Lactobacillus subspecies in mare's milk wine|354841 |BNCC

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Lactobacillus kefiranofaciens subsp

Subculture procedure	① Prepare 1-2 plates as described above (pre-conditioned in an anaerobic environment for 24 h for oxygen removal).② In a biosafety cabinet, open the vial and briefly flame the top with an alcohol burner, then immediately add sterile water to break the seal. Use sterile forceps to crush the vial.③ Aspirate 0.5 mL of sterile water (pre-conditioned in an anaerobic environment for 24 h for oxygen removal) into the freeze‑dried vial, thoroughly dissolve the bacterial powder, then transfer 200 μL of the suspension onto each Agar plate and spread evenly.④ Incubate the plates under the specified culture conditions. The strain is ready for use once growth appears.
Growth conditions	30 ° C; 5-7 days; anaerobic
Storage conditions	2-8 ℃
Safety level	1
morphology	The colonies measure 1-2 mm in diameter, are circular with regular margins, and are opaque. They exhibit a creamy-white pigmentation on the surface, have a raised center, and are smooth, glossy, and moist in texture, making them easy to lift. The organisms are Gram-positive (G⁺, appearing blue-violet) and rod-shaped (bacilli). Purity: pure.
Sharing mode	Public welfare sharing

Lactobacillus kefiranofaciens subsp.

Storage conditions: 2~8 ℃

No. 354841

Product format: freeze dried, 200ul

Validity : 6 years

Biosafety level : 1 , handle in ultra-clean 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:37 ℃, anaerobic, MRS medium, 5-7 days, MRS medium: peptone 10.0g, beef paste 10.0g, yeast powder 4.0g, glucose 20.0g, magnesium sulfate 0.2g, sodium acetate 5.0g, triammonium citrate 2.0g, dipotassium hydrogen phosphate 2.0g, manganese sulfate 0.04g, Tween 80 1.0g, agar 20.0g (not included in liquid medium), distilled water 1.0l. pH 5.7±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 6 days liquid medium become turbid,strain layer is obvious;colony is typical on the streaked plate
colony morphology: (above)	size: 0.5-1mm shape: round edge: neat transparency: opaque color: off-white bulge: middle convex surface: bright and smooth texture: moist
Conclusion:	good viability, no abnormal colony morphology, qualified

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