Bacillus subtilis|133116 |BNCC

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Bacillus subtilis subsp. subtilis

Subculture procedure	① Prepare 1 test tube containing 5–10 mL liquid medium and 2 plates; ② Open in a safety cabinet, flame the top with an alcohol lamp, then quickly add sterile water to break it; subsequently crush it with forceps; ③ Pipette 0.5 mL liquid medium into the freeze-dried vial, dissolve thoroughly, then return to the liquid test tube and mix well; ④ Pipette 0.2mL of the bacterial suspension onto a Agar plate and spread evenly. Repeat twice to obtain two plates; ⑤ Incubate all liquid tubes and plates under the specified conditions. The culture is ready for use once the bacterial strain grows.
Growth conditions	30°C, 18-24h, aerobic.
Storage conditions	2-8°C
Safety level	1
morphology	Colony diameter: 0.5–1mm; circular, with neat edges; opaque, grayish-white on the upper surface; flat morphology; smooth surface; easily liftable; G+ (blue-violet); bacillus
Sharing mode	Public welfare sharing

Bacillus subtilis subsp.

Storage conditions: 2~8 ℃

No. 133116

Product format: freeze dried,200ul

Validity period: 6 years

Biosafety level: 1, handle in ultra-clear table or safety cabinet

Receiving notice: if any abnormality is found on the day of receipt, please contact the customer service within 24 hours. If it is overdue, it is deemed that the bacteria are well. The freeze dried culture shall be used up once and shall not be retained. The bacterial viability will resume after 1-2 generation of recovery and can be used normally. Handling instructions of agar slant and suspension liquid is enclosed. Please operate in strict accordance with this instruction, otherwise the replacement of bacteria are not be available in case of aberrant growth and loss of viability.

Growth conditions: 30 ℃, aerobic, tryptone soybean medium, 18-24h. Tryptone soybean medium: tryptone 17.0g, soybean peptone 3.0g, sodium chloride 5.0g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar 20.0g (not included in liquid medium), distilled water 1.0L,pH 7.3 & plusmn;0.2. Sterilization at 121 ℃ for 15min.

Recovery steps:
(1) Prepare a test tube containing 5-10mL of liquid culture medium and 2 plates;
(2) Open the ampoule in the biosafety cabinet, heat the tip of ampoule in a flame, quickly drop sterile water to creak it, then break it with forceps;
(3) Draw 0.5mL of liquid culture medium into a freeze dried ampoule, fully rehydrate and transfer the solution to the liquid test tube, mix evenly;
(4) Inoculate a plate with 0.2mL of suspension liquid, repeat the step to obtain two plates;
(5) Put all the liquid test tubes and plates under the above culture conditions for cultivation, and the strains can be used when they grow.

Recovery record: According to the recovery instructions, the results of the recovery are reported as follows:

item	test results
viability:	good viability，in 18h liquid medium become turbid, obvious strain layer occurs on the plate; colony is typical on marked plate
colony morphology: (above)	size: 1-2mm ; Shape: round ; Edge: irregular ; Transparency: opaque color: light yellow ; Protuberance: flat ; Surface: gray and rough ; Texture: dry
Conclusion:	good viability, no abnormal colony morphology, qualified

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