# Methods Manual for Salt Lake Studies/Phytoplankton

Authors: PSJ Coleman,

### Overview

An estimate of total biomass of planktonic algal species may be obtained by counting plankton in several samples of the brine. Microalgae are settled, and to a limited extent preserved, by the settling agent. A subsample of the cells in the concentrated algal mass is counted. This figure is extrapolated to give a rough idea of the total algal biomass and the main phyla present.

### Reagents and equipment required to settle algae and concentrate sample

* Algae sample
* Lugols Media for algae settling
* Measuring cylinder and vacumn pump (or siphon)
* Haemocytometer, cover slip and transfer pipette
* Microscope

### Method of concentrating the sample

Add approximately one millilitre of Lugol's Media to an empty 250 millilitre measuring cylinder. Fill the 250 mL measuring cylinder to the 200mL mark with a sample from the field. Make sure that the media and the sample are well mixed. This solution must sit undisturbed for a day before the next step. If too much sample is added, some of the solution can be poured out if the excess is less than 25 millilitres. If a larger amount has been added, pouring some away will upset the ratio of sample to Lugol's Media, so start again.
After the solution of sample and Lugol's Media has been left for a day the top 180 millilitres must be removed without disturbing the remaining 20 millilitres. This is done by using a vacuum pump attached to a water trap, a venturi pump, or by simple siphoning. It is important that the bottom 20 millilitres is not disturbed while the upper 180 millilitres is being removed.
Mix the remaining 20 mL of sample thoroughly and place a drop on a haemocytometer slide, using the following instructions, prior to viewing under the microscope.

### Lugols Media for algae settling

Chemicals needed:
* Iodine I$_2$,
* Potassium Iodide KI,
* Acetic Acid (Glacial)
Add 10g of I$_2$ and 20g of KI to a 500mL beaker. Add about 150mL of distilled water and mix well to dissolve the chemicals. Make up to 200mL and add 20gm of acetic acid and mix again. Store in a glass reagent bottle. Do not use until several several days have passed.

### Preparing the haemocytometer slide for viewing

The haemocytometer is a glass slide normally used to count blood cells. It may be used to count algae cells in the same manner. The diagram shown here and discussed in this text is an Improved Neubauer Haemocytometer. Refer to diagram 1 for the general structure of the haemocytometer.
The two rails that support the slide coverslip are moistened with water to stop the coverslip from moving. The coverslip is placed on the rails equal distance between the two slides. The coverslip may tend to slide off the haemocytometer, so use your fingertip to hold it in place.
The bottom 20 millilitres of the concentrated sample is then well mixed and, using a transfer pipette, a small sample of the concentrated algae is sucked into the tube. A drop of this is placed on the small (2 by 4mm) half cone notch on the side of the haemocytometer. The sample will draw up the cone to fill the area between the raised platform of the haemocytometer and the coverslip. Sometimes the drawing or capillary action does not start by itself, so move the coverslip slightly to start the action by pushing gently on the side, not top, of the coverslip. Be careful as the coverslip is fragile and may crack if handled roughly. This process is repeated for the other side of the haemocytometer. This entire process should only take about one minute. There should be an even cover of brine across both raised platforms and no excess brine on the top of the coverslip.
Not all haemocytometers have a notch in the side. In these, place a drop of the concentrated sample on each stage of the haemocytometer slide and gently slide the coverslip into place.

### Viewing a haemocytometer slide

Haemocytometers have two main counting areas. A single counting area is shown in the diagram 2. Each counting area is divided into nine equal portions of 1mm by 1mm. These portions are referred to as CELLS.
To do a simple count, the number and type of microalgal cells present in a single haemocytometer cell is totalled and recorded in your laboratory daybook.
The number of counting cells examined is limited to a maximum of 18 cells. If too many algal cells are present counting all 18 cells may be too time consuming and less than 18 areas may be examined. So, the number of cell areas counted should always be scored and recorded in the neighbouring column to the count, for use in the final plankton density calculation.
If you wish to know which groups of plankton are the dominant species, the algae may be identified with the aid of the literature to the taxonomic level you require and the groups counted separately. You may find drawing up a matrix with separate columns for each group of microalgae you are scoring simplifies this type of count.

### Calculation of the plankton count

Once you have your microalgae counts, the number of counting area cells you counted and the concentration factor then:
${\text{plankton count}}={\frac {\text{1}}{\text{how many times concentrated}}}\times {\frac {\text{no of plankton}}{10^{-4}}}\times {\frac {\text{1}}{\text{no of cells (squares) counted}}}$
The concentration factor is normally 1:10 and plankton cells are expressed as plankton x 10$^3$ per mL
Therefore, more normally :
${\text{plankton count}}={\frac {\text{no of plankton}}{\text{no of cells}}}\times 10^{3}$

### Calculating algal biomass

Wet algal biomass (biovolume) may be calculated (as a volume) from the shape and size of typical plankton present in your count, multiplied by the plankton count. Typical plankton volume calculations are illustrated in Diagram 3. Click on the thumbnail to obtain a larger version of the diagram. The groups of plankton illustrated in the diagram include those groups most commonly found in saline lakes.
The biovolume is reported as mm$^3$/L and is obtained by multiplying the calculated cell volume (in cubic micrometres) for each plankton group or species by the count for that group or species per millilitre, and summing all the groups/species (Eaton et al, 1995):
${\text{V}}_{t}=\sum _{i=1}^{n}({\text{N}}_{i}\times {\text{V}}_{i})$
where:
${\text{V}}_{t}={\text{total plankton cell volume, mm}}^{3}/{\text{L}}$,
${\text{N}}_{i}={\text{count of the i}}^{th}{\text{species/mL}}$, and
${\text{V}}_{i}={\text{average volume of cells of the i}}^{th}{\text{species, }}\mu m^{3}$

### Alternative biomass methods

Reference to methods manuals such as Standard Methods for the Examination of Water and Wastewater (Eaton et al, 1995) will provide information on:
* obtaining dry biomass using gravimetric methods, and
* measuring chlorophyll$_a$ and multiplying by 67 to obtain an estimate of dry biomass.
Should you be using samples of hypersaline brines, remember that you may need to wash any filter pads rather more thoroughly that you would for a similar sample of marine or freshwater origin. Poor rinsing will result in the inclusion of a mass of salts in your biomass, invalidating the results.
Rinsing so thoroughly is not always appropriate. For example, the green Alga Dunaliella salina has no cellulose cell wall and is likely to lyse when exposed to fresh water. Filtering this species requires very gentle suction or even gravity filtration, and no exposure to deionised water. To correct for the inevitable quantity of salt that will be trapped in the filter, one could pass an equivalent quantity of filtered brine through a second filter and treat it in exactly the same manner as the filter used to concentrate the biomass sample.