The appropriate volume of glycerol is added to a suspension of log-phase bacteria and vortexed to dissociate the cells and ensure even mixing of the bacteria with the glycerol. 4įreezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool. Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast extracts, and tripticase soy. Non-permeable additives used as cryopreservants, such as polysaccharides, proteins, and dextrans, adsorb to the surface of microorganisms and form a viscous layer that protects membranes, making these agents particularly useful for cryopreservation. The most commonly used cryoprotectants are dimethylsulfoxide (DMSO) and glycerol, which are typically used at 5–15% (v/v). Additives that are mixed with the bacterial suspension before freezing lower the freezing point and protect cells during freezing to minimize the detrimental effects of increased solute concentration and ice crystal formation. 3 Furthermore, ice crystal formation can damage cell membranes. This localized increase in salt concentration can denature biomolecules. 1-2Ĭryoprotectants: As water in cells is converted to ice, solutes accumulate in the residual free water. For most bacteria, a density of 10 7 cells/mL will result in adequate recovery if all conditions are properly maintained. Also, the greater the cell density, the better the recovery is after thawing the cells. Freezing and thawing cells at an appropriate rate and maintaining the frozen stocks at the proper storage temperature help to minimize damage from the freezing process. The vial is then sealed tightly and stored in the dark at 4☌.Īs mentioned above, the temperature at which frozen bacteria are stored affects how long they can be stored while remaining viable. The wire with the bacteria is then plunged deep into the soft agar several times, and the vial is incubated at 37☌ for 8–12 hours with the cap slightly loose. After the agar has solidified, a single colony is picked from an actively growing culture using a sterile, straight wire. coli) and then transferring the warm liquid agar to screw-cap vials using the appropriate aseptic technique. Stab cultures are prepared by first sterilizing strain-compatible agar (e.g., lysogeny broth agar for E. Some bacterial strains can be stored for up to 1 year at 4☌ in agar stab cultures, which are especially useful for transporting samples to other research facilities. Culture dishes should be wrapped with laboratory sealing film (plastic or paraffin) and stored upside down (agar side up) to minimize contamination and to keep both the culture and agar properly hydrated. Working bacterial stocks can be streaked onto agar plates and stored at 4☌ for daily or weekly use. If cultures will not be used for more than a few weeks, though, more long-term storage methods should be considered for maximum bacterial viability (Table 1). Bacterial cultures that are used regularly (i.e., daily/weekly) can be stored on agar plates or in stab cultures in a standard refrigerator at 4☌. Cell death during storage is inevitable but should be minimized as much as possible, which can sacrifice ease of use. The specific length of time that a culture will remain viable in a given storage condition is dependent upon the bacterial strain. Once the temperature is below the freezing point, however, cryoprotectants are essential to reduce cell damage caused by the freezing process. As a general rule, the viable storage period of bacteria increases as the storage temperature decreases. Although there are many ways to store bacteria, the ideal method is a function of bacterial compatibility, experimental purpose, and cell viability. Bacterial samples are critical for research, diagnostic, and teaching purposes. MHRA 'TSB', All Acronyms, 19 April 2023, Bluebook All Acronyms, TSB (Apr. TSB, All Acronyms, viewed April 19, 2023, MLA All Acronyms. Retrieved April 19, 2023, from Chicago All Acronyms. Facebook Twitter Linkedin Quote Copy APA All Acronyms.
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