The separation of strains is mainly carried out on Petri dishes, and the commonly used methods are dilution method and streaking method.

The purpose of strain separation is that a single microorganism grows a colony visible to the naked eye on the solid medium through propagation, and then according to the culture characteristics, the required strain is retrieved with an inoculation needle, and it is proved to be a single-shaped thallus under the microscope.

Changing the culture medium conditions is also helpful for strain isolation. No culture medium or culture conditions can meet the needs of all microorganisms, and all cultures have certain selectivity.

If the growth requirements of microorganisms are known, a specific environment suitable for the growth of microorganisms can also be designed, so that the microorganisms can be selectively cultivated from the mixed microbial population, although the microorganisms may be only a minority in the mixed microbial population.

Second, the method

1, dilution inverted plate method

Firstly, the microbial suspension is diluted in series (for example, 1: 10, 1: 100,1000, 1: 1000).

If diluted properly, scattered single colonies can appear on the surface of the plate or in agar medium, which may be formed by the reproduction of bacterial cells. Then pick a single colony, or repeat the operation for more than a few times, and you can get pure culture.

2, coating plate method

Because adding microbial suspension to the hot culture medium first and then pouring the plate will easily lead to the death of some heat-sensitive bacteria, and the dilution pouring plate method will also affect the growth of some strict aerobic bacteria because of the lack of oxygen fixed in agar, so the pure separation method commonly used in microbiology research is the coated plate method.

The method is as follows: firstly, the melted culture medium is poured into a sterile Petri dish to make a sterile plate; After cooling and curing, a certain amount of microbial suspension is dropped on the surface of the plate; Then the bacterial liquid is evenly dispersed on the whole surface of the flat plate with a sterile glass daubing rod; After culture, single colony was selected.

3, flat line method

The simplest method to separate microorganisms is plate scribing method, that is, in aseptic operation, a small amount of substances to be separated are dipped with an inoculation ring, and continuous scribing is carried out on the surface of aseptic plate, so that the number of microbial cells decreases with the increase of scribing times and gradually disperses. If the lines are appropriate, the microorganisms can be dispersed one by one, and a single colony can be obtained on the surface of the plate after culture.

Sometimes this single colony is not all propagated by a single cell, so it must be separated repeatedly to get a pure species. Its principle is to dilute microbial samples from point to line for many times on the surface of solid medium to achieve the purpose of separation. There are many methods of drawing lines, and the common methods of drawing lines that are prone to single colony are diagonal line method, curve method, grid method, radiation method, four-grid method and so on.

4, dilution shake tube method

It is special to separate strict anaerobic bacteria with solid culture medium. If the microorganism does not die immediately after being exposed to the air, it can be prepared into a flat plate by the usual method and then cultured in a closed container. Oxygen in the container can be removed by chemical, physical or biological methods.

For those anaerobic microorganisms that are more sensitive to oxygen, the pure culture can be separated by dilution shake flask culture, which is an improved form of dilution inverted plate method.

Firstly, a series of test tubes filled with sterile agar medium were heated to melt the agar, and then cooled and kept at about 50℃. The substances to be separated are diluted with these test tubes in gradient, and the test tubes are shaken quickly. After condensation, a layer of sterilized mixture of liquid paraffin and solid paraffin was poured on the surface of agar column to separate the culture medium from the air.

After culture, colonies were formed in the middle of agar column. To pick and transplant a single colony, it is necessary to take out the liquid paraffin-paraffin cap with a sterile needle, then insert a capillary between the agar and the tube wall, blow sterile oxygen-free gas, suck out the agar column, put it in a Petri dish, slice the agar column with a sterile knife, and observe and transplant the colony.

Extended data

Among the above methods, plate separation method is widely used for the separation and purification of laboratory microorganisms, and dilution method is often used for the separation and purification of liquid culture medium.

Generally speaking, characteristic strains can be cultivated by properly improving the conditions of culture medium.

For example, in order to obtain thermophilic microorganisms, they can be cultured at 50℃~60℃. Sometimes adding corresponding inhibitors can also improve the isolation effect of strains. For example, adding a few drops 10% phenol to the suspension of soil samples can inhibit the growth of molds and bacteria, which is beneficial to the separation of actinomycetes. Adding a certain amount of penicillin or streptomycin to the culture medium can inhibit the growth of bacteria and is beneficial to the separation of mold.

Baidu Encyclopedia-Strain Isolation