Determination of the number of microorganisms - microscopic direct counting method!
Fundamental
Microscopic direct counting method is a simple, fast and intuitive method of placing a small amount of the suspension of the sample to be tested on a special glass slide (also known as a bacteria counter) with a definite area and volume, and counting directly under a microscope. At present, the commonly used bacteria counters at home and abroad include: blood cell counting board, Peteroff-Hauser bacteria counter and Hawksley bacteria counter, etc. They can all be used to count suspensions such as yeast, bacteria, and mold spores, and the basic principles are the same. The latter two 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 small cells such as bacteria can be observed and counted with an oil immersion objective lens. In addition to using these bacteria counters, there is also an estimation method of directly observing 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 advantage of microscope direct counting method is that it is 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 bacteria. At present, there are some methods to overcome this shortcoming, such as combining living bacteria staining microchamber culture (short time) and adding cell division inhibitors to achieve the purpose of counting only living bacteria.
In this experiment, a hemocytometer was used as an example to perform direct counting under a microscope. For the usage methods of the other two bacteria counters, please refer to the instructions of each manufacturer. Direct counting under a microscope with a hemocytometer is a commonly used microbial counting method. The counting board is a specially made glass slide, on which four slots form three platforms; the wider platform in the middle is divided into two halves by a short horizontal slot, and each side of the platform is lined with a grid. Each grid is divided into nine large squares, and the middle large square is the counting chamber. The scale of the counting room generally has two specifications, one is that a large square is divided into 25 middle squares, and each middle square is divided into 16 small squares; the other is that a large 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, there are 400 small squares in each large square. The side length of each large square is 1 mm, and then the area of each large square is 1 mm 2. After the cover glass is covered, the height between the cover glass and the slide glass is 0.1 mm, so the volume of the counting chamber is 0.1 mm (one ten-thousandth of a milliliter).
