Determination of the number of microorganisms - the direct counting method of the microscope!
Bacterial population growth is manifested by an increase in cell number or an increase in cellular mass. The methods for determining the number of cells include direct microscope counting method, plate colony counting method, photoelectric oil ratio method, maximum probability method, and membrane filtration method. The methods of measuring cell matter include the determination of cell dry weight, the determination of certain cellular components such as nitrogen, RNA and DNA content, and the determination of metabolites. In short, there are many methods for measuring the growth of microorganisms, each with its own advantages and disadvantages, and should be selected according to the specific situation. This experiment mainly introduces the microscope direct counting method commonly used in production and scientific research.
1. Purpose Requirements
1. Clarify the principle of blood cell count.
2. Master the method of counting microorganisms using a blood cell counter.
2. Basic principles
Microscope direct counting method is a simple, fast and intuitive method to directly count a small amount of suspension of the sample to be tested on a special glass slide with a certain area and volume (also known as a bacteria counter). Methods. At present, the commonly used bacteria counters at home and abroad are: blood cell counting board, Peteroff-Hauser bacteria counter and Hawksley bacteria counter, etc. They can be used for the counting of yeast, bacteria, mold spores and other suspensions, and the basic principle is the same. The latter two types of bacteria counters have a total volume of 0.02mm3 after being covered with a cover glass, and the distance between the cover glass and the slide is only 0.02mm, so the oil immersion objective lens can be used to observe and observe small cells such as bacteria. count. In addition to these bacteriometers, there is also an estimation method for the ratio of the area of the smear to the area of the visual field observed directly under the microscope, which is generally used for bacteriological examination of milk. The advantages of microscope direct counting method are intuitive, fast and easy to operate. However, the disadvantage of this method is that the measured result is usually the sum of dead and live cells. At present, there are some methods to overcome this shortcoming, such as the combination of viable bacteria staining microchamber culture (short time) and the addition of cell division inhibitors to achieve the purpose of counting only viable bacteria.
In this experiment, a hemocytometer was used as an example for direct microscopic counting. For the use of the other two types of bacteria counters, please refer to the instructions of each manufacturer. Counting directly under the microscope with a hemocytometer is a commonly used method for counting microorganisms. The counting plate is a special glass slide, on which three platforms are formed by four slots; the wider platform in the middle is divided into two halves by a short transverse slot, and there is a grid on each side of the platform. Each grid is divided into nine large squares, and the large square in the middle is the counting room. The structure of the blood cell counting plate is shown in Figure l5-1. The scale of the counting room generally has two specifications, one is a large square divided into 25 middle squares, and each middle square is divided into 16 small squares (Figure 15-2); the other is a large square. The square is divided into 16 middle squares, and each middle square is divided into 25 small squares, but no matter what kind of counting board it is, there are 400 small squares in each large square. The side length of each large square is 1mm, and the area of each large square is 1mm2. After covering with a cover glass, the height between the cover glass and the slide glass is 0.1mm, so the volume of the counting chamber is 0.lmm3 (one thousandth of a milliliter). Figure 15-1 The structure of the blood cell counting board (1) Figure 15-2 The structure of the blood cell counting board (2) A. Front view; B. Longitudinal section view; The enlarged grid, the large square in the middle is the counting chamber 1. Blood cells Counting plate; 2. Cover glass; 3. When counting in the counting chamber, usually count the total number of bacteria in five squares, then calculate the average of each square, and multiply by 25 or 16 to get The total number of bacteria in a large square is then converted into the total number of bacteria in 1ml of bacterial solution. Let the total number of bacteria in the five squares be A, and the dilution ratio of the bacterial solution to be B. If it is a counting plate with 25 squares, the total number of bacteria in 1 mL of bacterial solution = A/5×25×104× B=50000A·B(pieces) Similarly, if it is a counting plate with 16 medium squares, the total number of bacteria in 1mL bacterial solution=A/5×16×104×B=32000A·B (pieces)
3. Equipment
1. Bacteria
Saccharomyces cerevisiae
2. Instruments or other utensils
Hemocytometer, microscope, coverslip, sterile capillary dropper.
4. Operation steps
1. Preparation of bacterial suspension
Saccharomyces cerevisiae was prepared into a bacterial suspension of appropriate concentration with sterile physiological saline.
2. Microscope counting room
Before adding samples, microscopically inspect the counting chamber of the counting plate. If there is dirt, it needs to be cleaned and dried before counting.
3. Add sample
Cover the clean and dry hemocytometer with a coverslip, and then use a sterile capillary dropper to drop a small drop of the shaken Saccharomyces cerevisiae suspension from the edge of the coverslip, and let the bacterial solution automatically move along the gap by capillary osmosis. Entering the counting room, the general counting room can be filled with bacterial liquid. When sampling, firstly shake the bacterial solution; when adding samples, no air bubbles should be generated in the counting chamber.
4. Microscope counting
After adding the sample, stand still for 5 minutes, then place the hemocytometer on the microscope stage, first find the location of the counting chamber with a low-power microscope, and then switch to a high-power microscope for counting. Adjust the intensity of the microscope light appropriately. For microscopes that use mirrors for lighting, pay attention not to deviate from one side of the light, otherwise it will not be easy to see the square lines of the counting room clearly in the field of vision, or only vertical lines or horizontal lines will be seen. If it is found that the bacterial solution is too concentrated or too diluted before counting, it is necessary to readjust the dilution before counting. Generally, the sample dilution requires about 5 to 10 bacteria in each small cell. Each counting chamber selects 5 middle cells (optional 4 corners and one middle cell in the center) for counting. The cells located on the grid line are generally only counted on the upper and right lines. In the case of yeast budding, when the size of the bud reaches half of the mother cell, it is counted as two bacterial cells. To count a sample, calculate the bacterial content of the sample by calculating the average value from the two counting chambers.
5. Wash the blood cell count
After use, rinse the blood cell counting board with water in the tap, do not scrub with hard objects, and dry it by yourself or with a hair dryer after washing. Microscopic examination to observe whether there are residual bacteria or other sediments in each small cell. If it is not clean, it must be washed repeatedly until it is clean.
