Direct Counting Method for Microorganisms Under Microscope
Direct microscopic counting using a hemocytometer is a common method for microbial enumeration. This method features intuitive operation and rapid detection. An appropriately diluted bacterial suspension (or spore suspension) is added to the counting chamber between the slide and coverslip of a hemocytometer for microscopic counting. Since the volume of the counting chamber is fixed (0.1 mm³), the total number of microorganisms per unit volume can be calculated according to the microbial count observed under the microscope. As this method counts both living and dead microbial cells, it is also known as the total bacterial counting method.
A hemocytometer is a specially designed glass slide with four grooves forming three platforms. The middle platform is further divided into two halves by a short transverse groove. A grid pattern is engraved on each half of the middle platform, and each grid consists of nine large squares. The central large square serves as the counting chamber, where microbial counting is performed. The structure of the hemocytometer is shown in Figure Ⅷ-1.
There are generally two specifications for the graduation of counting chambers. In the first type, one large square is divided into 16 medium squares, and each medium square is further divided into 25 small squares (Figure Ⅷ-2). In the second type, one large square is divided into 25 medium squares, and each medium square contains 16 small squares (Figure Ⅷ-1, C). Regardless of the specification, the total number of small squares in one large square remains consistent: 16 × 25 = 400 small squares, as shown in Figure Ⅷ-2.
Each large square has a side length of 1 mm, giving an area of 1 mm². After covering with a coverslip, the height between the slide and the
coverslip is 0.1 mm; therefore, the volume of the counting chamber is 0.1 mm³.
During counting, the total number of bacteria in five medium squares is usually counted first. The average count of a single medium square is calculated and then multiplied by 16 or 25 to obtain the total bacterial count in one large square. Finally, the total microbial count per milliliter of bacterial suspension can be converted accordingly.
