Cell counting using a Haemacytometer

Larger cells or long chains of cells are more appropriately counted using a Sedgwick-Rafter cell or settling chamber. A haemacytometer is used for cell densities >10⁴ cells/mL. The size of these chambers can vary with manufacturer but we use a Neubauer brand which consists of two chambers, each with a volume of 0.1 mm³, containing a marked counting grid 1 mm² in area .

Be extremely careful when handling haemacytometers as they are fragile if dropped and very expensive items of equipment costing up to $Aus 200.00 each

Method

1. Algal Sample – Non-motile cells which do not need fixing can be counted as soon as the sample is collected. However, if there will be a delay between sample collection and counting, or if the cells are motile then the sample will need to be preserved. The most common fixative used for marine microalgae is Lugol’s Solution. The recipe for the acidic form is given here but note in the case of microalgae with calcium carbonate scales, such as the coccolithophorids, the acid will destroy the organisms and a basic solution should be prepared instead.
   For cultures add 1 drop of 1-2% Lugol’s solution to 1 mL sample, for field samples 10 drops per 200 mL of sample or until the colour of weak tea. Overuse of Lugol’s will cause some delicate flagellate species to overstain, lose flagella or blow up entirely.
   Lugol’s is made by dissolving 100 g Potassium Iodide (KI) in 1 L of distilled water, then 50 g crystalline Iodine (I₂) is dissolved in this solution and then 100 mL glacial acetic acid is added. Lugol’s should be stored in the dark as the iodine is light sensitive and will degrade. It should also be stored with a tight fitting lid and kept away from the general culture environment.

Note: Sample dilution or concentration: The haemacytometer can be used where cell densities are in the range 5 x10⁴ – 10⁷ cells / mL. It is more likely that cultures will be less rather than more dense than this range but occasionally very dense cultures, such as nanoplanktonic flagellates and some cyanobacteria, may need to be counted. It is both inefficient and very difficult to accurately count these cultures without first diluting the sample (contrast with the Sedgwick-Rafter Cell where cell density range is 30 – 10⁴ cells / mL). Therefore dilute with a known volume of culture media and then fix. Alternatively a Lugol’s Dilution Solution (LDS) may be used where 100 mL of 0.2 mm filtered seawater or distilled water (depending on whether the culture is marine or freshwater) is prestained with concentrated Lugol’s until it is a weak tea colour. Then a known volume of culture can be added to a known volume of the LDS. Using LDS also means that all cells are exposed to the optimum concentration of Lugol’s whereas adding concentrated Lugol’s could destroy some cell types when it mixes into the sample. For example a culture with a density above 1 x10⁷ cells / mL will have >1000 cells in the counting region of a haemacytometer (see detailed explanation below) and a 1 in 5 dilution (1 mL of culture + 4 mL of LDS) will allow the sample to be counted more readily.

If the culture is dilute, concentrate by centrifuging or settling in a flat bottom measuring cylinder (allow 1 hour of sinking for each 10 mm of cylinder height; therefore overnight is a practical solution). For either method once concentrated, remove and discard up to 90% of the clear supernatant (upper portion of the liquid) without disturbing the settled biomass. Homogenize the remaining sample and count, bearing in mind the need to integrate the concentration factor = final count x (settled volume / initial volume).

2. To fill haemacytometer chambers, place the thick coverglass over both grids and take a Pasteur pipette and fill its tip by capillary action with sample. Hold the pipette at an angle of ~45⁰ (higher or lower to control flow rate) 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. The chamber surface in the Neubauer brand is a flat mirror-like rectangle and the sample must cover this rectangle but not flow over its edges. It is useful to rest your hand on a bench and steady the pipette tip with a finger.
   —       If flooding occurs, rinse haemocytometer and coverslip with distilled water, and repeat procedure.
   —       Refill the pipette for each chamber. The time taken to fill the chamber should be short, to minimize setting of cells in the pipette.

3. Allow cells to settle (~1 min) and check grid under the microscope (x 20 objective) for satisfactory distribution of cells, i.e. evenly spread.

4. The Haemacytometer grid in detail: The grid is divided into 9 large squares, each 1 mm x 1 mm, by triple lines. Each large square is divided into 25 medium squares, each 0.23 mm on a side, and each medium square is further divided into 16 small squares, each 0.05 mm on a side.
   For all haemacytometers, the fundamental measurement is the average number of cells per 1 mm square, so the centre large square is usually counted. To obtain the total number of cells in this large square, the number of cells in each of the 25 medium squares are counted, recorded then added (see sample cell count)

Note: When counting cells bordering on triple rulings, the convention is to count only those cells touching the top and left-hand side rulings of each square.

5. After counting each of the two haemacytometer chambers, the haemacytometer and coverslip are rinsed with distilled water. Usually the procedure is repeated twice more to give a total of 6 counts.
6. To obtain the cell density, calculate the average cell count and multiply by the conversion factor (for Neubauer = x10⁴)

Sample Cell Count

Tisochrysis lutea CS-177

Age; 8 days

Growth conditions; 25⁰C, light intensity 50 μmol. photons m^-2 s ^–1 , 12:12 light: dark cycle

Each block 1-6 represents the total number of cells in the large centre square.

Mean  =

 

Where = total no.
= sum of all totals

& n= no. of counts

Cell Count = x ccf (chamber conversion factor for Neubauer = x104) 

Standard deviation S= where variance =
                                        =

% Error = 

from above example

cell count ; = 174.3 x 104 x ccf
              = 1.74 x 106 cells/mL


standard deviation =

                     =
                     =9.99
%error = x 100 = 5.7%

Note: %error should be below 10%