1. Marine Biotechnology Lab
Monitoring algal populations

2. Monitoring algal Monitoring is essential to:
A regular check of microalgae cultures.
Monitoring is essential to:
Prevent crashes .
To keep high quality standards.

3. The main parameters to be monitored are:
Color.
Density.
pH .
Contaminant levels.
As an example, a change in color to grey and a pH level lower than 7.5 may indicate a high degree of bacterial contamination.
A lighter color than normal may reveal insufficient nutrients or poor lighting.

4. Algal cell count in cultures is useful for a number of other reasons as it is possible:
To determine the growth curve for each species of algae under local conditions.
To quick identification of possible troubles (e.g. presence of foam, changes in color, etc.).
To determine optimal utilization time, i.e. the age at which the algal population reaches the peak of the log-phase.
To adjust environmental conditions to maximize production.
To control possible contaminants and try countermeasures.

5. Haemocytometer The haemocytometer (counting chambers)
is a device used to count cells. It was originally designed for the counting of blood cells.
Also they can be used to calculate the cell density of an algal culture providing the cells are relatively small.

6. This device consists of a thick rectangular slide with two counting areas.
The size of these chambers can vary with manufacturer but we use a Neubauer brand.
With its special cover slip in place, each area forms a chamber 0.1 mm deep.
The total area of each chamber is 9 mm2. Each chamber is divided into 9 large squares, each 1mm x 1mm (1mm2).
The four corner squares are subdivided into 16 medium squares, whereas the central one is subdivided into 25 medium squares, and each square is further divided into 16 small squares.

8. Algal Sample
Non-motile cells which do not need fixing can be counted as soon as the sample is collected.
If the cells are motile the sample will need to be fixative by Lugol’s Solution.

10. Sample dilution or concentration:
The haemocytometer can be used where cell densities are in the range 5 x cells / mL .
If you have very dense cultures may need diluting the sample to be counted.
Homogenize the remaining sample and count, bearing in mind the need to integrate the dilution factor.

11. Procedure
Prepare clean Neubauer slides and covers; clean slide and cover-glass with Kleenex paper.
Attach the cover glass to the counting chamber, pressing it carefully into place. When the cover glass is properly attached, colored bands, called Newton’s rings, appear between the two glass surfaces.
Mix well the sample.
Take a pipette and fill its tip with sample.

12. Hold the pipette at an angle of ~450 and place the tip at the leading edge of the coverslip.
With very gentle pressure, allow the sample to flow quickly and evenly into the chamber, exactly filling it.
Take care not to overfill beyond the ruled area. It is useful to rest your hand on a bench and steady the pipette tip with a finger.
Leave the counting chamber on the bench for 1-3 minutes before counting to allow the cells to settle, then Check under microscope.

14. Counting of Algae
Count the number of cell in the four corner squares .
Counting cells that are on a line:
Cells that touch the bottom and right lines of a square should not be counted, cells on the top and left side should be counted.

15. Count the cells in the square and those which touch the top and left border. Do not count the ones touching the right and lower border.

16. Record your results in a table .
After the counting, the haemocytometer and coverslip are rinsed with distilled water.
To obtain the cell density, calculate the average cell count and multiply by the conversion factor (for Neubauer = x104)

17. Calculation
The cell density per milliliter or the number of cells/ml = average cell count × 104 (which is called the chamber conversion factor for Neubauer).
Total cells = Cell density (cell/ml) X Volume (ml)

18. Example

20. Important Notes:
If the sample was diluted; multiply the above value by the dilution factor, where the
dilution factor = total volume/sample volume.
Dilution of 5 ml sample in 95 ml of water will give a dilution factor = 100/5 = 20.
If the sample was concentrated; multiply the above value by the concentration factor, where
the concentration factor = settled volume/initial volume.
Settling of 1 liter sample to 100 ml will give concentration factor of 100/1000 = 1/10.

21. Plotting of the Growth Curve.
Determined the cell number each day of your experiment.
Plot the cells number against time in days.