04

Phytoplankton - collection,
estimation, classification and
diversity

Gireesh R., Molly Varghese and V.J. Thomas

Central Marine Fisheries Research Institute, Kochi-682 018

Introduction The distribution of some commercially important fish

Phytoplankters are microscopic, unicellular and photosynthetic and shellfish species and their larvae depend on certain
organisms which freely float in water bodies. They are phytoplankton species which act as indicators. The diatom
composed of both eukaryotic and prokaryotic species which species Fragilaria oceanica and Hemidiscus hardmannianus
colonizes upper euphotic part of the water column ranging have been considered as indicators of oil sardine, Sardinella
from freshwater to ocean conditions. They exhibit remarkable longiceps in the west coast of India. The abundance of
adaptation to remain in floating condition. Phytoplankton cells coccolithophores is another indicator for herring fishery
can range in size from about 1 µm to 1 mm. Like terrestrial in European waters while Fragilaria antarctica indicates
plants, these tiny primary producers require sunlight, nutrients abundance of krill in Antarctic waters. Some dinoflagellates,
and carbon dioxide for their growth and multiplication. The due to their luminescence help to locate and identify fish
cells of these organisms contain chlorophyll pigments to shoals during night.
harvest solar radiations. Phytoplankters photosynthesize in the
presence of sunlight using sufficient nutrients, fixing carbon Collection
dioxide and releasing oxygen. Hence they play significant In coastal waters, estuaries or lagoons, surface samples are
role in maintaining carbon budget of atmosphere as well collected usually with a clean bucket of measured volume.
as in seawater and help mitigate global warming. Physical The subsurface samples from different depths are collected
process such as wind and current play significant role in their with water samplers such as Van Dorn samplers, Niskin
distribution especially in estuarine and marine conditions. bottles, Meyer’s water sampler or Friedinger water sampler.
Phytoplankton act as primary link in energy pathway to The samples can be obtained using a weighed flexible or
higher trophic level through various food chains. It supports rigid plastic tube. The sampler is sent down vertically up to a
half of global primary production which directly or indirectly measured depth and then closed at the top to trap a column
supports almost all marine life. Phytoplankters are a major of water. In oceanic waters, large size Niskin bottles (5, 8 or
food source for variety of organisms such as zooplankters, 20 litres) are used along with CTD probe to collect samples.
larvae and juveniles of fishes and invertebrates. Today world The samples are collected in a container.
over the fishery depends on Potential Fishery Zone which is an
Fixing and preservation
attribute of pigment characteristic of phytoplankton measured
through remote sensing technique. For enumeration of phytoplankton, the cells must be
preserved at the earliest. Formalin is the widely used fixative
Phytoplankton usually undergoes a fairly predictable annual and preservative of phytoplankton cells. Formalin stored in
cycle, but some species may develop exponentially and form amber coloured bottles can be kept in cool temperature. For
so called blooms. Sometimes, this may cause adverse effect, 100 ml water sample, 2 ml of formalin is sufficient. If kept in
especially on coastal environment. Not all blooms are toxic. light coloured bottles, a white precipitate will develop due to
Blooming can cause oxygen depletion and hence it acts as a exposure of sunlight and form toxic paraformaldehyde.
threat to other marine life especially sedentary organisms like
shellfishes. Blooms of certain harmful species produce toxin. If Lugol’s iodine solution is another good preservative especially
such species are consumed by shellfish or other species, toxin for diatoms and nanoplanktons and except coccolithophorids.
may accumulate and affect organisms of higher trophic levels To prepare Lugol’s solution 10 g of potassium iodide and 5
which is a concern for human health. g iodine are dissolved in 20 ml of distilled water and to this

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 Gireesh R., Molly Varghese and V.J. Thomas

50 ml of distilled water and 5 g of sodium acetate or 5ml of identification of phytoplankton cells. The ideal microscope
19% acetic acid is added. About five drops are sufficient for should have magnifications of 10x, 40x and 100x and also
250 ml sample. with oil immersions and phase contrast. Many species are
transparent under light microscope. So different techniques are
Osmic acid is also added as preservative at the rate of 5-6 used to improve the contrast of cell identification. Commonly
drops per 100 ml sample. This is prepared by adding 200 mg used microscopes for identification and enumeration are
osmium tetroxide in 10 ml of distilled water. Standard Compound Microscope (10x or 12x Ocular and 10x,
20x, 40x or 100x Objectives) and Inverted Microscope. The
Gluteraldehyde solution is prepared by mixing 8 gm of advantage of inverted microscope is that it allows viewing the
gluteraldehyde in 100 ml distilled water and is applied to organisms settled at the bottom of the chamber.
sample in the ratio 1:1.
Enumeration and estimation of phyto-
Concentration of phytoplankton cells plankton
The collected samples are concentrated by the use of plankton
concentrator, centrifuge or settling method. The concentrated Biomass can be calculated either by direct count method (no.
phytoplankton samples are stored, especially diatoms, in of cells/ m3) or chlorophyll estimation by spectrophotometric
polythene bottles. method. In the direct count method, the cells have to be
identified and counted and then expressed as numbers per m3
Plankton concentrator
of water. For this, Sedgwick Rafter can be used. The sample
The samples can be filtered immediately using plankton is spread uniformly as a thin layer and cells are counted.
concentrator. In this method, sample is passed through a PVC Diluting the stock plankton sample is ideal to avoid clumping
tube or Perspex tube fitted with nylon net attached at one or clustering of organisms. The cells are counted individually
end. form one corner of the counter. Replicate cell counts are
necessary for accuracy and to avoid any statistical error. The
Centrifuge
total number of phytoplankton cells present in a litre of water
Centrifuge is a simple device by which phytoplankton can is calculated using the formula,
be concentrated from samples without causing damage
to cells. In this method, 10-20 ml of aliquot is centrifuged N=nv/V
in an electrical centrifuge at 1500-2000 rpm for 15 to
30 minutes. The supernatant water is decanted until the Where N is the total number of phytoplankton cells per lite of
volume reduced to 1/10 to 1/30 of initial sample. Later, it water filtered, n is the average number of phytoplankton cells
is suspended in remaining water and add few drops of 1% in 1 ml of sample and v is the volume of plankton concentrate
potassium aluminium sulphate to ensure the precipitation (ml) and V is the volume of water filtered. The unit is expressed
of phytoplankton. Finally the samples are preserved using as N of cells/litre or N × 103 / m3.
neutralized formalin or lugol’s iodine for further examination
under microscope. In the spectrophotometric method, the common and
most abundant pigment in all photosynthetic organisms,
Settling method
Chlorophyll a is generally used for estimating phytoplankton
The water sample can be kept in measuring cylinder for biomass. A known volume of water collected from surface or
settlement after preservation. Later, settled portion can be subsurface is filtered immediately through a synthetic fiber
separated by siphoning out water from the top. or glass fiber filter (Millipore, Whatman GF/F filter paper with
0.45 µm pore size) or sample can be brought to laboratory by
Staining keeping it in an ice box and filter it later. While filtering two to
The process of staining phytoplankton is species specific. three drops of magnesium carbonate should be added. After
Neutral red and Evans blue are commonly used stains the filtration, filter should be removed for further extraction
for whole plankton. Flurochromes are used to enhance or should be folded and kept in desiccators at -20ºC until the
fluorescence quantum yield, particularly in cyanobacteria. analysis can be done. The filter is placed in a 15 ml glass or
Fluorescein developed by Bentley-Mowat, enhances the centrifuge tubes and 15 ml of 90% acetone is added and then
green fluorescence and is widely used in marine plankton. The should be shaken vigorously. These tubes should be closed
stock solution is prepared by mixing fluorescein hydrate in and kept overnight for 24 hours in a refrigerator in dark.
0.5% acetone and is used as 0.01% solution in freshly filtered The tubes are removed from the refrigerator and allowed
seawater. Equal volume of solution and samples are mixed to warm up in the dark nearly to room temperature. The
and used under fluorescent microscope. samples are centrifuged at 5000 to 6000 rpm for 10 minutes.
The supernatant is decanted into a glass spectrophotometer
Identification cuvette (10 cm length) without delay. The spectrophotometic
A high quality microscope is essential for enumeration and reading of samples are taken at wave lengths 630, 664, 647

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Phytoplankton – collection, estimation, classification and diversity

and 750 nm. Correction factor for each extinction can be taken and green algae.
by using a blank solution of 90% acetone. The quantity of
chlorophyll pigments in the water can be measured by using Diatoms (Bacillariophyceae)
following formulas. Diatoms are ubiquitous algae (jewels of the plant world)
and have very important role in aquatic vegetation of world
Chlorophyll a = 11.85 E664 - 1.54 E647 - 0.08 E630 forming part of the plankton. They are the best known
group of phytoplankton and most important in terms of
Chlorophyll b = 21.03 E647 - 5.43 E664 - 2.66 E630 their contribution (approximately 40%) to oceanic primary
productivity. They are unicellular, filamentous or some
Chlorophyll c = 24.52 E664 - 1.67 E664 - 7.60 E647 forms colonies and have chlorophyll a, b, β-carotene and
fucoxanthin as main light harvesting pigments. The markings
Where E is the absorbance at different wavelengths (turbidity of cell wall, structure and position of raphae and nodules are
corrected by 750 nm) and the unit of Chlorophyll is expressed the characteristic features for identification of species. The
as µg/mL. diatom cell is known as frustules and characteristic feature
is possession of silica cell wall. This structure is highly
Chlorophyll mg/m3 = C×v/V×10 ornamental, which is species specific and often used as means
of identification. It is composed of two overlapping halves like
Where, v is the volume of acetone in cuvette, V is the volume pill box or a pair of petri dish. The outer layer is called epitheca
of water filtered in litres and C is the chlorophyll pigment. while inner is hypotheca. Edges of these two halves together
forming girdle. If one half of cell is seen, it is valve view, while
Apart from the spectrophotometric method, high performance only the girdle, then it is girdle view. Diatoms are divided into
liquid chromatography is also used to analyze the pigment order centrales and pennales based on symmetry. Centrales
concentration present in water. But in this case large are radialy symmetrical about a central point while pennales
volume of water is necessary to be filtered. Now a days, are bilaterally symmetrical with respect to long axis of the cell.
application of remote sensing has an important role in
predicting phytoplankton population structure. The spectral Centrales: Valves are circular, polygonal or irregular in outline
property of water is used as tool for determining the pigment and with ornamentation on the wall; ornamentation is radial
concentration. The colour of water is determined by volume or concentric about a central point. Valve have raphae or
scattering in a water body (transmittance). However, in pseudoraphae. Protoplast with many chromatophores.
taxonomic point of view, the application of fluorescence Centrales are more often seen in open sea.
mcicroscopy or remote sensing is limited to determination of
phytoplankton functional groups only. Centrales are divided into three suborders, 9 families, 14
subfamilies and 35 genera.
Classification
Phytoplankters are classified as microplankton (200-20 Sub order Discoidae: Cells shortly cylindrical, valves circular,
μm), nanoplankton (20-2 μm) and picoplankton (2-0.2 μm) hyaline, aerogated or with radiating striations. Eg. Cyclotella,
according to their size. The first two size classes can be Melosira, Stephanodiscus
identified by optical microscopy while the third is determined
by fluorescence microscopy. Sub order Solenoidae: Cells elongate, cylindrical or
subcylindrical, complex girdle with numerous bands. Eg.
Classification of algae has always been changing. W.H. Rhizosolenia
Harvey (1836) was the first who classified algae into three
groups based on colour: (a) Chlorospermae (green algae and Sub order Biddulphiodeae: Cells box shaped, valves
fresh water forms) (b) Melanospermae (brown algae) and (c) with two or more poles provided with horns or bosses. Eg.
Rhodospermae (red algae). Thereafter, a lot of workers have Biddulphia, Triceratium
described algal classification based on different criteria. The
classification proposed by F.E. Fritsch (1933) is still widely Pennales: Valves are bilaterally symmetrical or asymmetrical
recognized and accepted by algal taxonomists. Based in surface view. The cell wall ornamentation is also bilateral
on pigment and morphological characters, Fritsch (1935, with respect to a long line, along the long axis of cell. Valve
1945) classified algae into 11 classes viz. Chlorophyceae, always with a raphae or pseudoraphae. Protoplasts with
Cryptophyceae, Phaeophyceae, Rhodophyceae, Xantho- one or two chromatophores. Pennales are more common in
phyceae, Dinophyceae, Bacillariophyceae, Chloromonadinae, coastal waters.
Eugleniae, Chrysophyceae and Myxophyceae.
Pennales are divided into three suborders based on presence
The phytoplankton composed of mainly diatoms, or absence of raphae. These are further classed into 5 families,
dinoflagellates, cocolithoides (prymenophyceae), cyanophytes 10 subfamilies and 28 genera.

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 Gireesh R., Molly Varghese and V.J. Thomas

Suborder Araphidae: only pseudoraphae present Green algae are microscopic, uniccelluar, some filamentous or
colonial, flagellates or nonflagellates and have chlorophyll a,
Family Fragilarioidaea: Valves mostly straight. Eg. Asterionella, b and - carotene as light harvesting pigments. Mostly fresh
Fragilaria, Synedra, etc water and saline forms restricted to coastal waters. They are
widely distributed in tropical waters and few species are found
Suborder Monoraphidiodeae: Shows the beginning of in Arctic and Antarctic oceans. Picoplankton cannot identify
raphe, no central nodule easily due to lack of distinct morphological characters. Green
algae are subdivided into two sub-classes Chorophyceae and
Family Eunotioideae: Raphae on one or both valves. Eg. Charophyceae on the basis of difference in structure, and
Cocconies, Acnanthes, etc reproductive organs and methods of reproduction. Plankton
forms are mainly comes under the chlorophyceae and majority
Suborder Biraphidiodeae: Shows raphae on both valves, is fresh water forms. This subclass further divided into 14
central nodule is present. Eg. Pleurosigma, Navicula, etc orders and 22 families.
Dinoflagellates (Dinophyceae) Cyanobacteria (earlier Blue-green algae)
Dinoflagellates are unicellular, flagellates, naked or covered The large part of seas consists of prokaryotic unicellular or
with cellulosic plates (theca). They possess two flagella, filamentous organism known as cyanobacteria, as it appear
one longitudinal while other in furrow and form significant bluish or blue green, formerly called as blue green algae,
blooms (known as red tides), which are often toxic. They however, the name is less used today. Unlike the common
have chlorophyll a, c, phycobilins or fucoxanthin as main bacteria, they carry out photosynthesis and referred as part
light harvesting pigments. Several of them are luminescent of phytoplankton. Unicellular or multicellular organisms,
and produce light. The perforation in the thecal plates are filamentous or nonfilamentous with or without heterocysts
the characteristic features of dinoflagellates and help in the and cell contain phycocyanin pigment. Cyanobacteria is
identification. divided into 5 orders (Chroccocales, Chaemosiphonales,
Pleurocapsales, Nostocales and Stigonematales)
Coccolithophores (Prymnesiophyceae )
Mostly occur in marine waters. Size ranges between 5 to 20 Diversity
µm. Some have flagella while others are devoid of them. They Due to small size, rapid growth rate and spatio-temporal
are characterized by possessing two flagella and a fine whip- variation of species in relation to environmental conditions,
like structure called haptonema. The cells are covered with phytoplankters are very sensitive to stress imparted by them.
scales. One of the important group is coccolithophores. They The degree of stress imposed on phytoplankton reflects
are two flagellated and filamentous forms with calcified cells. the change at community level (group or functional). The
classwise, orderwise, familywise, genuswise and specieswise
Green algae (Chlorophyceae) numbers of algae listed out from the published information
with respect to India is given below:

Region No. of species Reference

East coast of India 249 131 Dinoflagellates (7 orders, 19 families, 30 genera), Geetha Madhav and
111 Diatoms (2 orders, 17 families, 43 genera), 7 Kondal Rao, 2004
Cyanophytes (1 order, 2 families, 4 genera)
Andaman Sea 227 58 Dinoflagellates, 164 Diatoms (2 orders, 8 failies), 2 Kartik et al., 2012
Cyanophytes, 2 Silicoflagellates
South east coast of India 185 16 Dinoflagellates, 166 Diatoms (Centrals 94; Pennales Sahu et al., 2012
72), 2 Cyanophytes, 1 Silicoflagellates
Mangalore coast (west coast of 73 22 Dinoflagellates, 46 Diatoms Karolina et al., 2009
India)
South East Arabian Sea 105 25 Dinoflagellates, 75 Diatoms (Centrals 55; Pennales Robin et al., 2013
20), 1 Cyanophytes, 2 Silicoflagellates, 2 Green algae
South West Coast of India 67 17 Dinoflagellates (7 genera), 49 Diatoms (Centrals 40; Robin et al., 2010
Pennales 9), 28 Genera 1, Cyanophytes (1 genus)
Nethravathi-Gurupura estuary 80 54 Diatoms (20 orders, 26 families, 33 genera), 5 Shruthi and Rajasekhar,
Dinoflagellates (4 orders, 4 families, 4 genera), 15 2014
Cyanobacteria (6 orders, 6 families, 9 genera), 6 Green
algae (5 orders, 5 families, 5 genera)
Tumkur Lake 171 Chlorophyceae, 46 species; Bacillariophyceae, 52 Ravishankar et al., 2009
species; Desmidiaceae, 22 species; Euglenophyceae, 27
species; Cyanophyceae, 24 species

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Phytoplankton – collection, estimation, classification and diversity

Suggested reading Halllegraeff, G.M., G.M. Anderson and A.D. Cembella. 1995. Manual on Harmful
Marine Micro algae, IOC Manuals and Guides No. 33, pp. 551, UNESCO,
Desikachary, T.V. 1959. Cyanophyta. Indian Council of Agricultural Research, New Paris.
Delhi, pp. 686. Subrahmanyan, R. 1958. Plankton organisms of the Arabian sea of the west coast
Desikachary, T.V. and P.M. Sreelatha. 1989. Oamaru Diatoms (Ed. J. Cramer), pp. of India. Indian Journal of Botanical Society, 37: 435-441.
330. Tomas, R. 1997. Identifying marine phytoplankton, pp. 858, Academic press,
Gopinathan, C.P. 1984. A systematic account of the littoral diatoms of the California.
southwest coast of India. Journal of Marine Biological Association of India, Venkataraman, G. 1939. A systematic account of some south Indian diatoms.
26: 1-31. Proceedings of Indian Academy of Sciences, 10: 293-368.

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