Common Questions and Answers Related to Microbial Control Strain Experiments

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1) The strain is stored in the third generation at -80°C; what generation will it be once it is taken out for activation? Answer: What is preserved is the third generation after revival; if it is activated using a flat plate or broth, it is equivalent to one transfer, making it the fourth generation. If it remains in ceramic beads, then it stays at its original generation. If it is stored on one inclined plane, this inclined plane remains the third generation; if it is transferred to another inclined plane, it becomes the fourth generation. The principle of passage: Whenever a strain is activated and revived through reproduction in any form, it is considered to be one generation passed. But if there is only a change in physical location without reproduction, it is not generation transfer. 2) Is it necessary to select a single colony when subculturing and storing the strain in ceramic beads? Answer: It is not necessary to select a single colony, but it is essential to choose colonies with identical characteristics; that is, they should be purified from the same colony to ensure that their properties are exactly the same. The principle is to choose as many as possible – it’s better to have too many than too few. 3) Are there specific standards specifying whether strain cryopreservation should be carried out at -70°C or -80°C? Answer: There are standards. SN/T 2632-2010 \"Specifications for Routine Preservation of Microbial Strains\" is a standard related to the preservation of strains; it is worth studying. Various preservation methods are described in this standard, but ceramic beads represent a relatively new method of preservation and are not included therein for now. GB4789.28-2013 recommends that long-term frozen storage be carried out at temperatures not higher than -70°C. 4) How can the storage duration of strains be extended? Answer: Numerous experiments have shown that strains are best preserved under conditions of low temperature, dryness, lack of oxygen, and limited nutrients; the closer these conditions are to those in which preservation takes place, the longer the strain can be preserved. For example, glycerol cryovials retain their quality for a longer period at -80°C than at -20°C. Lyophilized bacterial powder can be stored at -20°C for a longer period than at 2–8°C. 5) How to purify the strain? Answer: Purification is the process of picking a single colony from a plate containing suspicious colonies and then streaking it onto an appropriate non-selective plate (such as TSA, NA, etc.) to isolate a single colony. 6) Since we only keep the first-generation strain, do we need to carry out subculturing for preservation? Will the certification instructor consider this a mistake? Answer: Under normal circumstances, certified instructors do not ask such questions. The best storage method is to store the glycerol vials containing the first-generation strain in an ultra-low temperature freezer for long-term preservation. The second-generation strain is stored on an agar slant as a working reserve strain. After transfer using the second-generation working stock strain, it becomes the third-generation working strain. 7) Why use the third generation as the working strain? Answer: After multiple passages, the probability of variation in the strain **increases**; therefore, the number of passages should be kept within five. The first-generation strain is usually relatively unstable, and it is necessary to pass it through three generations to achieve stability. Therefore, in practical work, the third-generation strain is usually chosen as the working strain. 8) Do quality control strains have to be destroyed if they have been passaged more than five times? Answer: Not necessarily. Passaging the strain for more than five generations only raises the possibility of genetic variation; however, if it is passed to the sixth, seventh generation, and on, and its properties remain stable after verification through morphological, biochemical, and DNA characteristics, then these strains from beyond the fifth generation can still be used normally. 9) What is the meaning of the strain number? Answer: Taking Staphylococcus aureus CMCC (B) 26003 as an example, CMCC is the abbreviation for the China Medical Microbiological Culture Collection Center; other common microbiological culture collection centers include ATCC (American Type Culture Collection), CICC (Industrial Microbiology Culture Collection Center), and FSCC (Food Safety Microbiology Culture Collection Center) ； B stands for bacteria, with F corresponding to fungi ； 26003 is CMCC’s preservation number. 10) What are the differences between Staphylococcus aureus ATCC6538 and ATCC25923 as well? Answer: Both ATCC6538 and ATCC25923 are strains from the American Type Culture Collection (ATCC); their different numbers indicate that these two strains were originally isolated from different sources. However, it is necessary to conduct a specific analysis to determine whether biochemical properties are the same for different numbers. For example, in actual biological tests, it has been found that Staphylococcus aureus ATCC6538 has a stronger hemolytic ability than ATCC25923. 11) Why is it necessary to drain the bacterial solution after adding it and mixing well when storing the strain in a ceramic bead storage tube? Answer: The principle behind preserving bacteria using ceramic beads is that these beads have a porous structure; bacteria adhere to them. By removing the liquid, it is prevented from causing damage to the bacteria due to ice crystals formed during freezing and thawing cycles, thereby protecting the bacteria. If the bacterial solution is not dried, the porcelain beads cannot be preserved in such a way that repeated freezing and thawing are possible. 12) At what angle are TSA slants usually stored, and can they be kept in the refrigerator? Answer: For normal bacteria, TSA slants should be stored at 2-8°C, that is, in a refrigerated condition. However, for strains that are sensitive to low temperatures such as Vibrio, Pseudomonas aeruginosa, and Aeromonas hydrophila, they can be stored at room temperature of 20-25°C for a short period; for long-term storage, glycerol tubes or ceramic beads must be used. 13) Can liquid culture medium be used for the recovery of lyophilized strains? Answer: It is possible, but when using liquid rehydration, only turbidity of the culture medium can be observed; it is not possible to determine whether there is contamination. It is recommended to use plate medium for resuscitating lyophilized strains, as this facilitates the visual observation of colony morphology to determine whether the resuscitated strain is contaminated and to preliminarily assess its purity. 14) Can colored media be used for the recovery of lyophilized strains? Answer: It is not recommended to use chromogenic media for resuscitating lyophilized strains. Lyophilized strains suffer some damage during the lyophilization process, and their revival requires an optimal growth environment; therefore, media for revival should not be selective media containing antibacterial components that hinder the revival of the strains (chromogenic media are considered selective media). 15) Is it necessary to maintain an anaerobic environment when cryopreserving Clostridium perfringens? Answer: No, it’s not necessary. Although Clostridium perfringens is an anaerobic bacterium, it is not highly sensitive to anaerobic conditions, and it can be frozen using standard methods for freezing bacterial strains. If strictly anaerobic bacteria are to be cryopreserved, special cryopreservation methods are required.