The counting method of live bacteria is also called the indirect counting method. The direct counting method measures the total number of dead and living cells, while the indirect counting method measures only the number of viable cells. The value obtained by this type of method is often smaller than the value obtained by the direct counting method. Living bacteria counting methods generally include plate colony counting, liquid dilute selection maximum probable number method (most probable number, MPN) measurement, and membrane filtration counting method.
Plate colony count
This method is based on the fact that each dispersed living cell has The ability to reproduce and form a colony during birth. Therefore, the number of bacteria is the number of viable bacteria contained in the sample to be tested. After the single-cell microbial test solution is diluted by a 10-fold serial, the dilution solution of a certain concentration is quantitatively inoculated on the agar plate medium for culture. The number of colonies grown is the number of viable cells contained in the dilution solution, and the supply can be calculated. Measure the number of viable cells in the sample. However, it should be noted that due to various reasons, a single colony on the plate may not be formed by a single bacterial cell. Therefore, when the number of bacteria in a unit sample is expressed, it can be expressed in terms of the number of bacteria formed in the unit sample, that is, CFU/ml Or CFU/g (CFU stands for colony-forming unit).
Measurement by the maximum probable number method (MPN) of dilute liquid selection
Take a quantitative (1 ml) single-cell microbial suspension and use the culture medium Make quantitative 10-fold serial dilutions, repeat 3 to 5 times, and place the serial dilution tubes of different dilutions at an appropriate temperature for cultivation. Under the premise of proper dilution, bacterial growth will appear in the dilution tube with relatively high bacterial concentration, and there will be no bacterial growth in the dilution tube with higher dilution from the dilution tube with higher dilution. In the order of the dilution degree from low to high, the dilution degree of the last 3 dilution tubes with relatively high dilution degree and bacteria growth is called the critical order. Obtain the exponent from the series of three consecutive critical series of 3 to 5 repetitions, check the corresponding repetitive maximum probability table to find the maximum possible number, and then multiply it by the lowest dilution of the critical series where growth occurs. Relatively reliable sample concentration of viable bacteria.
Membrane filtration counting method
When determining the number of viable bacteria in water and air, due to the low bacteria concentration, the sample to be tested can be first ( A certain volume of water or air) is filtered and concentrated through a microporous membrane (such as a nitrocellulose membrane), and then the membrane is cultured on a suitable solid medium, and the bacteria can be counted after they grow.
Cautions for use
This method can cause pollution due to unskilled operation, or cause unstable results due to excessive medium temperature and damage to cells. Nevertheless, because this method can measure the microbial count in the sample, it is still an effective method for measuring the bacterial count commonly used in teaching, scientific research and production. The amount of bacteria, yeast, buds and spores contained in soil, water, milk, food and other materials can all be determined by this method. But it is not suitable for measuring filamentous microorganisms in samples, such as actinomycetes or filamentous fungi or filamentous cyanobacteria and other vegetative bodies.
In addition to the above two commonly used counting methods, there are membrane filtration method and turbidimetric method. The membrane filtration method is to pass a certain volume of lake water, sea water or drinking water through the membrane filter when the number of bacteria in the sample is very low. Then the filter membrane is dried, stained, and processed to make the membrane transparent, and then the number of bacteria on the membrane (or in a certain area) is calculated under a microscope; the principle of turbidimetry is that the cell concentration in the bacterial suspension is within a certain range. The turbidity is directly proportional to the optical density. The more bacteria, the greater the optical density. Therefore, with the help of a spectrophotometer, at a certain wavelength, the optical density of the bacterial suspension can be measured, and the optical density (O.D.) is used to express the amount of bacteria. The experimental measurement must be controlled within the linear range where the bacterial concentration is proportional to the optical density, otherwise it will be inaccurate. Microbial counting method is developing rapidly, and there are various methods such as rapid, simple and automated instruments and devices.