TEST METHOD Plankton Counts

Revised 30 May 2007 (additional advice on brine-affected objectives)

RISK ASSESSMENT

Assess Safety, Health, Quality and Environmental aspects of each specific step.

OVERVIEW

This task is designed to provide an estimate of total biomass and give an indication of the dominant species. The count does not attempt to be definitive but does provide an indication of the health of the waterbodies.

The algae 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.

 TASK SAFETY REQUIREMENTS

Safety boots are mandatory

 SPECIFIC JOB STEPS RISK ASSESSMENT RISK CONTROL
Reagents and Equipment Required
  • Algae sample
  • Lugols Media for algae settling (see later in this instruction for preparation)
  • Measuring cylinder and vacumn pump (or siphon)
  • Haemocytometer, cover slip and transfer pipette
  • Microscope
   
Add approximately one millilitre of Lugol's Media to an empty 250 millilitre measuring cylinder. Fill the 250 mL measuring cylinder to 200mL 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.

After the solution of sample and Lugols 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, or by simple siphoning. It is important that the bottom 20 millilitres is not disturbed while the upper 180 millilitres is being removed.

Too much sample is added. The solution can be poured out if the excess is less than 25 millilitres but if it is more, the whole process must be repeated.
Mix the remaining 20 mL of sample thoroughly and place a drop on a haemocytometer slide, using the following instructions, for viewing under the microscope.    
Lugols Media for algae settling

Chemicals needed:

  • Iodine I2,
  • Potassium Iodide KI,
  • Acetic Acid (Glacial)

Add 10 g of I2 and 20 g 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 20 gm of acetic acid and mix again. Store in a glass reagent bottle. Do not use for several days.

   
Preparing the haemocytometer slide for viewing

The haemocytometer is a glass slide normally used to count blood cells. It will be used to count algae cells in the same manner. The sort that is provided is an Improved Neubauer Haemocytometer. Refer to diagram 2 for the general structure of the haemocytometer and diagrammatic description of the following steps.

   
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 It shall be held firmly in place.

Diagram 1 - Microscope

Diagram 2 - Haemocytometer

SPECIFIC JOB STEPS RISK ASSESSMENT RISK CONTROL
The bottom 20 millilitres from the sample above is then well mixed and, using a transfer pipette, a small sample of the concentrated algae is sucked into the the tube. A drop of the concentrated algae 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. The drawing or capillary action does not start by itself

The coverslip may crack as it is fragile.

The half cone notch shall be partially covered by the cover slip. The coverslip can be moved slightly to start the action by pushing gently on the side of the coverslip.

Care shall be taken not to touch the top of the coverslip.

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.    
Using the Microscope

The parts of the microscope are labelled on diagram1 on the previous page. Place slide on the stage. Rotate nosepiece so that the x10 objective is in use. Start with the objective down near the slide and coarsely focus upwards by turning the focus control towards the operator.

The objective could smash into the slide. Never focus downwards.
Use the fine focus control to gain the clearest image. Adjust the eyepieces so that the image unites. The eyepieces adjust both for distance between pupils and individual focus. Make sure that the image through the fixed eyepiece is sharp, then adjust the other eyepiece to match.    
It is often easier to view through a binocular microscope if you don't actually touch the eyepieces, but view from a slight distance.    
Close the condenser iris just enough to give the specimen some contrast. It should be about one third closed. Too much closure results in reduction of depth of field. Remove an objective and look down the tube to check on closure.
Viewing the Haemocytometer Slide    
The haemocytometer has two main counting areas. A single counting area is shown in diagram 3    
Each counting area is divided into nine equal portions of 1mm by 1mm. These portions are referred to as CELLS.    
The number and type of algal cells in a haemocytometer cell is totalled and recorded in the laboratory daybook    
The number of cell areas counted are scored in the neighbouring column.    
The algae are identified with the aid of the literature and photographs available, to a taxonomic level depending upon the client's needs.    
The number of counting cells examined is limited to a maximum of 18 cells. Too many algal cells are present to count. Less than 18 areas can be examined for any one pond if the individual total of any single type of algae is greater than thirty for that pond sample.

In cases where there is a very high density of algae, only one counting area need be counted.

When all the samples have been counted, the samples can be disposed of down the laboratory sink.    
It is the responsibility of the operator to clean up the work area, wash the relevant containers and dispose of unwanted material.    
The counting sheet shall be totalled and algal densities calculated using the calculation provided below.Once this is done the sheet is filed in the laboratory    

Therefore:

 

The concentration factor is normally 1:10 and plankton cells are expressed as plankton x 103 per mL

Therefore, more normally :

 

Diagram 3 - Haemocytometer Counting Area

SPECIFIC JOB STEPS RISK ASSESSMENT RISK CONTROL
Maintenance of the Microscope

The microscope must be kept clean and dry. Refer to each microscope's manual for details of specific maintenance requirements. General advice is presented here.

   
Dust should be removed from the eyepieces and objectives using a blow-brush. Stubborn dirt adheres to the lenses. Stubborn dirt or fingerprints should be removed using a softly wadded piece of laboratory lens cleaning tissue. If necessary, dampen the lens by breathing on it.

Solvents should NEVER be used to clean a microscope lens or the "blooming" will be removed, resulting in distorted images and "halos" around specimens in the field of view.

Wipe down the body of the microscope occasionally, using a damp cloth.    
If an objective should come into contact with brine take immediate steps to prevent damage. Salt will corrode the objective Unscrew the objective from the nosepiece.Unscrew the outer housing of the objective.Wipe off any brine, paying particular attention to the thread where the objective screws in to the nosepiece.Wipe over with another lens tissue, dampened with fresh water.Repeat with several changes of dampened lens tissue until salt has been removed. Leave until completely dry before reassembling.
If dirt should enter the body of the microscope, unscrew the objectives and use a blow-brush to remove any loose debris.    
Regularly check that the condenser iris and lens are in working order.    

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