Microscopic Direct Counting Method for Microorganisms
Direct counting using a hemocytometer under a microscope is a common method for enumerating microorganisms. This method features the advantages of being intuitive and rapid. A properly diluted bacterial suspension (or spore suspension) is loaded into the counting chamber between the hemocytometer slide and coverslip 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 based on the number of microorganisms observed under the microscope. As this method counts both live and dead microbial cells, it is also known as the total count method.
A hemocytometer is a special glass slide consisting of three platforms formed by four grooves. The middle platform is further divided into two halves by a transverse groove. A grid is etched on each half of the platform. Each grid is divided into nine large squares, and 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 two common types of graduation specifications for counting chambers. One type has a large square divided into 16 medium squares, and each medium square is further subdivided into 25 small squares (Figure Ⅷ‑2). The other type has a large square divided into 25 medium squares, and each medium square is subdivided into 16 small squares (Figure Ⅷ‑1C). Regardless of the specification of the counting chamber, the total number of small squares within each large square remains the same: 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 placing the coverslip, the height between the slide and coverslip is 0.1
mm; therefore, the volume of the counting chamber is 0.1 mm³.
For counting, the total number of microorganisms in five medium squares is usually counted. The average value for each medium square is then calculated and multiplied by 16 or 25 to obtain the total count within one large square. Finally, this value is converted to the total number of microorganisms in 1 mL of the bacterial suspension.
