Maintenance and Preservation of Organisms
Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination. Similarly, a microbiology laboratory has to maintain quality control (QC) stocks obtained from the ATCC or commercial vendors. QC strains are required for the testing of culture media, kits, and reagents.
Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination.
Since repeated subculturing is time-consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze-drying).
Periodic Transfer to Fresh Media
Strains can be maintained by periodically preparing a fresh culture from the previous stock culture. The culture medium, the storage temperature, and the time interval at which the transfers are made vary with the species and must be ascertained beforehand. The temperature and the type of medium chosen should support a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible. Many of the more common heterotrophs remain viable for several weeks or months on a medium like nutrient agar.
The transfer method has the disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants.
Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold rooms. This method is applied for a short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continue slowly, nutrients are utilized and waste products are released into medium. This results in, finally, the death of the microbes after some time.
Molds can be stored on potato dextrose agar (PDA) slants at 4°C for 6 months to 1 year.
Preservation of organisms by overlaying culture medium with mineral oil is a simple and most economical method of maintaining pure cultures of bacteria and fungi. For example, PDA slants may be overlaid with sterile mineral oil and stored at room temperature for the longer-term storage of fungi.
In this method, sterile liquid paraffin is poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved from months to years (varies with species).
The advantage of this method is that we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture. The simplicity of the method makes it attractive, but changes in the characteristics of a strain can still occur.
Freezing at -70°C
Long-term storage of aerobes and anaerobes can be accomplished by freezing at -70°C. Frozen, nonfastidious organisms should be thawed, reisolated, and refrozen every 5 years; fastidious organisms should be thawed, reisolated, and refrozen every 3 years. Acid-fast bacilli (AFB) may also be frozen at -70°C in 7H9 broth with glycerol. Viruses may be stored indefinitely at -70°C in a solution containing a cryoprotectant, such as 10% dimethyl sulfoxide (DMSO) or fetal bovine serum.
Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the liquid nitrogen at -150°C) helps the survival of pure cultures for long storage times.
In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol or dimethyl sulfoxide (DMSO) that prevent cell damage due to the formation of ice crystals and promote cell survival.
This liquid nitrogen method has been successful with many species that cannot be preserved by lyophilization and most species can remain viable under these conditions for 10 to 30 years without undergoing a change in their characteristics, however, this method is expensive.
Freeze-drying is a process where water and other solvents are removed from a frozen product via sublimation. Sublimation occurs when a frozen liquid goes directly to a gaseous state without entering a liquid phase.
It is recommended to use slow rates of cooling, as this will result in the formation of vertical ice crystal structures, thus allowing for more efficient water sublimation from the frozen product. Freeze-dried products are hygroscopic and must be protected from moisture during storage. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into a dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures are then sealed and stored in the dark at 4°C in refrigerators.
Freeze-drying method is the most frequently used technique by culture collection centers. Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.
Advantage of Lyophilization
- Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area.
- Small vials can be sent conveniently through the mail to other microbiology laboratories when packaged in a special sealed mailing container.
- Lyophilized cultures can be revived by opening the vials, adding the liquid medium, and transferring the rehydrated culture to a suitable growth medium.