Determination of microbial population - direct microscopic counting method!
I. Purpose Requirements
1,To clarify the principle of haematocrit plate counting.
2,Master the method of microbial counting using haematocrit plate.
II. Basic principles
Microscope direct counting method is a small amount of suspension of the sample to be tested on a special slide with a defined area and volume (also known as the bacteriological counter), in the microscope directly counting a simple, rapid, intuitive method. At present, domestic and foreign commonly used bacteriological counters are: blood cell counting plate, Peteroff-Hauser bacteriological counters and Hawksley bacteriological counters, etc., which can be used for yeast, bacteria, mould spores and other suspensions of counting, the basic principle is the same. The latter two types of counters have a total volume of 0.02mm3 after the coverslip is put on, and the distance between the coverslip and the carrier slice is only 0.02mm, so smaller cells such as bacteria can be observed and counted with an oil immersion objective. In addition to the use of these counters, there is also an estimation method of the ratio of smear area to field of view area observed directly under the microscope, which is generally used for bacteriological examination of cow's milk. The advantages of the direct microscope counting method are intuitive, rapid and easy to operate. However, the disadvantage of this method is that the result is usually the sum of dead and live organisms. There are some methods to overcome this disadvantage, such as the combination of live bacteria stained microchamber culture (short time) and the addition of cytokinesis inhibitors to achieve the purpose of counting only live organisms.
Operation steps
1,Bacterial suspension preparation
Use sterile saline to make bacterial suspension of brewer's yeast with appropriate concentration.
2,Mirror counting chamber
Before adding samples, the counting chamber of the counting plate should be inspected first. If there is any dirt, it needs to be cleaned and blown dry before counting.
3,Adding samples
Clean and dry blood cell counting plate covered with a coverslip, and then use a sterile capillary burette to shake the brewer's yeast suspension from the edge of the coverslip to drop a small drop, so that the bacterial liquid along the gap by capillary penetration into the counting chamber automatically, generally counting chamber can be filled with bacterial liquid. When taking samples, the bacterial solution should be shaken well first; when adding samples, the counting chamber should not have air bubbles.
4,Microscope counting
After adding the sample, let it rest for 5min, then put the blood cell counting plate on the microscope carrier stage, first find the location of the counting chamber with low magnification, and then change it to high magnification for counting. Adjust the intensity of the light of the microscope appropriately, for the microscope with a reflector light should also pay attention to the light should not be biased to one side, otherwise the field of view is not easy to tongue clear counting chamber grid line, or only see the vertical line or only see the horizontal line. If the bacterial liquid is found to be too thick or too thin before counting, it is necessary to re-adjust the dilution before counting. General sample dilution requirements of about 5 to 10 bacteria in each cell is appropriate. For each counting chamber, the organisms in 5 middle compartments (4 corners and one middle compartment in the centre) are selected for counting. The organisms located on the grid lines are usually counted only on the upper and right lines. In the case of yeast budding, when the budding body reaches half the size of the mother cell, it is counted as two organisms. Counting a sample from the two counting chambers in the average value to calculate the bacterial content of the sample.
5. Cleaning the haematocrit plate
After use, rinse the blood cell counting plate with water under the tap, do not use hard objects to wash and brush, dry it by yourself or use a hair dryer to blow it dry after washing. Microscopically inspect the plate to see if there are any residual organisms or other precipitates in each compartment. If it is not clean, the washing must be repeated until it is clean.
