Microbiological microscope direct counting
Experimental principle
Direct counting under a microscope using a hemocytometer is a commonly used microbial counting method. 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 cover glass of the hemocytometer, and count under the microscope. Since the volume of the counting chamber is fixed (0.1mm2), it can be converted into the total number of microorganisms per unit volume according to the number of microorganisms observed under the microscope. Because this method counts the sum of live and dead bacteria, it is also called the total bacteria count method.
A hemocytometer is usually a special glass slide on which four grooves form three platforms. The platform in the middle is divided into two halves by a short horizontal groove. A grid is engraved on the platform on each side. Each grid is divided into nine large squares. The large square in the middle is the counting room. Microorganisms are counted in the counting chamber.
The scale of the counting room generally has two specifications, one is that a large square is divided into 16 middle squares, and each middle square is divided into 25 small squares (Figure Ⅷ-2); the other is a large square The square is divided into 25 middle squares, and each middle square is divided into 16 small squares (Figure Ⅷ-1, C). But no matter what kind of counting board, the number of small squares in each large square is the same, that is, 16×25=400 small squares.
The side length of each large square is 1 mm, and the area of each large square is 1 mm2. After the cover glass is covered, the height between the slide glass and the cover glass is 0.1 mm, so the volume of the counting chamber is 0.1mm3.
When counting, usually count the total number of bacteria in the five middle squares, then obtain 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 Converted to the total number of bacteria in 1ml bacterial solution.
Laboratory equipment
Hemocytometer, microscope, coverslip, sterile capillary;
Experimental Materials
Saccharomyces cerevisiae suspension
Experimental procedure (microbial microscope direct counting method)
1. dilution
Properly dilute the Saccharomyces cerevisiae suspension. If the bacterial solution is not thick, it does not need to be diluted.
2. Microscopic counting room
Before adding samples, perform a microscopic inspection of the counting chamber of the counting plate. If there is dirt, it needs to be cleaned before counting.
3. add sample
Cover the clean and dry hemocytometer plate with a cover glass, and then use a sterile narrow-mouth dropper to drop a small drop of the diluted Saccharomyces cerevisiae solution from the edge of the cover glass (not too much), so that the bacteria solution is close to the gap along the gap. Capillary osmosis enters the counting chamber by itself, and the general counting chamber can be filled with bacterial liquid. Be careful not to have air bubbles.
4. microscope count
After resting for 5 minutes, place the hemocytometer on the stage of the microscope, first use a low power lens to find the location of the counting chamber, and then switch to a high power lens for counting. If it is found that the bacterial solution is too thick or too thin before counting, it is necessary to re-adjust the dilution before counting. The general sample dilution requires about
5-10 cells are suitable. Each counting chamber selects the bacteria in 5 middle grids (optional 4 corners and central grids) for counting. The bacteria on the grid line are generally only counted on the upper and right lines. In case of yeast budding, when the size of the bud body reaches half of the mother cell, count two bacteria. Count a sample to calculate the bacterial content of the sample from the values counted in the two counting chambers.
5. Cleaning the Hemocytometer
After use, rinse the blood cell counting plate on the faucet with water jets, do not wash it with hard objects, and dry it by yourself or with a hair dryer after washing. Microscopic examination, observe whether there are residual bacteria or other sediments in each cell. If it is not clean, it must be washed repeatedly until it is clean.
