The dead grass spores are isolated, cultured and purified.

1. Bacteria extraction (1) preparation sample dilution solution called sample 10g, put in a triangular bottle containing 90 ml of sterile water and brought into the glass beads, vibration of about 20min, so that soil samples mixed with water, cell dispersion.
Use a 1 ml sterile straw to draw 1 ml of soil suspension from it and mix it well into a large test tube with 9 ml of sterile water, then use a sterile straw to suck 1 ml from this test tube and add another 9 ml of sterile water. In the test tube, mix well to make a soil solution of varying dilutions of 10-1, 10-2, 10-3, 10-4, 10-5, 10-6.
(2) coating with sterile straws from 10-4, 10-5, 10-6 three-tube soil dilution each take 0.1 ml pairs into the written dilution plate, with sterile glass coating stick gently coated on the medium surface uniform, room temperature static 5 to 10min, so that the fungus adsorption into the medium.
(3) culture is placed in a greenhouse culture tank at 37 degrees C, cultured 24h, solid culture base composition: distilled water 1L, glucose 20g, protein 15g, sodium chloride 5g, beef paste 0.5g, agar 20g.
, strain separation with the bacteria needle to pick a single place, bacteria to solid culture.
culture 24h.
solid culture base composition is i.m.
, strain bio-chemical identification 1, Terranean staining (1) production of bacteria culture routine smear, drying, fixed.
to use active long-term young cultures for Terratin dyeing; to avoid decoloration does not completely cause false positives; flame fixation should not overheat (to the slide is not hot hand).
(2) initial dyeing drops plus crystalline purple (just to cover the membrane as appropriate) dyeing 1-2min, washing.
(3) media dyeing with iodine liquid washed away residual water, and with iodine liquid covered with about 1min, washing.
(4) Decoloring The residual water on the slide is absorbed with filter paper, the slide is tilted, and on a white background, 95% ethanol is added to the dropper flow until the ethanol is not purple, and is washed immediately.
Terrain dyeing results are correct, ethanol decolorization is the key link of Terratin dyeing operation, decolorization is insufficient, negative bacteria were mistakenly dyed positive bacteria, excessive decolorization, positive bacteria were mistakenly dyed negative bacteria, decolorization time is generally about 20-30s.
(5) re-dyeing with red dye about 2min, washing.
(6) mirror inspection dry, with oil mirror observation.
bacteria are dyed blue-purple is Glocylane-positive bacteria, is dyed red Glocylane-negative bacteria.
2, spore staining: the improved Schaeffer-Fulton staining method (1) preparation bacteria suspension: plus 1-2 drops of sterile water in a small test tube, with the inoculation ring from the slope to pick 2-3 rings of bacteria in the test tube and fully smooth, to make a thick bacteria solution.
(2) add dyeing solution: add 5% peacock green water solution 2-3 drops in a small test tube, with inoculation ring stirring to make the dye and bacteria liquid fully mixed.
(3): Immerse the test tube in a beo bowl in a boiling water bath and heat 15-20min.
(4) smear: use the inoculation ring from the bottom of the test tube to pick a number of ring bacteria liquid on a clean slide, to make a coating, dry.
(5) fix: Fix the smear 3 times through the alcohol lamp flame.
(6) decoloration: wash with water until there is no peacock green color in the water flowing out.
(7) re-dyeing: after dyeing 2-3min with red dye, pour out the dyeing liquid, do not wash with water, directly with absorbent paper to absorb dry.
(8) mirror inspection: first low times, then high times, and finally with oil mirror observation.
results: the buds were green, and the bud sacs and bacteria were red.
4. Strain preservation (l) label to take a variety of sterile oblique interview tubes, will be filled with the name of the strain and vaccination date of the label, affixed to the test tube slope directly above, from the test tube mouth 2 to 75px.
(2) slope inoculation Transfer the species to be stored with an inoculation ring to the solid culture base test tube slope with a sterile operation method.
(3) culture bacteria 37 degrees C constant temperature culture 18 to 24h (4) preservation slope long, can be directly placed in the 4 degrees C refrigerator storage.
In order to prevent cotton plugs from being subjected to moisture germs, tube-mouth cotton is wrapped in psoria paper, or replaced with sterile plugs, which can also be used to melt solid paraffin fused cotton plugs or glue plugs.
5, bacteria active 1, the first level of expanded culture will be protected by the dead grass spores inoculated into the LB medium, the selection of 50mL conical bottle, built-in 15mL medium, placed at 37 degrees C, shaker shock culture 24h.
2, the second-level expansion of the culture LB culture base, the selection of 250mL conical bottle, built-in 70mL culture base, the amount of bacteria, placed at 37 degrees C, shaker culture 24h.
3, three-level expansion of the culture LB culture base, the selection of 500mL conical bottle, built-in 150mL culture base, the amount of bacteria is 5%, 10%, 15%, 20%, placed at 37 degrees C, shaker culture 12h, determine the OD value, and every two hours to test, thereby determining the growth of bacillus spores to the next stage of vaccination to lay the foundation.
6. Bacteria number determination 1, flat plate culture count Introduction Plate culture count is based on microorganisms under highly diluted conditions, its formation on the solid culture base of a single bacterium is a bacterial reproduction of this culture characteristic design of the counting method, that is, a bacterium represents a bacterium.
count, first of all, the sample to be tested for a series of dilution, as far as possible to spread the bacteria in the sample, so that a single presence (otherwise a bacterium does not represent only one bacteria), and then take a certain dilution, a certain amount of dilution inoculated into the plate, so that it is evenly distributed in the plate medium.
cultured, a single bacterium grows and reproduces to form a single bacterium, and the number of bacteria in the sample can be calculated by counting the number of places.
2, operating steps (1) sample thinner preparation accurately called 10 ml of samples to be tested, put into a 250 ml triangular bottle containing 90 ml of sterile physiological saline and small glass beads, oscillating 20min by hand or on a shaker bed To disperse the bacteria, set aside 20S to 30S, i.e. into 10-1 diluent, and then use 1 ml of sterile straw, absorb 10-1 dilution lml, move into a test tube filled with 9 ml of sterile physiological saline water, blow Suck 3 times, let the bacteria mix evenly, that is, into 10-2 dilution, and then another sterile straw to absorb 10-2 dilution 1 ml, into a test tube containing 9 ml of sterile physiological saline water, also blow 3 times, that is, into 1 0-3 dilution, and so on, continuous dilution, to make 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 and a series of diluted bacteria.
(2) Count bacteria number plate coating culture method: first melt the medium and pour it into the sterile plate while hot, to be solidified after the number, and then use a sterile straw to absorb 0.1 ml of bacteria liquid pair inoculated on different dilution numbers on the agar plate (three repetitions per number).
then use a sterile spatula to spread the liquid evenly on the plate, each dilution with a sterile spatula, replace the dilution need to burn the spatula to sterilise.
can also be applied from low to high concentrations without replacing the scraper.
will be coated flat on the table 20min to 30min, so that the liquid penetrates into the culture base, and then the plate inverted, put 35 degrees C to 37 degrees C, culture 24h to 48h, count the fall.
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