Soil microbial inoculation experiment procedure

Soil microbial inoculation experiment process- China Microbial Species Inquiry Network

Related knowledge point inoculation method: commonly used in the oblique inoculation method, plate inoculation method, liquid inoculation method, puncture inoculation method of test tube deep solid medium.
Inoculation tools: commonly used inoculation needles, inoculation rings, inoculation hooks, inoculation loops, inoculating shovel or inoculation, glass coating rods, etc.
The principle of soil is the base of microbial life. In order to obtain pure culture of a certain microorganism, it is generally based on the conditions of nutrition, pH, oxygen and other conditions of the microorganism, and supply appropriate culture conditions, or add some inhibitors, and eliminate Other undesirable microorganisms are used to separate and purify the microorganisms in various ways until a pure strain is obtained.
Equipment sample: fresh soil.
Others: sterile culture dishes, sterile straws, sterile triangular glass rods, electronic balances, markers, inoculation rings, alcohol lamps, matches.
Reagents: 10,000 U/mL streptomycin solution, 10% phenol medium: sterilized beef extract peptone agar medium, Gao's No. 1 medium, potato sucrose medium.
Sterile water: A glass bead containing 45 mL of sterile water triangle flask and a test tube containing 9 mL of sterile water.
Experimental procedure (1) Experimental procedure 1
Prepare soil dilutions:
1. Weigh 5g of soil, put it into a triangular flask of 45mL sterile water, and shake it for 10min, which is the soil suspension diluted 10-1.
2. Take another 5 mL test tube containing 9 mL of sterile water, and write 10-2, 10-3, 10-4, 10-5, and 10-6 with a marker. Take the soil solution diluted to 10-1, shake it for 0.5 min, pipette 1 mL of soil suspension into a 10-2 sterile water test tube with a sterile pipette, and gently pipette it several times in the test tube. Mix well and form a 10-2 soil dilution. The same method was successively diluted to 10-4, 10-5, and 10-6 soil dilutions.
Draw the diluent Take a pipette and aspirate 0.1 mL of 10-4, 10-5, 10-6 soil dilution solution by aseptic method, and add it to the prepared plate medium.
Coating The mixture was thoroughly mixed on a culture machine with a glass spatula and placed in an incubator.
Calculate the amount of bacteria per gram of soil.
That is, the number of colonies of the fungus in a certain culture dish is multiplied by the dilution factor of the culture dish inoculum.
A single colony was picked and separated by scribing.
(2) Experimental procedure 2
Making a plate Draw up the diluent Take a pipette and aspirate 0.1 mL of 10-4, 10-5, 10-6 soil dilution solution by aseptic method, and add it to the empty culture dish.
Mix the bacteria gently. Rotate the culture dish, mix the bacteria and medium thoroughly, place them on a flat tabletop, solidify and pour them into a constant temperature incubator.
Calculate the number of actinomycetes per gram of soil.
A single colony was picked and separated by scribing.
(3) Experimental procedure 3
Made into a flat plate.
After solidification, a corresponding loop of the bacterial liquid (10-1) was used to scribe the plate on the plate.
1. Continuous scribing method: The inoculation loop with the sample picked up is continuously streaked on the surface of the plate medium. After completion, it was placed in a greenhouse of 28 to 300 C.
2, the partition line method: use the inoculating ring to pick up the soil dilution 1 ring aseptically, first make the first parallel scribe line 3-4 on the side of the medium, then rotate the culture dish about 600C angle, and The residue on the inoculation ring is burned off, and after cooling, the second underline is performed by the first underlined portion, and the third and fourth scribing are sequentially performed in the same manner. After the scribing is completed, the lid is placed and placed in a greenhouse for cultivation.
A single colony was picked and inoculated on the slant medium, and if it was not pure, it was transplanted and purified, and finally a pure culture was obtained.