Common medium formula for Bacillus spores

, broth agar medium10g protein, beef paste 15g, NACL 15g, distilled water 1000ml, agar 18gAdjust PH7.2, 0.1MPA20min sterilizationIf the medium is made with a liquid, remove the agar100 mL/group, of which 20 mL liquid medium/250 mL (10 glass beads inside); Or (J-agar medium: trypsin 5g, yeast paste 15g, hydrophosphate potassium 3g, glucose 2g, agar 20g, distilled water 1000mlAdjust PH7.3-7.5Mpa 30min sterilizationII, hydrogen peroxide enzyme determination1, reagent: 3-10% hydrogen peroxide2, inoculation and culture: generally the test species will be inoculated on the oblique agar slope, 30 degrees under culture 1-2 days3, test method: take a clean slide, add a drop of 3-10% of the hydrogen peroxide water on it, pick 1 ring 1-2 days of moss, in the hydrogen peroxide solution, if there are bubbles appear (O2), as positive as hydrogen peroxide, no bubble is negativeHydrogen peroxide can also be added directly to the slope to observe the production of bubbles3, oxygenated determination 1, medium: tyrosin hydrolysis 20g, glucose 10g, NACL5g, HOCH2SO3Na1g, HSCH2COONa2g, agar 15g, distilled water 1LAdjust PH7.2, sub-install test tube, 0.06Mpa, 30min sterilization, medium does not swing slope2, inoculation and observation: with a small ring of broth, puncture inoculated into the above-mentioned medium (piercing tube bottom), generally with 30 degrees culture 3-7 days observation resultsIf Bacillus spores grow as aerobic bacteria on the surface of the agar column, and anaerobic bacteria grow along the puncture lineIV, tyrosin hydrolysis (used to detect whether different kinds of spores have the characteristics of decomposition of tyrosin)1, medium (1) skimmed milk preparation take fresh milk, boiled to remove the upper fat, and then by centrifugal degreasing (3000r/min, 10min), remove the upper fat, that is, skimmed milk (2) preparation of milk plate Take 50 ml of skimmed milk into a triangular bottle, another 1.5g agar containing 50 ml of distilled water in another 3 triangular bottles, and then separate the two liquid sterilization, 0.06Mpa-20min, with cold to 45-50 degrees Celsius, the speed of mixing the two liquids to pour the plate, that is, milk plate Turn the plate upside down overnight to dry the surface 2, vaccination and observation will test the bacteria at three points on the milk plate, generally in 30 degrees culture 1, 3, 5, 7 and 14 days, such as the colony around and below transparent, indicating that tyrosin has been decomposed medium 1.1 soluble starch 3g/L Protein 10g/L Yeast Paste 3g/L KH2PO41.5g/L K2HPO42g/L MgSO4 0.1g/L PH 7.4-7 6.(Enrichment) 1.2 Broite (NA) Medium (Nutritional Agar): Protein 10g/L Beef Paste 3-5g/L NaCl5g/L PH7.2-7.4 (For Save Counting or Separation Purification) Nutritional agar: malt juice medium 1:1 mixed use, colonies not easy to spread, morphological characteristics typical 1.3 protein 10g/L yeast paste 5g/L glucose 3.5g/L NaCl5g/L K2HPO4 0.5g /LMgSO4 3g/L MnSO4 25mg/L PH 7.2-7.4 (separation purification) 1.4 amylase strain screening medium: soluble starch 3-5g/L protein 10g/L Beef paste 3-5g/L NaCl 5g/L PH7.2-7.4 (for amylase-producing bacteria separation screening) 1.5 protease strain screening medium 1.5.1 thinita medium: ★★ 2.1.5.1.1 glucose 1g/g/1 glucose L, yeast paste 1g/L, tyrosine 4-5g/L, K2HPO4 1g/L, KH2PO4 0.5g/L, MgSO4 0.1g/L Distilled Water 1000ml, pH7.0-7.2 Also: glucose 0.5g/L, tyrosine 10g/L, NaCl 5g/L K2HPO4 0.5g/L, KH2PO4 0.5g/L, Agar 20g/L pH7.2-7.4 2.1.5.1.2 beef paste 3g/L or yeast-soaked powder 5g/L 0g/L NaCl 4-5g/L PH7.2-7.4 First wetthet the tyrosin with a small amount of 2% sodium hydroxide, stir the rod, add the appropriate amount of distilled water, heat and stir in a boiling water bath to completely dissolved (10-15min), fill 1000mL distilled water, add other ingredients, to mix PH- sterilizing 2.1.5.1.3 Yeast paste 0.05g/L tyrosine 4g/L KH2PO4 0.36g/L Na2HPO4 1.1g/L MgSO4 0.5g/L NaCl 0.16g/L FeSO4 0.002g/L CaCl2 0.002g/L Zcl2 0 .014g/LpH6.5-7.0 4c/o tyrosin mother liquid preparation: 4g tyrosin dissolved in 0.1N 30-35mL sodium hydroxide solution, water bath dissolved 20min, dissolved after adding 60-70 c hot water dissolved to 100mL.200mL medium plus 20mL ★★2.1.5.2 Skimilk Medium: Beef Paste 3g/L (Yeast Dip Powder 5g/L) Protein 10g/L NaCl5g/L Skimmed Milk Powder 10-12g/L, PH7.2 (Ph7.2) For the screening of proteases to produce bacteria) dissolved the skimmed milk (powder) with water into a 10c/o solution, heated and sterilized 20-30min at 115 degrees C, and mixed with the bovine protein solid medium 1:9 to pour the flat 2.1.6 cellulose-producing bacteria medium: CMC-Na2g/L Protein 10g/L Yeast Paste 5g/L NaCl5g/L PH7.2-7.4 (screening for cellulose-producing bacteria) 2.1.7 Bud-forming medium: soil leachate 1000mL protein 5g/L beef paste 6g/L PH7.2-7.4 Protein 1g/L Yeast Paste 0.7g/L Glucose 1g/L (NH4) 2SO40.2g/L MgSO40.2g/L K2HPO4 1g/L PH7.2-7.4 (separation purification) Spore medium: protein 10g/L beef paste 3-5g/L NaCl5g/L .Cornmeal 0.5g/L Yeast Paste 0.1g/L PH7.2-7.4 2.1.8 Protease Fermented Medium: Cornmeal 30g/L BeanCake powder 20g/L Bran 10g/L dihydrophosphate sodium 4g potassium dihydrophosphate 0.3g/L sodium carbonate 1.0g/L PH7.4-7.6 eight layers of gauze, kraft paper tied well 2.1.9 production amylase fermentation medium: cornflour 40g/L bean cake powder 20g/L protein 5g/L ammonium sulfate 4g/L phosphate dihydross a sodium 8g/L phosphate dihydropotassium 3g/L PH7.4-7.6 eight layers of gauze, kraft paper tied Enzyme-producing media (protein amylase): protein 10g/L yeast paste 5g/L glucose 1g/L soluble deposit powder 5g/L Dihydro potassium phosphate 2g/L MgSO4 0.5g/L CaCl2 0.2g/L PH7.0-7.4 2.2 separation purification (or compound strength) 2.2.1 frozen dry tube And bacteria re-ification: activation: use sterile straw to absorb 0.5mL sterile water dripping into the tube, light-slinging to dissolve the dry bacteria into bacteria suspension, do gradient dilution after taking 0.1-0.2mL coating or directly line in the broth (malt juice) culture flat dish, or transfer to the oblique surface (activated) 37 degrees C 24-36h re-strong: pick and choose to grow fast, the colonies are grown in 4.5mL sterile water test tube, oscillation shake situated in 80 c water bath heating 15-20min, the gradient diluted after coating in the bud cell to form a medium flat dish or the bacteria suspended direct cross, 37 degrees C 24h Repeatedly two to three times, choose flat dish on the large and fast growth of the colonies transferred to the slope 37 degrees C 18-20h, 4 degrees C refrigerator preservation 2.2.2 commercial microbiology preparations in the selection of bacteria 1g (1mL) commodity preparations added to 99mL sterile (physiological salt) water / 250mL triangular bottle (with glass beads), placed in the shaker oscillation 20-30min, 80-85 oC water bath heating 15-20min, gradient dilution into 10-3, 10-4, 10-1-10,10-10-10-10-10-10 0-7, according to the living bacteria content of two to three gradients selected for mixed bacteria or coated 35 degrees C-37 degrees C 24 h, pick a ring growth fast, the colonies of different typical colonies are transferred to 5mL sterile water test tube, mixer mixed 5-10min, to make bacteria suspension gradient dilution after mixing or coating, or directly take bacteria suspension direct line, 37 c.28 Combined with mirror inspection until the size of the colonies is basically the same, transferred to the bevel 37 degrees C 18-20h, in the 4 oC refrigerator to save Or the gradient bacteria liquid 1mL added to the plate, and then injected with starch and tyrosin medium, 30-37 degrees C 24-48h, pick D/d large (starch culture medium iodization liquid, observation transparent circle) transferred to the bevel backup or using dash method purification And through the staining mirror observation, from the colony form and cell morphology to identify 2.2.3 Natural Screening 2.2.3.1 Enriched Culture: take 5g mud or intestinal solution 0.5g, add 45 (50) mL sterile water / 250mL triangular bottle (with glass beads), oscillate 15-20min, take 5mL liqueuring or 5mL mL water sample added 45mL bud-promoting medium /250mL triangulation bottle, 75 c-80 degrees C water bath 15-20min, down to 35 degrees C-37 degrees C 150-200r/min oscillation 24h, dyeing mirror test, two to three consecutive training backup Or take 1g of mud (earth) placed in 20mL broth medium/250mL triangular bottle, eight layers of gauze seal, 75c-80C water bath treatment 15-20min, then 150-200r/min oscillation 24h Mirror detection of spores, pick the colonies with spores for dash purification culture, to obtain single colonies 2.2.3.2 separate purification take the concentrated bacteria liquid in a water bath of 75 c-80 degrees C 15-20min, gradient dilution 10-3, 10-4, 10-5, 10-6, 10-7, choose three suitable gradient mixtures Or coated in a nutritional agar malt medium or directly coated on a cheesein medium for a primary sieve, or a dash separation (the dash method first places the plate at 30 degrees C (24-48 h) or 37 degrees C (18-24h), aseptic inspection, surface condensate drying) Two flat dishes of each gradient, the plate is inverted at 35 C-37 degrees C 24-48h Number the single colonies that grow, choose fast growth, large colonies, dry surface, rough, opaque colonies transferred to the bevel Pick a little moss smear, do bud cell dyeing to determine whether it is bacillus spores