Isolation and purification of microorganisms and colony counting on dilution plates

Summary

The isolation, purification and counting of microorganisms are generally used for (1) certain product testing, such as rhizobial agents and other product testing, biological product testing; (2) determination of the bacterial content of the soil; (3) testing of the degree of contamination of foodstuffs and water sources.

Operation method

Isolation and purification of microorganisms and colony counting on dilution plates

Principle

I. Under natural conditions, microorganisms often exist in a community state, and this community is often a mixture of different kinds of microorganisms. In order to study the characteristics of a certain microorganism or to cultivate and use a large number of microorganisms, it is necessary to obtain a pure culture from these mixed microbial communities, and this method of obtaining a pure culture is called the isolation and purification of microorganisms. Dilution plate counting is based on microorganisms in highly diluted conditions on the solid medium formed by a single colony is propagated from a single cell this culture characteristics of the design of the counting method, that is, a colony represents a single cell. When counting, (1) first of all, the sample to be tested will be made into a uniform propagation dilution, as far as possible to make the microbial cells in the sample dispersed, so that it becomes a single cell, otherwise a colony is not just a cell; (2) and then take a certain dilution, a certain amount of dilution inoculated to the plate, so that it is uniformly distributed in the plate within the medium; (3) after the cultivation of the growth and reproduction of individual cells to form colonies The number of bacteria in the sample can be calculated by counting the number of colonies. The number of bacteria counted by this method is the number of colonies grown on the culture medium, so it is also known as viable bacteria counting.

Materials and Instruments

Bacillus thuringiensis Agar
Beef Paste Peptone Agar Medium Koch's 1 Agar Medium Martin's Agar Medium
Scales, sample bottles, test tube racks, alcohol lamps, inoculation rings.

Move

I. Preparation of sample dilution

1. accurately weigh the sample to be tested 10g, into the 90mL sterile water and put a small glass beads in a 250mL triangular bottle, by hand or placed on a shaker for 20min, so that microbial cell dispersion, let it stand for 20-30s, that is, into the 10-1 diluent; and then 1mL sterile pipette, suck the 10-1 diluent 1mL, transferred to the test tube containing 9mL of sterile water, blowing 3 times, so that the bacterial liquid mixed evenly, that is, into the 10-2 dilution; and then change a sterile pipette, sucking up the 10-2 dilution of 1mL, transferred to the test tube containing 9mL of sterile water, also blowing 3 times, that is, into the 10-3 dilution And so on, continuous dilution, made of 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 and so on a series of diluted bacterial solution. 2.

2. When using dilution plate counting, the choice of dilution of the bacteria to be tested should be determined according to the sample. When the sample contains a large number of bacteria to be tested, the dilution should be high, and vice versa. Usually, when determining the number of bacteria contained in the bacterial agent, 10-7, 10-8, 10-9 dilution, when determining the number of soil bacteria, 10-4, 10-5, 10-6 dilution, when determining the number of actinomycetes, 10-4, 10-5, 10-9 dilution, and 10-7 dilution. 6 dilutions are used for determining the number of actinomycetes, 10-3, 10-4, 10-5 dilutions are used for determining the number of fungi, 10-2, 10-3, 10-4 When determining the number of fungi, 10-2, 10-3, 10-4 dilution is used.

Second, plate inoculation culture

1. Mixed plate culture method: the sterile plate will be programmed with 10-7, 10-8, 10-9 number, each number set up three repetitions, with a sterile pipette according to the requirements of aseptic operation to suck 1 × 10-9 dilution of each 1mL into the The same method was used to absorb 1mL of each 10-8 dilution into the three plates numbered 1×10-8, and then 1mL of each 1×10-7 dilution into the three plates numbered 1×10-7, and the same method was applied to the three plates numbered 1×10-7. 7 in 3 plates, the pipette can be changed without changing from low concentration to high concentration. Then pour the melted and cooled to 45-50 ℃ bacterial medium into each of the 9 plates, gently rotate the plate, so that the bacterial liquid and the medium mixed uniformly, condensed and then inverted, incubated at 30 ℃. Until the colonies grow out can be counted.

2. Smear plate counting method: smear plate counting method is basically the same as the mixing method, the difference is that the culture medium will be melted and poured into the sterile plate while it is still hot, to be numbered after solidification, and then use a sterile pipette to suck up 0.1mL bacterial inoculation on the agar plate with different dilution numbers, each number is set up for three repetitions. Then use a sterile spatula to spread the bacterial solution evenly on the plate, use a sterilized spatula for each dilution, and cauterize the spatula when changing the dilution. The spatula should be sterilized by cauterization when changing the dilution. When applying from low concentration to high concentration, the spatula can not be changed. The coated plate is placed flat on the table for 20-30min, so that the bacterial liquid penetrates into the medium, and then the plate is inverted and incubated until the colonies grow out and can be counted.

Third, the isolation method

1. Mixed bacteria method: according to the "dilution plate counting method" in the "mixed plate culture method" to carry out, the use of dilution for 10-4, 10-4, 10-6 three, each do three Dilutions of 10-4, 10-4, and 10-6 were used, and 3 replicates were made for each. 2.

2. Smear method: Perform the "Smear Plate Counting Method" in the "Dilution Plate Counting Method", using three dilutions of 10-3, 10-4, 10-5, and three replicates of each. -3. Scribing method: Use the "Dilution Plate Counting Method" in the "Smear Plate Counting Method".

3. Scribing method: Use a sterilized inoculation ring to dip 1 ring of 10-1 dilution on the solidified plate for scribing (Figure). The line can be drawn in the following two ways, one for the cross-hatching method, is on one side of the plate to do the first "Z" line. Turn the petri dish at an angle of 70°, burn the inoculation ring over the fire and cool it down, then make a second "Z" through the first scribed portion. The third and fourth scribes are made in the same way. The other is the border demarcation method, which starts from a point at the edge of the plate, and makes a close wave demarcation continuously until the center of the plate. Turn the petri dish 180 °, and then from the other side of the plate (not burn the inoculation ring) the same line to the center of the plate. Line method essentially belongs to a "point to line" dilution method, more suitable for the purification of materials containing a relatively single bacteria, such as soil microorganisms are highly mixed samples are less commonly used.

Cultivation

Invert the plate inoculated with soil suspension and incubate at 28～30℃ until colonies grow (24～36h).

V. Bacterial purification

Select a representative single colony with good separation on the plate to inoculate the slant, and at the same time make a smear for inspection, if found to be impure, the colony should be picked for further delineation and separation, or made into a bacterial suspension and then do dilution and separation, until a pure culture is obtained.

Counting

Select the number of colonies per dish 30-300 in the mixed bacteria method and smear method, respectively, according to the "dilution plate counting method" in the "mixed plate counting method" and "smear plate counting method" in the formula. The formula in "Dilution plate counting method" and "Smear plate counting method" are used. This is the number of viable bacteria per gram of the original soil sample, if you want to convert to the number of bacteria per gram of dry soil, you should also divide this value by the mass fraction of dry soil in the soil sample (mass of dried soil / mass of the original soil sample).

Caveat

1. The entire procedure should be carried out under sterile conditions.

2. Scorch the inoculation ring on an alcohol lamp until red hot before use.

3. Dip a small amount of bacterial solution and make a line on the petri dish first, do not rotate the inoculation ring.

4. After sterilization, the medium should be cooled down to 55℃ and the inverted plate operation should be carried out in time. If this is not possible, the medium should be kept warm in a hot box at about 55℃ to prevent the agar from solidifying.

5. After the experiment, all used culture medium and culture solution should be autoclaved and then disposed of at the medical waste to prevent environmental pollution.

Common Problems

1. Generally speaking, the number of colonies on the plate is between 30-300, multiply the number of colonies by the dilution and then calculate CFU according to the amount of bacterial liquid you coated the plate, if there are two dilutions of the number of colonies are between 30-300, then calculate the number of colonies of different dilutions, and then average. If the number of colonies in three plates of the same dilution are similar, then calculate CFU and average separately, if there is a big difference between one and the other two, it is recommended to take the average of those two small differences or repeat the coating of the plate once again. 2.

2. If you want to count the total number of bacteria, it is better to use a counting plate, the general blood cell counting plate can not be used with oil microscope because it is too thick, it can only be used for yeast counting, and bacteria should be used with bacterial counting plate.

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