Measurement of Microbial Quantity - Microscopic Direct Counting Method!
The growth of bacterial populations is characterized by an increase in the number of cells or an increase in cellular material. There are several methods for measuring cell numbers, including direct microscopic count, plate count, turbometry by spectrophotometer, most probable number MPN, and membrane filtration. The methods for measuring cellular substances include measuring cell dry weight, measuring the content of certain components of cells such as nitrogen, RNA and DNA, and measuring metabolic products. In summary, there are many methods for measuring microbial growth, each with its own advantages and disadvantages, and the selection should be based on specific requirements in the work. This experiment mainly introduces the commonly used microscope direct counting method in production and scientific research work.
Purpose requirements
1. Clarify the principle of using a blood cell counting board for counting.
2. Master the method of using a blood cell counting board for microbial counting.
2, Basic Principles
Microscopic direct counting method is a simple, fast, and intuitive method of directly counting a small amount of suspension of a sample under a microscope by placing it on a special glass slide with a certain area and volume (also known as a bacterial counter). At present, commonly used bacterial counting devices at home and abroad include blood cell counting plates, Peteroff Hauser bacterial counting devices, and Hawksley bacterial counting devices, all of which can be used for counting suspensions of yeast, bacteria, fungal spores, etc., with the same basic principle. The latter two types of 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 carrier slide is only 0.02mm. Therefore, an oil immersion objective can be used to observe and count smaller cells such as bacteria. In addition to using these bacterial counters, there is also an estimation method for directly observing the ratio of smear area to field area under a microscope, which is generally used for bacteriological examination of milk. The advantages of direct counting with a microscope are intuitive, fast, and easy to operate. But the disadvantage of this method is that the measured results are usually the sum of dead and live cells. At present, there are some methods that can overcome this drawback, such as combining live cell staining chamber culture (short time) and adding cell division inhibitors to achieve the goal of only counting live cells.
Using a blood cell counting board as an example for direct counting under a microscope. The usage methods of the other two bacterial counters can be found in the manuals of various manufacturers. Direct counting under a microscope using a blood cell counting plate is a commonly used method for microbial counting. The counting board is a specially made glass slide with four grooves forming three platforms on it; The wider platform in the middle is divided into two halves by a short horizontal groove, and there is a grid on each side of the platform. Each grid is divided into nine large grids, and the large grid 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 that a large square is divided into 25 middle squares, and each square is further divided into 16 small squares (Figure 15-2); Another method is to divide a large square into 16 middle squares, and each square is further divided into 25 small squares. However, regardless of the size of the counting board, there are 400 small squares in each large square. If the side length of each square is 1mm, the area of each large square is lmm2. After covering with a cover glass, the height between the cover glass and the slide is 0.1mm, so the volume of the counting room is 0.1mm3 (one thousandth of a milliliter). Figure 15-1 Structure of Blood Cell Counting Board (I) Figure 15-2 Structure of Blood Cell Counting Board (II) A Front view; B. Vertical section view; The enlarged grid has a large square in the middle as the counting room 1. Blood cell counting board; 2. Cover glass; When counting in the counting room, the total bacterial count is usually calculated for five squares, then the average value of each square is obtained, multiplied by 25 or 16 to obtain the total bacterial count in a large square, which is then converted into the total bacterial count in the lml bacterial solution. Assuming the total bacterial count in five square grids is A and the dilution factor of the bacterial solution is B. If there are 25 counting plates in the square grid, the total bacterial count in 1mL of bacterial solution is A/5 × 25 × 104 × B=50000A · B. Similarly, if there are 16 counting plates in the square grid, the total bacterial count in 1mL of bacterial solution is A/5 × 16 × 104 × B=32000A · B
