First, the basic principle of using the blood cell counting board to count directly under the microscope, is a commonly used microbial counting method. The advantage of this method is intuitive and fast. The appropriately diluted bacterial suspension (or spore suspension) is placed in a counting chamber between the hemocytometer slide and the coverslip and counted under a microscope. Since the volume of the counting chamber is constant (0.1 mm2), it can be converted into the total number of microorganisms per unit volume based on the number of microorganisms observed under a microscope. Since this method calculates the sum of living cells and dead cells, it is also called total bacterial counting method.
The hemocytometer, usually a special glass slide, consists of four slots on three platforms. The middle platform is separated into two halves by a short horizontal groove. Each side of the platform is engraved with a square grid. Each square grid is divided into nine large squares. The large square in the middle is the counting room. The counting of microorganisms is carried out in the counting chamber. The structure of the hemocytometer is shown in Figure VIII-1.
The scale of the counting room generally has two specifications. One is that a large square is divided into 16 squares, and each square is divided into 25 small squares (Fig. VIII-2); the other is a large square. The square is divided into 25 squares, and each square is divided into 16 small squares (Fig. VIII-1, C). But no matter which size of the counting board, 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 length of each large square is 1mm, and 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 the five squares, and then find the average of each square, then multiply 16 or 25 to get the total number of bacteria in a large square, and then Convert to the total number of bacteria in 1 ml of bacterial solution.
The following is an example of a large square grid with 25 squares as the example: the total number of bacteria in the five squares is A, and the dilution factor of the bacteria is B. Then, the total bacteria in a large square The number is 1ml=1cm3=1000mm3,
(ie the total number of bacteria in 0.1mm3) = A*B*25/5
Therefore, the total number of bacteria in 1ml of bacterial liquid = A * 25 * 10 * 1000 * B / 5
=50000A·B(a)
Second, equipment Saccharomyces cerevisiae suspension, blood cell counting plate, microscope, coverslip, sterile capillary.
Third, the operation steps 1. Dilute the Saccharomyces cerevisiae suspension properly, if the bacterial solution is not concentrated, it may not be diluted.
2. The microscopic inspection chamber performs a microscopic examination on the counting chamber of the counting plate before loading. If there is dirt, it needs to be cleaned before counting.
3. Add a sample and cover the cleaned and dried blood cell counting plate with a cover slip. Then use a sterile fine-mouthed dropper to drip the diluted Saccharomyces cerevisiae solution from the edge of the coverslip by a small drop (not too much), so that the bacterial liquid along the edge The gap enters the counting chamber by capillary permeation, and the counting chamber can be filled with the bacteria liquid. Be careful not to have bubbles.
4. After the microscope count is stationary for 5 minutes, the blood cell counting plate is placed on the microscope stage, and the position of the counting chamber is first found by a low power microscope, and then replaced by a high power microscope for counting. If the bacterial liquid is too concentrated or too thin before counting, it is necessary to readjust the dilution and then count. Generally, the dilution of the sample requires about 5-10 cells per cell. The cells in the 5 median (optional 4 angles and the central median) were counted in each counting chamber. The cells on the grid line are generally only on the upper and right lines. In the case of yeast sprouting, when the size of the bud reaches half of the mother cell, it is counted as two cells. The sample is counted from the values ​​counted in the two counting chambers to calculate the bacterial content of the sample.
5. After cleaning the blood cell counting plate, flush the blood cell counting plate on the water head with water column. Do not wash with hard objects. After washing, dry it by yourself or dry it with a hair dryer. Microscopic examination to see if there are residual bacteria or other sediments in each cell. If it is not clean, it must be washed until it is clean.
Fourth, the experimental report
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