Cell counting using a Sedgwick-Rafter chamber

These counting chambers have been developed for the counting of large cells or where the cultures form long chains or colonies and are suitable where the cell density range is < 10,000 cells / mL.

The settling chamber has a volume of 1 mL. To calculate the cell density of a dense algal culture where the cells are relatively small (~ 5-50 µm) and either single cells or short chains, a haemacytometer is more appropriately used.

Method

    1. While the Sedgwick-Rafter chambers are nominally 1 mL, variations in manufacture can create variations in chamber volume. Therefore, it is recommended that prior to use that the chamber volume is calibrated. The volume is calibrated by weight by filling the chamber with deionised water by the Chamber filling method below. The average is calculated from 10 measurements. The chamber measurements of 50 mm long, 20 mm wide and 1 mm deep should also be checked. Any variation from the nominal values that is greater than 5% should be accounted for as a calibration factor in any cell counts.

Fig. 1 Filling a Sedwick-Rafter Cell

Chamber filling method: To fill counting chamber, place the thick coverslip diagonally across the cell. Use a pipette to carefully fill the chamber from one corner. Once filled adjust the coverslip to cover the chamber, avoiding bubbles within the chamber.

 

  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 Lugols 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 Sedgwick-Rafter can be used where cell densities are < 104 cells / mL. Where culture are very dense and a Haemocytometer is not suitable, such as for some cyanobacteria, it is both inefficient and very difficult to accurately count these cultures without first diluting the sample (contrast with). 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 0.22 μm filtered seawater or distilled water (depending on whether the culture is marine or freshwater) is prestained with concentrated Lugols 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.
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).
  1. Mix the sample gently but well and sub-sample the calibration volume of culture with a wide-tipped pipette. Fill the chamber to the method described above. The time taken to fill the chamber should be short to minimise the settling of cells in the pipette.
    —       If flooding or bubbles occur, rinse chamber and coverslip with deionised water, dry with lint-free absorbent paper and repeat procedure.
  2. Allow cells to settle for (~15-30 min) or as little time as necessary after focusing through the water column at several points in the chamber to ensure there are no planktonic cells. A benefit of Lugol’s is that it increases cell density and shortens settling time. Once the cells have settled, the chamber should also be checked at a low magnification for an even spread of settled cells and to confirmno cells have floated to the top of the chamber. Cultures that are naturally buoyant either from gas-vacuoles (eg. some cyanobacteria) or oil droplets (eg. Botryococcus) are difficult to count by this method.
  1. For very dilute samples of large cells or colonies, the entire chamber can be counted to give a cell/colony count. For more dense cultures, the chamber is divided into a grid of 50 squares long x 20 squares width. Cell/colony counts are made for each square for one or two long transects in the chamber. Counting is made to a minimum of 30 counts or until the average and standard deviation of counts per square is stable. Cyanobacteria trichomes can be counted per square as above. Where cyanobacteria cell counts are desired then the number cells per trichome can be counted again for a minimum of 30 trichomes or until the average and standard deviation are stable.
Note: When counting cells bordering on grid lines, our convention is to count only those cells touching the top and left-hand side rulings of each square.

Sedgwick-Rafter Cell grid indicating algal cells to be included in counts

Fig. 2 Sedgwick-Rafter Cell grid indicating edge and empty algal cells to be included/excluded in counts

  1. After counting of the chambers, the chamber and coverslip are rinsed with deionised water.
  1. The cell or colony concentration (C) to unit per mL for a transect count is calculated using the equation below, followed by adjustments for any dilution or concentration factors.

C = (N x 1000 mm3) / (L x D x W x S)

Where:

N = number of cells/colonies counted

L = length of transect strip (mm)

W = width of transect strip (mm)

D = chamber depth (mm)

S = number of transects counted

 

General tip for sample storage tubes and choice of loading pipette: In experiments with a large number of treatments and/or replicates where there may be several days or even weeks between sample collection and counting, it is recommended to store preserved count tubes out of the sun and preferably in a fridge. Typical subsamples may be 1 – 5 mL and may be further diluted if the culture is dense as previously indicated. The lower the count-tube volume and the longer the storage time the greater is the risk of tube desiccation and loss of unknown volume compromising derived cell counts.  Cells in the sample may stick to the tube bottom and sides, most notably observed in plastic containers but also in glass tubes. Gentle aspiration may be necessary to homogenize the sample. Our experience with several cyanobacteria species shows that this is sometimes not enough with species producing  sticky  exudates and  a known volume of a “non-stick” solution may be useful in pre-coating the tube bottom before samples are taken.

 

References:

Guillard R. R. L. and Sieracki M. S. (2005) in Andersen R. A, “Algal Culturing Techniques”, Elsevier/Academic Press; Burlington, Massachusetts pg 244-249.

Hotzel G. and Croome R. (1999) in “A phytoplankton methods manual for Australian rivers”, Land and Water Resources Research and Development Corporation; Canberra pg 17-27.

Woelkerling W. J., Kowal R. R. and Gough S. B. (1976) “Sedgwick-Rafter cell counts: a procedural analysis” 48:2; 95-107; Hydrobiologia