Bảo quản chủng vi khuẩn (2 bài)


Storing Bacterial Samples for Optimal Viability (Bảo quản vi khuẩn sống sót tối ưu)

 Bài số 1

Introduction

Bacterial samples are critical for research, diagnostic and teaching purposes. Although there are many ways to store bacteria, the ideal method is a function of bacterial compatibility, experimental purpose and cell viability. As a general rule, the viable storage period of bacteria increases as the storage temperature decreases. Once the temperature is below the freezing point, however, cryoprotectants are essential to reduce cell damage caused by the freezing process. The specific length of time that a culture will remain viable in a given storage condition is dependent upon the bacterial strain. Cell death during storage is inevitable but should be minimized as much as possible, which can sacrifice ease of use. 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°C. 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).

Table 1. Approximate time bacterial cultures remain viable in different storage conditions.
Condition Temp (°C) Time (approx.)
Agar plates 4 4-6 weeks
Stab cultures 4 3 weeks-1 year
Standard freezer -20 1-3 years
Super-cooled freezer -80 1-10 years
Freeze dried ≤4 15 years+

Short-term Storage

Working bacterial stocks can be streaked onto agar plates and stored at 4°C for daily or weekly use. 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. Some bacterial strains can be stored for up to 1 year at 4°C in agar stab cultures, which are especially useful for transporting samples to other research facilities. Stab cultures are prepared by first sterilizing strain-compatible agar (e.g., lysogeny broth [LB] agar for E. coli) and then transferring the warm liquid agar to screw-cap vials using the appropriate aseptic technique. After the agar has solidified, a single colony is picked from an actively growing culture using a sterile, straight wire. The wire with the bacteria is then plunged deep into the soft agar several times, and the vial is incubated at 37°C for 8-12 hours with the cap slightly loose. The vial is then sealed tightly and stored in the dark at 4°C.

Long-term Storage

As mentioned above, the temperature at which frozen bacteria are stored affects how long they can be stored while remaining viable. 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. Also, the greater the cell density, the better the recovery is after thawing the cells. For most bacteria, a density of 107 cells/mL will result in adequate recovery if all conditions are properly maintained.1-2

Cryoprotectants: As water in cells is converted to ice, solutes accumulate in the residual free water. This localized increase in salt concentration can denature biomolecules.3 Furthermore, ice crystal formation can damage cell membranes. 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. The most commonly used cryoprotectants are dimethylsulfoxide (DMSO) and glycerol, which are typically used at 5-15% (v/v). 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.4 Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast extracts and tripticase soy.4

Freezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool. 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. After aliquoting the suspension into cryogenic screw-cap vials, the cells are snap-frozen by immersing the tubes in either ethanol-dry ice or liquid nitrogen and then stored in freezers (‑20 to -80°C) or liquid nitrogen (-150°C).5 Repeated thawing and refreezing of the bacterial stocks will reduce cell viability and should be avoided. When recovering strains with antibiotic selection markers, culturing them on selective media will ensure that the bacterial stocks were not contaminated.

Freeze drying: Bacteria can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze drying the suspension. Not all bacteria can be successfully freeze-dried.6-8 Certain strains might not survive the process or die rapidly once freeze dried. The best way to determine if a strain is amenable to freeze drying is to empirically evaluate its stability post-freeze drying while maintaining a live culture as a backup. Once freeze dried, it is best to store the bacteria at or below 4°C.

Conclusion

Storing bacterial samples requires careful consideration about how they will be used, availability of the storage unit and space within the unit. The time it takes to prepare a sample for storage, how precious the sample is and whether the strain is compatible with the desired storage condition must also be considered. Furthermore, the storage unit must be carefully monitored, as inconsistent temperature fluxuations can often occur in institutional freezers commonly used in research facilities.9 Aseptic technique must be strictly observed during sample handling and stock preparation to avoid cross-contamination between strains and contamination from air movement and handling. Finally, good record keeping is critical for maintaining bacterial samples. Downloadable forms available online can help keep track of all events surrounding the handling of a specific sample. With careful planning and handling, you can ensure that your bacterial sample remains viable for the entire study period and for future studies.

Cited References

1. Simione, F.P. and Brown, E.M. (1991). ATCC Preservation Methods: Freezing and Freeze Drying. American Type Culture Collection, Rockville, Maryland.
2. Simione, F.P. (1992). Key issues relating to the genetic stability and preservation of cells and cell banks. J Parenter Sci Technol 46:226-32.
3. De Paoli, P. (2005). Biobanking in microbiology: From sample collection to epidemiology, diagnosis and research. FEMS Microbiology Reviews 29:897-910
4. Huba’lek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology 46: 205-29.
5. Moore, L.W. and Rene, V. (1975). Liquid nitrogen storage of phytopathogenic bacteria. Phytophathology 65:246-50.
6. Miyamoto-Shinohara, Y., et al. (2008). Survival of freeze-dried bacteria. J Gen Appl Microbiol 54(1):9-24.
7. Miyamoto-Shinohara, Y., et al. (2006). Survival curves for microbial species stored by freeze-drying. Cryobiology. 52(1):27-32.
8. Miyamoto-Shinohara, Y., et al. (2000). Survival rate of microbes after freeze-drying and long-term storage. Cryobiology. 41(3):251-5.
9. Su, S.C., et al. (1996). Temperature variations in upright mechanical freezers. Cancer Epidemiol Biomarkers 5(2):139-40.

Bài số 2

A Guide to Bacteria Preservation:  Refrigeration, Freezing and Freeze Drying

Between stock cultures, mutant strains, and genetically engineered variants, the number of individual bacterial cultures which any one lab can accumulate can be numerous.  Indeed, the number of variations created in the process of engineering one plasmid can be astounding.  And most labs will hold on to all those and other variations as you’ll never know what you might need tomorrow.  Consequently, preserving all those bacterial cultures and genetic variants is something to be approached with thought.

A bacterial culture in a capped tube is in a closed environment.  Though the culture may start healthy, given time the number of viable cells will decrease to zero.  The goal of preserving the cultures is to slow that death rate so that when the culture is revisited, some of the cells are still viable and available for culturing.  The reasons the cells die can be numerous, but in every instance are based on the inherent chemistry of the cells and their environment.  If the deleterious chemical reactions can be slowed or halted, then the overall culture will remain viable for a longer period of time.

There are two basic approaches to slowing the rate of deleterious reactions in a culture of bacteria.  The first is to lower the temperature which decreases the rate of all chemical reactions.  This can be done using refrigerators, mechanical freezers, and liquid nitrogen freezers.  The second option is to remove water from the culture, a process which can be tricky and involves sublimation of water using a lyophilizer.

Following is a brief discussion of the major options for preserving bacteria.  The strengths and weakness of each option is reported.

Refrigeration

Bacteria can survive for a short period of time at 4°C.  For strains that are used daily or weekly, cultures grown on agar slants or plates can be stored in a refrigerator assuming that precaution has been taken to avoid contamination.  Cultures should be prepared using standard techniques and then sealed before storing.  For slants, we recommend using screw capped tubes.  For cultures on Petri dishes, the plates need to be sealed with Parafilm.  Sealing the plates not only helps to prevent molds from sneaking into the plates, but it slows the agar from drying.  For anything over a week or two, cultures can be stored as stabs in small, flat-bottomed screw capped vials.  In this technique, vials are filled with a small amount of agar medium (e.g., 1 ml) and sterilized.  Bacteria are then introduced into the solidified agar with a sterile needle.  The culture is incubated overnight with loose caps and then stored at 4°C with tight caps.  Cultures stored in stabs are more resistant to drying and contamination, but they will lose viability more quickly than frozen stocks.  The length of time a stab can remain viable is dependent upon the strain.  Some manuals claim that stabs are good for a year however it is unwise to make that assumption unless it is tested.

Freezing

Freezing is a good way to store bacteria.  Generally, the colder the storage temperature, the longer the culture will retain viable cells.  Freezers can be split into three categories:  laboratory, ultralow, and cryogenic.  The problem faced by bacteria (and other cells) stored in freezers is ice crystals.  Ice can damage cells by dehydration caused by localized increases in salt concentration.  As water is converted to ice, solutes accumulate in the residual free water and this high concentration of solutes can denature biomolecules.  Ice can also rupture membranes, though this problem is more often associated with cells lacking walls, such as cultured animal cells.  To lessen the negative effects of freezing, glycerol is often used as a cryoprotectant.  Glycerol is produced by many fish and insects to defend against cold temperatures by depressing the freezing point of the cells, enhancing supercooling, and by protection from ice.  With bacteria, adding glycerol to final concentration of 15% will help to keep cells viable under all freezing conditions (see this link for a protocol or this link for a ready to use freezing tube).  The following are some specifics for each freezer category.

Laboratory freezers are those that can pull temperatures down to -20 to -40°C.  These are single stage systems (one compressor) and often called general purpose freezers.  Bacteria can be stored for moderate periods of time, e.g., 1 year, in general purpose freezers.  It is best to use freezers without frost-free temperature cycling as this can wreak havoc on cells and other temperature sensitive biomolecules.  General purpose freezers are inexpensive and found in most labs, thus they are readily available for storing cultures.  The downside is that they are not sufficiently cold for long-term storage.

Ultralow freezers are two stage systems (two compressors each having a different refrigerant) which pull down to around -86°C.  Ultralow freezers are very prevalent, but space in them can sometimes be limited and competitive.  Ultralow freezers also are much more expensive to purchase, run and maintain.  The upside is that cells stored at -80°C tend to remain viable for several years.  The lower temperature generated by ultralow freezers substantially reduces chemical reactions within the culture.  However, molecular motion still occurs in frozen cells and thus the viability of the culture will decline.  It is important to regularly monitor cultures to assess their level of viability.

Cryogenic freezers are very cold and rely on liquid nitrogen or specialized mechanical systems to operate.  For biological samples, cryogenic storage should be below -130°C.  At this temperature, the molecular motion of water is halted and cells are trapped in a glass-like matrix.  Bacteria stored in cryogenic freezers retain their viability for many years.  In our laboratory bacterial and yeast cultures have been maintained at -140°C for 15 years without significant loss of viability.  Storing cells in cryogenic freezers is the most effective and, as compared to freeze drying, the easiest method for long-term storage.  The downside is cost and potential vulnerability of stocks to power outages, mechanical failures, and failed deliveries of liquid nitrogen.  Additionally, tubes should never be stored in tanks submersed in liquid nitrogen.  Screw cap tubes leak and will pull the nitrogen into the tube along with contaminants (see link for more information).  Liquid nitrogen vapor phase freezers will effectively avoid this problem, but these freezers are very expensive (upwards of $10K) and require large volumes of liquid nitrogen.  An alternative is mechanical cryogenic freezers that can go as low as -150°C, but these are also very expensive to purchase (about $20K).  Both cryogenic freezers will cost several hundred dollars a month to operate.

Freeze Drying

In an aqueous system, such as a living cell, water not only serves as the medium for enzymatic reactions, but also spontaneous negative reactions such as free radical formation.  Removing water halts both enzymatic and non-enzymatic reactions.  Freeze drying is one method of removing this water.  Many bacteria can be preserved very effectively by freeze drying.  By freezing the cells in a medium that contains a lyoprotectant (usually sucrose) and then pulling the water out using a vacuum (sublimation), cells can be effectively preserved.  This method is laborious and requires specialized equipment, but it has the advantage of generating stock cultures that are unaffected by power outages and empty liquid nitrogen tanks.  Furthermore, if cultures are routinely shipped to other labs, freeze dried cultures do not require special handling.  The downside on freeze drying is that not all cultures react the same way thus some experimentation is required to optimize the process for each strain.  For any lab which is serious about producing and maintaining a culture collection, then freeze drying should be included as a major method for preservation.

Details on freeze drying bacteria can be found on the webpage Bacteria Freeze Drying Protocol.

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