BIOLOGY Volume 2 - A Guide to General Biology - 2004

12. MICROBIOLOGY AND BIOTECHNOLOGY

12.6. Measuring the growth of bacteria and fungi in culture

12.6.2. Total cell count

Haemocytometer

The number of bacterial or Yeast Cells in a liquid culture, such as a broth, can be counted directly using a Microscope. This method is convenient for counting yeast cells, which are much larger than Bacteria. An oil-immersion objective is required when counting bacteria (see section 5.11.2).

Usually, a special Glass slide known as a haemocytometer (Fig. 12.10) is used for counting, and the method itself is known as haemocytometry. To ensure firm contact between the slide and the coverslip, the coverslip should be pressed down firmly on either side of the chamber (without breaking the coverslip!). The coverslip is then gently slid until coloured Interference lines appear (Fig. 12.10). A sample of the liquid, applied with a pipette, is drawn under the coverslip by capillary action. The chamber is left for 2–3 min to allow the cells to settle, which facilitates counting. The microscope is focused on the grid, and the number of cells is counted across the entire grid or in a representative section of it. At least 600 cells should be counted. If cells lie on a boundary, they can be assigned to a square bounded by two sides (e.g., the top and right boundaries) and excluded from the square bounded by the other two sides; however, one should not attempt to count half-cells. Each small square has a known area, and the depth of the liquid beneath the coverslip is constant (usually 0.02 or 0.1 mm). Consequently, the volume of each compartment can be calculated, and the number of cells in a given volume determined. If the number of cells in each square exceeds 30, the sample should be diluted. Remember to account for the dilution factor in your calculations.

On the grid shown in Fig. 12.10, each small square measures 0.05 mm × 0.05 mm = 0.0025 mm², with a total grid area of 1 mm². If the gap between the coverslip and the grid is 0.02 mm, the total volume above the grid is 1 × 0.02 mm = 0.02 mm³. The number of cells in a given sample volume can thus be calculated. Remember that 1000 mm³ = 1 cm³, and 1000 cm³ = 1 dm³ (1 l).

Methylene blue dye can be used when counting yeast cells. In this case, only dead cells take up the blue stain. Living cells actively pump the dye out of The Cell. Thus, the Number of viable cells can be determined by counting only colourless or pale blue cells.

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Fig. 12.10. Haemocytometry.

Turbidity measurement

The method known as nephelometry is straightforward in principle. The greater the number of cells in suspension, the higher its turbidity, or "cloudiness", the degree of which can be measured.

The simplest approach is to use commercially available standard tubes (Brown's tubes). These contain barium sulphate Suspensions of varying concentrations, ranging from clear (tube 1) to opaque (tube 10). A sample of the microorganism suspension under study is placed in an identical tube, and one of the standard tubes that best matches the turbidity of the suspension is selected. A table is provided with the set of standard tubes, allowing cell concentrations to be calculated from the turbidity for a wide range of different microorganisms.

Alternatively, turbidity can be measured as the fraction of light transmitted (or optical density) by the suspension using a colorimeter or spectrophotometer. Cell mass is directly proportional to optical density. It is best to use red light (a red filter), as it does not interfere with the yellowish colour of many culture media.

Errors can occur when measuring turbidity if the growing cells form clumps (see the section on viable cell counts). The most accurate results are obtained when the population density is neither too high nor too low. To achieve an optimal cell density (10⁶ - 10¹⁰ cells · cm^-3), sample dilution is sometimes required.