Microbial strain purchase, acceptance, contamination
1.1 Collection of strains
The first is to purchase strains for laboratory use from national legal institutions, the second is to purchase commercially derived strains, and the third is to exchange strains in scientific research. No matter what kind of source, they are collected uniformly. The collection is strictly implemented in accordance with the specifications. It is required that the packaging is reliable, the collection is fast, and there is no leakage and no pollution. Ensure the qualification of strains and environmental safety. There must be a strain passage mark in the collection. Select qualified suppliers of qualified standard strains, and each batch of standardsstrainIt must be accompanied by the supplier's certificate of conformity or test report or instructions to prove that the standard strain purchased is qualified.
1.2 Standard strains and acceptance
When the laboratory receives the standard strain, it should first conduct a sensory inspection for conformity, record the strain number and the source route information of the standard strain to ensure clear traceability. At the same time, the name and quantity of the standard strain, the date of production, the date of receipt and whether it is damaged should also be recorded.
1.3 Resurrection of freeze-dried standard strains
1.3.1 open the product packaging: first wipe the outer packaging with 70% alcohol cotton, and then open it for use.
1.3.2 resurrection: select the appropriate medium and culture conditions (according to the strain instruction manual, see appendix) for resurrection. The first activation of the strain is preferably on a non-selective agar medium, unless special circumstances or special recommendations, liquid medium is generally not used. The number of passages of lyophilized strains shall not exceed 5 generations, and the lyophilized standard strain purchased from the Standard Strain Collection Center is the F0 generation.
1.4 Identification method and basis of working strains
Take the above culture with aseptic inoculation ring, separate individual colonies on the corresponding medium plate (nutrient agar, soybean tryptone agar) or the corresponding bacterial identification plate (such as eosin methylene blue, McConkay, BP, etc.), and culture under suitable conditions (if the microorganisms are anaerobic, the culture conditions should be anaerobic conditions). Take fungi and yeasts in the same way to SDA (Sabro medium) plate or rose red sodium medium plate, culture at 23-28 ℃ for 7 days; after culture, observe whether there is a typical colony state, then pick a single pure colony, perform Gram staining, microscopic inspection, observe its staining characteristics and morphology to determine the species.
1.5 Pollution treatment
If other colonies are found growing on the plate, it indicates that the operation is contaminated or the strain is impure. To
The contaminated culture was sterilized, the cause was found, and pure colonies were re-isolated and selected.
1.6 Strain preservation
All strains are kept in special refrigerators or other storage methods by laboratory personnel (double double lock). To establish a strain registry, record the collection, storage, preparation, use and disposal of strains in detail; each strain must have a test and identification report (for specific identification methods, see the method on the strain quality report identification certificate);
The specific preservation methods are generally as follows:
After resuscitation, the broth and sterilized glycerin are mixed in a ratio of 15% glycerol (broth 8.5mL + glycerol 1.5mL), frozen at -30 ° C, (multiple tubes can be frozen with 2mL cryopreservation tubes), as the F1 generation of preserved and stored strains, or commercial strain preservation tubes can be used.
1.7 Passage of strains
1.7.1 recovery of standard strains:
① Strain operation should be carried out under sterile conditions to prevent bacterial contamination.
② For each use and resuscitation, simply roll the beads on a plate or place them in the broth for cultivation.
1.7.2 standard reserve strains must be confirmed every time they are transferred. (The confirmation method is generally their own unique form, and the form on the identification medium)
Methods 1.7.3 transfer of working strains:
①. Prepare nutrient agar medium (hemolytic Vibrio plus 3% sodium chloride), autoclave at 121 ° C for 15 minutes, then pack it in a test tube, and set aside after cooling.
②. Under sterile conditions, use the inoculation ring to pick the fungus moss on a fresh test tube for "rice" shaped scribing inoculation, and place it in an incubator at 36 ° C for 24 hours.
Use of 1.7.4 working strains
1.7.4.1 internal quality control: once a month positive control, medium acceptance of each batch;
1.7.4.2 external quality control: ability verification, laboratory comparison;
Period verification 1.7.5 working strains
Frequency of 1.7.5.1 period verification: Semi-annual period verification of the use of standard strains.
Method and basis for verification during 1.7.5.2 working strain: same as confirmation of working strain.
1.7.5.3 Verification records during the establishment of standard strains.
1.8 Identification and expiration date of strains:
1.8.1 identification: The calculation of the algebra of the strain is the 0th generation of the dry powder strain, and the transfer is once plus one generation. The specific identification is: the 0th generation of Listeria monocytogenes, the identification is DZ-0, the first generation is identified as DZ-01 The first generation has 12 sticks, then DZ-01-01,..., DZ-01-12; and indicate the date: If 2016.02.13 fill in this way.
1.9 Preservation of strains
1.9.1 put the bacteria in the test tube into the refrigerator at 2 to 8 ° C.
1.9.2 the passaged and cultured strains are stored in the refrigerator at 2 to 8 ° C. Each preserved strain should be marked with the name of the bacteria, the standard number, the passage time, and the date of passage.
1.10 Destruction of strains
1.10.1 strains should be destroyed after use or beyond the storage period.
1.10.2 the strains to be destroyed, sterilize them with high pressure steam (121 ° C) for 30 minutes.
1.10.3 sterilized, then cleaned and treated.
1.10.4 destroyed strains should be recorded. When destroyed, the person in charge of the laboratory shall supervise the destruction, and the custodian shall be responsible for the destruction.
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