Preservation of Cultures: Microbial species are isolated and characterized by microbiologists and deposited in culture collection centers. The cultures are maintained in viable condition and are referred to as the stock-culture collection. Maintaining an isolated pure culture for extended periods in a viable condition, without any genetic change is referred to as preservation. Different types of cultures are preserved in laboratories by avoiding contamination as well as genetic changes (mutations). The various objectives of preservation are as follows:
Academic use: In microbiology subject, microbes are studied along with their practical applications. Microorganisms are needed for laboratory classes, to study morphology, basic staining techniques, isolation, counting, biochemical reactions, reference strains for taxonomic studies, etc. For the fulfillment of such academic requirements, microorganisms are preserved in laboratories.
Fermentation industry: The fermentation industry is engaged in the production of various compounds, produced by several microorganisms e.g. antibiotics, enzymes, vitamins, amino acids, vaccines, antisera, etc. For the process of preparation of the above compounds, an innoculum (starter culture) is required, hence the fermentation industry must maintain microbial cultures.
Biotechnology field: Novel microorganisms are produced with the help of biotechnology e.g. insulin-producing Escherichia coli. Such type of genetically modified microorganisms is not found in the environment. Hence, it is preserved for their biotechnological applications.
Research purpose: Many microorganisms are used for the study of byproducts produced from them. Some microorganisms produce infections (pathogenic) in human beings and are used for vaccine production. Some microorganisms are genetically modified and studied for different pharmaceutical applications. Many microbes have the potential to degrade pollutants. These and several related fields of research need stock cultures of microorganisms, which are preserved for their important uses.
Several methods are used for maintaining microorganisms in a viable condition over long periods:
- Periodic transfer to fresh media.
- Storage at low temperature.
- Storage in sterile soil.
- Preservation by overlaying cultures with mineral oil.
- Lyophilization or freeze drying.
1. Periodic transfer to fresh media:
In all microbiology laboratories, microbes are preserved on agar slants. The slants are incubated for 24 hours or more and then stored in refrigerators. These cultures are periodically transferred to fresh media. The time interval at which the transfers are made varies with the microorganisms and the conditions of growth. It is a simple method and any special apparatus is not required. But the risk of contamination is which may change the genetic and biochemical characteristics of the cultures.
2. Storage at low temperatures:
Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms when the temperature is maintained at 4 oC. This method is only used for short time preservation of cultures and subculturing is necessary if the period exceeds four weeks.
In another very low-temperature method, liquid nitrogen has provided long-term preservation of cultures. Microorganisms are prepared as a dense suspension in a medium containing a protective agent (glycerol or dimethyl sulfoxide), which prevents cell damage due to ice crystal formation. The cell suspension is sealed into small ampoules or vials and then frozen at a controlled rate of – 150 oC. The ampoules or vials are then stored in a liquid nitrogen refrigerator (– 196 oC). The liquid nitrogen method has been successfully used for many species and cells remain viable for 10 to 30 years without changing their characteristics.
3. Storage in sterile soil:
This method is mainly applied for the preservation of sporulating microorganisms e.g. Bacillus, Streptomyces, Aspergillus, Penicillium species, etc. Pure cultures (spores) of microorganisms are kept in a sterile soil medium and preserved for several months under refrigeration.
4. Preservation by overlaying cultures with mineral oil:
Many bacterial species can be preserved by covering their growth on the agar slant with sterile mineral oil or liquid paraffin. The oil must cover the slant completely. In this method, we can remove some of the growth under the oil with a transfer needle and innoculate it in a fresh medium by preserving the original culture. It is a simple method and is mainly used for anaerobic microorganisms. This is a cost-effective method of preserving cultures of bacteria and fungi for 15 to 20 years but changes in characteristics still occur.
5. Lyophilization or freeze drying:
Freeze drying can preserve different types of microorganisms that would be killed by ordinary drying. In this process, a dense cell suspension is placed in small vials and frozen at – 60 to – 78 oC. The vials are immediately connected to a high vacuum line. The ice present in the frozen suspension evaporates (sublimes) under the vacuum. This results in the dehydration of bacteria with a minimum of damage to delicate cell structures (Fig.1). The vials are then sealed off under a vacuum and stored in a refrigerator.
Advantages of Lyophilization or freeze drying:
- Cultures preserved by the freeze-drying method have remained viable for more than 30 years.
- Sub-culturing is not required and the culture can be maintained without contamination.
- The lyophilized strain remains genetically stable.
- Minimal storage space is required. Many lyophilized cultures can be stored in a small area.
- The small vials can be easily sent to other microbiology laboratories through the mail.
- Lyophilized cultures are easily revived by opening vials and transferring the rehydrated culture to a suitable growth medium.
- This method is also employed for the preservation of sera, toxins, enzymes, and other biologicals.
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