Using a Microscope Counting Chamber

The specific grid pattern etched into a hemacytometer divides the space into nine large squares, each of which is 1 mm (Fig- 14.3). The central counting area (square 5) contains 25 middle-sized squares and each of these has 16 smaller squares. Whereas some microbiologists only count the central large square, others will tally the corner squares as well (squares 

and 9). Because the cover glass rests exactly 0.1mm above the bottom of the chamber, the volume of liquid under each of the large squares is 0.1 mm or 0.1 jiL. 

When enumerating samples expected to have no or very low population densities (e.g., wines from the bottling line), it is necessary to concentrate known volumes by membrane filtration ( 0.45 pm) prior to microscopic examination. In these cases, conveniently sized portions of the membrane may then be stained and examined microscopically (Kunkee and Neradt, 

Prepare the appropriate dilutions of the sample to be examined using 9mL dilution blanks (Fig. 14.1) with thorough mixing between dilutions. 

Place the hemacytometer cover slip over the counting grid of the hemacytometer. 

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To measure performance, to achieve reproducibility or to make comparative studies, a means of quantifying the cell population is needed. Classically, direct counts of cell numbers using a microscopic counting chamber (haemocytometer), usually in conjunction with a vital stain (e.g. Trypan blue) to distinguish viable and non-viable cells, is used. However, all vital stains are subjective and cannot give absolute values, and cell numbers take no account of differences in cell size/mass. The method is simple, quick and cheap, and requires only a small fraction of the total cells from a cell suspension. 

Count the cells using a microscope counting chamber (hemo-cytometer) and resuspend in H441 culture medium at a density of 1.67x10 cells per ml before transferring to a reagent reservoir. 

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