Experimental principles of microbial microscopy direct counting method
Direct counting under a microscope using a hemocytometer is a commonly used method for counting microorganisms. The advantage of this method is that it is intuitive and fast. Place the appropriately diluted bacterial suspension (or spore suspension) in the counting chamber between the slide and the coverslip of the hemocytometer, and count under a microscope. Since the volume of the counting chamber is fixed (0.1 mm2), it can be converted into the total number of microorganisms per unit volume based on the number of microorganisms observed under the microscope. Since this method counts the total number of live and dead bacteria, it is also called the total bacterial count method.
A hemocytometer is usually a special slide with four slots forming three platforms. The middle platform is divided into two halves by a short horizontal groove. There is a square grid engraved on the platform on each side. Each square grid is divided into nine large squares. The large square in the middle is the counting room. The counting of microorganisms takes place in the counting chamber. The structure of the hemocytometer is shown in Figure Ⅷ-1.
The scale of the counting chamber generally has two specifications. One is a large square divided into 16 medium squares, and each medium square is divided into 25 small squares (Figure Ⅷ-2); the other is a large square. The square grid is divided into 25 medium squares, and each medium square is divided into 16 small squares (Figure VIII-1, C). But no matter what kind of counting board it is, the number of small squares in each large square is the same, that is, 16×25=400 small squares, as shown in Figure Ⅷ-2.
The side length of each large square is 1mm, so the area of each large square is 1mm2. After the cover glass is covered, the height between the slide and the cover glass is 0.1mm, so the volume of the counting chamber is 0.1mm3.
When counting, usually count the total number of bacteria in five medium squares, then find the average value of each middle square, and then multiply by 16 or 25 to get the total number of bacteria in a large square, and then calculate Convert to the total number of bacteria in 1ml of bacterial solution.
