Determination of the number of microorganisms-Microscopic direct counting method!
Bacterial population growth is manifested by an increase in cell number or an increase in cellular material. Methods for determining the number of cells include direct microscopic count, plate count, turbidityestimation by spectrophotometer, most probable number MPN and membrane filtration. (membrane filtration) etc. Methods for measuring cellular substances include the determination of cell dry weight, determination of certain cell components such as nitrogen content, RNA and DNA content, and determination of metabolite products. In short, there are many methods for measuring microbial growth, each with its own advantages and disadvantages. The method should be selected according to the specific requirements of the work. This experiment mainly introduces the microscope direct counting method that is commonly used in production and scientific research work.
1. Purpose requirements
1. Clarify the principle of counting with a blood cell counting board.
2. Master the method of counting microorganisms using a blood cell counting board.
2. Basic principles
Microscope direct counting method is a simple, fast and intuitive method that places a small amount of suspension of the sample to be tested on a special slide with a determined area and volume (also called a bacteriometer) and counts it directly under a microscope. Methods. Currently, commonly used bacteriometers at home and abroad include: hemocytometer, Peteroff-Hauser bacteriometer, and Hawksley bacteriometer, etc. They can all be used to count yeast, bacteria, mold spores and other suspensions, and the basic principles are the same. The latter two bacterial 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. Therefore, an oil immersion objective lens can be used to observe and detect smaller cells such as bacteria. count. In addition to using these bacteriometers, there is also an estimation method that directly observes the ratio of the smear area to the visual field area under a microscope. This method is generally used for bacteriological examination of milk. The advantages of the microscope direct counting method are that it is intuitive, fast and easy to operate. However, the disadvantage of this method is that the measured result is usually the sum of dead bacteria and live bacteria. There are currently some methods to overcome this shortcoming, such as combining viable bacteria staining microchamber culture (short time) and adding cell division inhibitors to achieve the purpose of counting only viable bacteria.
This experiment uses a hemocytometer as an example to conduct direct counting under a microscope. For the use of the other two bacteriometers, please refer to the instructions of each manufacturer. Direct counting under a microscope using a hemocytometer is a commonly used microbial enumeration method. The counting board is a special slide with four grooves forming three platforms; the wider platform in the middle is divided into two halves by a short horizontal groove, and there is a grid grid on each side of the platform. Each square 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 board is shown in Figure 15-1. There are generally two specifications for the scale of the counting chamber. One is a large square divided into 25 medium squares, and each medium square is divided into 16 small squares (Figure 15-2); the other is a large square. The square grid is divided into 16 medium squares, and each medium square is divided into 25 small squares. However, 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 lmm, then the area of each large square is lmm2. After the cover slip is covered, the height between the cover slip and the slide is 0.lmm, so the volume of the counting chamber is 0.lmm3 (one ten thousandth of a milliliter). Figure 15-1 Structure of the blood cell counting board (1) Figure 15-2 Structure of the blood cell counting board (2) A. Front view; B. Longitudinal section; enlarged square grid, the large square in the middle is the counting chamber 1. Blood cells Counting board; 2. Cover slip; 3. When counting in the counting chamber, usually count the total number of bacteria in five squares, then find the average value of each square, and then 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. Suppose the total number of bacteria in the five middle squares is A, and the dilution factor of the bacterial solution is B. If it is a counting plate with 25 middle squares, then the total number of bacteria in 1mL of the bacterial solution = A/5×25×104× B=50000A·B (pieces) In the same way, if it is a counting plate with 16 squares, the total number of bacteria in 1mL of bacterial solution =A/5×16×104×B=32000A·B (pieces)、、
