culture water in an Erlenmeyer flask or any container deemed suitable.
Repeat the steps above, till 10 -100 individuals have been selected and put
into the 100mL of culture water in the same Erlenmeyer flask (Figure
6.5d).

g) A minimum of ten individuals might be enough for culturing zooplankton

that reproduces asexually (e.g. daphnia, rotifers), but not for copepods
108 | P a g e
 Zooplankton cultivation techniques

(sexual reproduction). To have a faster generation time for copepod, a

minimum of 50 to 100 individuals should be inoculated into 1L of culture
water.

h) The procedures above are suitable and easy for zooplankton that moves
slowly (rotifer and daphnia) but might be difficult for targeting copepod
even in a single droplet of water. Copepod tends to move very fast out of
view of the microscope. If this happens, you can reduce the number of
water droplets for rinsing, either by siphoning with the plastic pipette or
dabbing with tissue paper to dry the water droplet a bit.

i) Daphnia or rotifer species will require a minimum of one week of

generation time, while copepod species will require a minimum of two
weeks. If any of the cultures crashed, it is advisable to start the isolation
process again. Always remember to keep the leftover wild zooplankton
alive by aerating it and feeding it with fresh algae water or some
particulate food as a backup for isolation failure issues.

j) The procedure mentioned here will place more emphasis on rotifer,

copepod and daphnia (as the author’s working background revolved
around these three species).

(a)

Small droplets with

zooplankton distributed
evenly.
Figure 6.5a Collect 1 mL of zooplankton culture with the plastic pipette
and distribute it little by little on a petri dish. Each droplet might or might
not contain zooplankton. This is to spread out the zooplankton onto
smaller droplets on the petri dish.

109 | P a g e
 Zooplankton cultivation techniques

(b)

Petri dish for rinsing

the “picked” up
zooplankton.

Figure 6.5b After found the targeted species, collect it from the petri dish
from step 1 with a plastic pipette, then transfer it to another clean petri
dish. Add a drop of clean culture water for rinsing. Repeat this step two
to three times until you are sure, only the zooplankton is found in that
drop of water. If not, repeat a few more times.

(c)

Figure 6.5c Another option during the picking step, will be placing the
picked plankton on a 24-Well plate.

110 | P a g e
 Zooplankton cultivation techniques

(d)

A container with lid and

200mL culture water

Figure 6.5d After ensuring the targeted zooplankton has been rinsed and
cleaned of contaminants, it will be transferred into a container with pre-
filtered culture water. If this isolation is successful, this will be your stock
culture. Prepare at least three replicates for a single species of
zooplankton. Algae food to be added every two days.

6.3 CULTURE MAINTENANCE

This section is to elaborate further on the culture work after the isolation
steps discussed in Section 6.2. Copepod culture will be elaborated under
a different subtitle (section 6.3.2), as copepod requires some “tweaking”
to see its growth pattern.
The stock culture general culture conditions will be emphasized here.
The objective behind a stock culture is for safekeeping a monoculture
(prevent contamination and as backup).

6.3.1 Rotifer and Daphnia stock culture

After succeeding in your isolation steps, this will be your stock culture.
The stock culture can be maintained at room temperature, 28-30 ℃. No
extra lighting is required. Small stock cultures have to be kept in a 200mL
flask or a closed vial; in an enclosed space to prevent contamination from
external sources. Stock cultures used for safekeeping and upscaling
purposes are best maintained with live algae water (Figure 6.6a - 6.6c).
During the zooplankton stock culture process, it will be good to
disinfect this stock culture first. It can be done with a cocktail of
111 | P a g e
 Zooplankton cultivation techniques

antibiotics (Lavens & Sorgeloos, 1996a) on the broodstock. It can also be

done on the cysts of zooplankton. However, the cyst is not easy to be
identified or collected for beginners. Certain requirements (physical
stress) have to be met to stimulate zooplankton for cyst production. For
further details on disinfecting the zooplankton culture, can refer to the
work of Lavens & Sorgeloos (1996a). It is important to note that any
zooplankton feed used as live feed must be in a single culture
condition(monoculture). Any contamination within the zooplankton
culture will easily cause high mortality during the larviculture process.
So, if a monoculture condition cannot be achieved, it is better not to use
them. Monoculture requirement also applies to phytoplankton culture
(live algae water). Contamination might also come from the
phytoplankton culture used for feeding zooplankton. Precautionary steps
such as observing both plankton cultures under the microscope for
unwanted organisms are encouraged to be done from time to time. Live
algae water and particulate feed preparation will be elaborated in the
following sections 6.3.1 (a) and 6.3.1 (b).

(a) Live algae water preparation

The feed used during the culture period can either be live algae water or
particulate food. Ideal algae live food used for zooplankton culture are
usually Isochrysis sp., Rhodomonas sp., Nanochloropsis sp., Pavlova sp. and
other diatoms species. Particulate food such as yeast, rice bran, wheat
flour could also be used upon live algae culture shortage. But do take note
of the amount used to prevent water fouling.
If live algae water is used, it has to be cultured earlier before the start
of the zooplankton culture. Generally, a 200mL algae stock culture is
required to upscale to a 2L culture (four to five days period) and from
there upscale to a 20L algae culture (another four to five days period). So
to prepare a 20 L algae food, it will be about 10 days of the waiting period.
Live algae food will need to be prepared and upscaled regularly every five
days for sustaining the zooplankton growth throughout the culture
period. Algae culture procedure can refer to “Chapter 5: Phytoplankton
culture”.

112 | P a g e
 Zooplankton cultivation techniques

Live algae water in good condition, will show a dark colouration. It can
be added in moderation to colour the zooplankton culture water and wait
till the water transparency drop before new algae water is added. Usually
within a day or two before topping up, depending on zooplankton culture
density. Algae density is usually given at a concentration of 1000 cells/m
for optimal growth (Lavens & Sorgeloos, 1996b). Algae water with dark
colouration will usually meet the algae cell density requirement for
feeding. (P/S: most of the time, the algae colour in a zooplankton
container will only fade after two days.)

(b) Particulate food preparation

To prepare particulate food, weigh one gram of particulate food (baker

yeast, rice bran, wheat flour etc) and dilute it in 1L of water. 10 mL can
be fed into 100mL of culture water every day. The feed to culture water
ratio will be 1:10. So, for 1L of copepod culture, 100mL of pre-diluted
particulate food (1g/L of particulate food) will be added into the culture
water every day. This is just for starting up the culture. When the number
of individuals has increased, the amount of food added will have to
increase accordingly (Lavens & Sorgeloos, 1996b; Rotmann et al., 2011).
The daphnia and rotifer cultures have to be sub-cultured weekly. From
the 100mL culture water, 50mL will be used for a new subculture by
adding 50mL of fresh culture water. Another half will be used for
upscaling in a 2L culture container. If algae food with a dark colouration
is used, it can be topped up once every two days. However, particulate
food has to be topped up once every day. If there are leftover algae cells
within the zooplankton culture, the cells will still be viable for some time.
On the other hand, particulate food is not living cells; the minimum
concentration given here is to prevent water fouling.

6.3.2 Copepod stock culture

After succeeding in your isolation steps, this will be your stock culture.
To have a faster regeneration time for copepod, a minimum of 50 to 100
individuals should be inoculated into 1L of culture water (freshwater or

113 | P a g e
 Zooplankton cultivation techniques

seawater depends on copepod species). Copepods tend to be

cannibalistic, so a larger volume of culture water (more space) and a
larger number of individuals is always preferable for this taxon. As the
culture grows, it can be transferred to a larger volume of water. From my
personal experience, after about two weeks of culture in 1L of water, it
can be transferred to a 5L tank (Figure 6.6c). Unlike rotifer and daphnia,
during the stock culture of copepod, particulate food and algae water can
be used at the same time. Copepod is a detritus feeder. They can feed on
biofilm, bacteria and algae. So, if the culture water “contains more food”,
copepod will grow faster. However, rotifer and daphnia are suspension
feeders. Biofilm and bacteria on the substrate will cause fouling to the
culture water.
Food preparation and feeding requirements for copepod are the same
for daphnia or rotifer. Compared to rotifer and daphnia, copepod must be
allowed a minimum of two weeks to see the effect of the culture.
The most promising species in copepod culture will be the harpacticoid
species. Harpacticoid species have higher fecundity and short generation
time. This species is able to tolerate different environmental conditions
(eg. salinity ranges between 25ppt to 38ppt and temperature ranges in
between 17-30 ℃). To simplifiy things, the culture conditions for
copepod will be the same with daphnia and rotifer (lighting, culture
medium, temperature and feed type).

(a)

Figure 6.6a An example of stock culture container for rotifer/daphnia in a

200mL culture water. Any 1L container can be used for copepod culture.

114 | P a g e
 Zooplankton cultivation techniques

(b)

Figure 6.6b Starter culture for rotifer/daphnia in a 2L culture water. Any

containers can be used.

(c)

Figure 6.6c Starter culture for copepods in a 60L culture water. Any
containers can be used. For copepods, larger volume of water and food will
encourage faster growth.

6.4 DAPHNIA, ROTIFER AND COPEPODS

CULTIVATION

Starter culture is the precursor step for upscaling to a batch culture condition.
Batch culture is large-scale closed system culture for the mass production of

115 | P a g e
 Zooplankton cultivation techniques

an organisms by using a fixed volume of culture water under specific

environmental conditions.
This section will emphasize the methods for upscaling from starter
culture to batch culture.

6.4.1 Batch culture for Rotifer

From stock culture (200mL) to starter culture (2L), the rotifer is to be

maintained at a density of 50 individuals per mL and fed with algae water at
a density of 1.6 x 106 cells/mL. About 50mL of algae water will be added into
the 2L culture for feeding. During this period, a minima aeration can be
supplied for better growth.
Under optimal conditions, the rotifer will have reached a density of 200
individuals /mL (starter culture). Failing to get this density, probably your
culture is about to crash. A new subculture must be prepared and topped up
with new food. Then from two litres, the rotifer culture will be upscaled to a
fifteen litres culture bottle. Top up with fresh algae water daily and supply it
with mild aeration.
Add two litres of rotifer culture water from the starter culture into the
fifteen litres bottle and then top up with two litres of fresh algae culture
water. For faster growth effect, the feed used will be one part rotifer culture
to one part of algae water (1:1). Top up with two litres of fresh algae water
every two days or after the water turbidity has decreased. Increasing water
transparency is a sign the rotifer culture is growing well while nothing
changes might be the other way round.
About ten days, the fifteen litres rotifer culture will be ready to be
inoculated into the 500L container. For a faster growth rate, batch culture
inoculum should be one part rotifer culture water to one part algae water
(1:1). About fifteen litres of rotifer water will be topped up with fifteen litres
of fresh algae water. Fifteen litres of new algae water will be topped up daily
or two days once, depending on water transparency. This is dependent on
the rotifer growth rate under optimal conditions.
Besides algae water, the particulate feed can be used to aid the rotifer
growth. Feeding one per cent of yeast (1%) twice a day will be sufficient for
sustaining the rotifer growth. Different studies used different concentrations

116 | P a g e
 Zooplankton cultivation techniques

of yeast or other particulate matter. It is best to start at a minimum

concentration and slowly increase the concentration as deemed required by
your zooplankton culture objectives. For different particulate food
concentrations, can refer to Lavens & Sorgeloos (1996a). Batch culture
conditions do not vary much as compared to starter culture conditions. As
the volume of water and density increased, put more food and moderate to
vigorous aeration to properly aerate the water.
As for harvesting the rotifer culture, either siphon the culture content or
pour out the content slowly onto a mesh filter. It is recommended to use two
different mesh filter sieves. Place the 250 μm sieve on top, and the 40-60 μm
sieve below. The 250 μm sieve is for filtering larger debris while the 40-60
μm sieve is for capturing rotifer. The 40-60 μm sieve is advised to be placed
submerged in a tray of water. This is to prevent damaging the rotifer during
the sieving process.

6.4.2 Batch culture for daphnia

From stock culture (200mL) to starter culture (2L), daphnia should have a
density of fifteen individuals per mL. From two litres, upscale to fifteen litres
culture container. The procedures for upscaling and feeding are similar to
rotifer culture. However, do take note there is a difference in the growth rate
between daphnia and rotifer. For example, density can go up to fifteen
daphnias per mL at optimal conditions (good water quality, and enough food),
while for rotifer, the density can go up to 100-300 individuals per mL.
Particulate food for daphnia can follow the reference in Rottmann et al.
(2011). (P/S: particulate food reference for rotifer culture (Lavens &
Sorgeloos, 1996a) works for daphnia too). Batch culture conditions do not
vary much as compared to starter culture conditions. As the volume of water
and density increased, put more food and moderate to vigorous aeration to
properly aerate the water. Harvesting technique for daphnia is similar to
rotifer. Please refer to section 6.4.1.

6.4.3 Batch culture for copepod (harpacticoid)

From stock culture (1L) to starter culture (60L), the copepod is to be

maintained at a density of one copepod per mL. Density can go up to ten

117 | P a g e
 Zooplankton cultivation techniques

copepods per millilitre at optimal conditions. From starter culture (60L),

upscale this to 500-800L of filtered seawater in a 1000L tank for batch
culture purposes. Batch culture is only prepared when fish or crustacean
larvae require this as their feed. Otherwise, maintaining in starter culture
(60L) volume is sufficient. Extra food will be required to maintain such a
large volume of copepod culture.
During batch culture, the optimal culture density will be 20 to 70
copepods per mL. The population growth rate will be growing at a rate of
15 % per day. If live algae water were used as feed, the cell density should be
maintained at 5 x 104 to 2 x 105 cells/mL every two days or depending on the
water transparency after topping with algae water. Copepod generation time
will be a minimum of two weeks before the culture can be harvested.
Preparation of particulate food can refer to Lavens & Sorgeloos (1996b). As
the volume of water and density increased, put more food and moderate to
vigorous aeration to properly aerate the water. Harvesting technique for
copepod is similar to rotifer. Please refer to section 6.4.1.
Below is the schematic diagram on the overall key points of stock culture,
starter culture and batch culture for ease of understanding (Figure 6.7).

118 | P a g e
 Zooplankton cultivation techniques

Figure 6.7 summarises the overall key points of stock culture, starter
culture and batch culture for ease of understanding.

119 | P a g e
 Zooplankton cultivation techniques

References
[1] Cheban, L.M. Grynko, O.E., Marchenko, M.M. (2017). Nutritional Value
of Daphnia Magna (Straus, 1820) Under Conditions of Co-cultivation
with Fodder Microalgae. Biological sytems, 9(2), 166-170.
[2] Dhont, J., Dierckens, K., Stottrup, J., Stappen, G.V., Wille, M., Sorgeloos
P. (2013). 5-Rotifers, Artemia and copepods as live feeds for fish
larvae in aquaculture In: Advances in aquaculture hatchery
technology. Woodhead Publishing Series in Food Science, Technology
and Nutrition. pp. 157-202.
[3] Evjemo J. O., Reitan, K., I., Olsen, Y. (2003). Copepods as live food
organisms in the larval rearing of halibut larvae (Hippoglossus
hippoglossus L.) with special emphasis on the nutritional value.
Aquaculture, 227, 191-210.
[4] Hite, J.L., Penczykowski, R.M., Shocket, M., Griebel, K.A., Strauss, A.T.,
Duffy, M.A., Caceres, C.E., & Hall, S. (2017). Allocation, not male
resistance, increases male frequency during epidemics: a case study
in facultatively sexual hosts. Ecology, 98(11), 2773-2783.
[5] Hoff, F. h., & Snell, T. W. (1987). Plankton Culture Manual. Dade City,
FL: Florida Aqua Farms Inc.
[6] Lavens, P. & Sorgeloos, P. (1996a). “Manual on the Production and
Use of Live food for Aquaculture: 3.5.3. Rotifers: Culture Procedures."
FAO Fisheries Technical Paper.
http://www.fao.org/docrep/003/W3732E/w3732e06.htm
[7] Lavens, P. & Sorgeloos, P. (1996b). “Manual on the Production and
Use of Live food for Aquaculture: 5.2. Production of copepods." FAO
Fisheries Technical Paper.
http://www.fao.org/docrep/003/W3732E/w3732e06.htm
[8] Miles, D., Auchterlonie, N., Cutts, C., de Quero, C.M., (2001). Rearing of
the Harpacticoid Copepod Tisbe holothuriae and its Application for
the Hatchery Production of Altantic Halibut Hippoglossus
hippoglossus. Link Project FIN 23, SEAFISH Development
Aquaculture.,1-42

120 | P a g e
 Zooplankton cultivation techniques

[9] Parvin, M., Zannat, M.N. and Habib, M.A.B. (2007). Two important
techniques for isolation of microalgae. Asian Fisheries Science,
20,117-124.
[10] Rottmann, R.W., Graves J.C., Watson, C., Yanong, R.P.E. (2011).
Culture Techniques of Moina: The Ideal Daphnia for Feeding
Freshwater Fish Fry. Department of Fisheries and Aquatic Sciences,
Florida Cooperative Extension Service, Institute of Food and
Agricultural Sciences, University of Florida. pp 1-7.
[11] Støttrup J.G. (2003). Production and nutritional value of copepods,
in Støttrup,J.G. & McEvoy,L.A. (eds), Live Feeds in Marine Aquaculture,
Oxford: Blackwell Science. pp. 145–205.
[12] Tarrant A.M., Nilsson, B., Hansen, B.W. (2019). Molecular physiology
of copepods - from biomarkers to transcriptomes and back again.
Comparative Biochemistry and Physiology, 30, 230-247.

121 | P a g e