Publish Time: 2024-06-03 Origin: Site
Microbial isolation and purification are key processes to obtain single strains from mixed flora. This article takes the isolation of actinomycetes, fungi, and bacteria in soil as an example to introduce the wide application of plate separation method in the isolation and purification of microorganisms. Due to its simplicity and efficiency, the plate separation method can effectively obtain pure strains through different separation techniques and appropriate culture media, ensuring the accuracy of subsequent research and applications.
1. Dilution coating plate method
The dilution plate method is a method in which bacterial solution is diluted in a gradient and spread on the surface of a solid medium to eventually form a single colony. The principle is that in a bacterial solution with a high enough dilution, the microorganisms gathered together will be dispersed into single cells, thus forming a single colony on the surface of the culture medium.
The specific steps are as follows:
1) Invert the slab
Dissolve the beef extract peptone culture medium, Gao's No. 1 culture medium and PDA culture medium and cool them to 55~60°C. Add specific additives respectively and mix well before pouring them into petri dishes. Pour 3 dishes of each culture medium (repeat).
2) Prepare soil dilution solution
a. Weigh the soil: Weigh 10g of fresh soil and put it into a conical flask containing 90ml of sterile water and glass beads.
b. Mix the soil: Shake on a shaking table at 150r/min for 30 minutes to fully mix the soil and water and completely disperse the bacteria.
c. Gradual dilution:
Pipette 1 ml of soil suspension into the first test tube containing 9 ml of sterile water, shake and mix.
Take 1ml of the suspension from the first test tube, pour it into the second test tube containing 9ml of sterile water, and shake to mix.
Repeat this process to make soil solutions of 10 -1, 10 -2, 10 -3, -410, 10 -5, 10 -6and 10 -7dilutions in sequence and set aside for use.
3) Coating
a. Prepare the plate: Pour the sterilized culture medium into the petri dish and wait for the plate to cool before use.
b. Coating process: Coat the soil bacterial suspension of 10 -2and 10 -3dilutions on the plate to observe and culture the morphology of actinomycetes; apply the soil bacterial suspension of 10 -4and 10 -5dilutions on the plate to observe and culture the fungal morphology; apply the soil bacterial suspension of 10 -6and 10 -7dilutions on the plate to observe and culture the bacterial morphology.
c. Coating technique: first push it from bottom to top, then push it left and right, and then fully push it on the upper part of the culture medium, turn the culture medium, turn the lower part to the top, and repeat the above pushing operation. During the rotation process, the applicator should not extend out of the petri dish. Finally, it should be applied in a circular motion from the middle to the outside, and then from the outside to the inside to spread the coating evenly.
4) Cultivate
Place upside down in a 30°C constant temperature incubator and culture bacteria for 48 hours and fungi and actinomycetes for 168 hours.
5) Purification
Pick a single colony and isolate and purify it 3 to 4 times to obtain a pure strain of bacteria.
2. Flat marking method
Plate streaking is a method of obtaining single colonies by diluting a sample by streaking on the surface of a plate. The principle is that each streak will lead to a looser distribution of bacteria on the inoculation loop, ultimately achieving the isolation of a single colony.
The specific steps are as follows:
1) Division of line areas
Divide a flat plate into four areas A, B, C, and D, arranged in ascending order of area.
2) Line drawing process
Use an inoculation loop to draw lines in each area and gradually dilute the bacteria. Area A is the source area to be separated, areas B and C are the transition areas for preliminary marking and dilution, and area D (the largest area) is the key single colony harvest area.
3) Cultivate
After culture, single colonies in area D were observed and isolated and purified.
Medium selection
Medium type selection: Select the appropriate medium type, such as solid medium or liquid medium, according to the type of microorganism and growth needs.
Nutrients: Select appropriate carbon sources, nitrogen sources, minerals, vitamins and other nutrients according to the growth needs of microorganisms.
pH adjustment: Adjust the pH of the culture medium to suit the growth of microorganisms.
Culture conditions: Adjust culture conditions such as temperature, humidity and light according to the needs of microorganisms.
FAQ
Q1: Why is it necessary to burn the inoculating loop before each streaking on a plate?
A1: Burn the inoculation loop before each streak to kill the remaining bacteria and ensure the purity of the bacteria for the next streak.
Q2: How to ensure that isolated colonies are obtained when using plate streaking method to separate bacterial species?
A2: When drawing lines, estimate the number of bacteria in the sample and adjust the number of overlapping lines between the two intervals. Generally, a single colony rarely emerges from the first area, so the area of the plate should not be too large, leaving large areas for the subsequent areas.
Q3: How to determine whether a single colony on the plate is a pure culture?
A3: Streak or dilute the single colony again. If the single colony formed has a single shape, it is a pure colony.
Q4: Why should we invert the culture medium after inoculation?
A4: ① Prevent condensed water dripping from contaminating the culture medium; ② Prevent the evaporation of water in the culture medium; ③ Prevent the spread of bacterial colonies to some extent.
Q5: What are the key points of aseptic operation?
A5: ① Keep the mouth of the test tube, the mouth of the bottle or the slit of the petri dish close to the flame area of the alcohol lamp (maintain a sterile environment); ② The mouth of the test tube is not upward to reduce contamination; ③ Remove the leftover items from the inoculation in a timely manner to prevent contamination; ④ Mark the inoculations of different strains to avoid mixing.