Simple Pharmanotes: Preservation methods for pure cultures.

Introduction:

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.
The cultures maintained in viable conditions are called “Stock culture Collection”.
Preservation here refers to maintenance of the pure culture to keep them viable for extended duration of the time without any genetic change.

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 genetic changes.

Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms.
This method is applied for 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 continues slowly, nutrients are utilized and waste products released in medium.
This results in, finally, the death of the microbes after sometime.

This method is applied for spore forming microbes like Bacillus, Streptomyces, Penicillium etc.
Pure cultures are kept in sterile soil medium and preserved for many months under refrigeration.

This is a simple and most economical method of maintaining pure cultures of bacteria and 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 for 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.

Also called “Freeze drying”.
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 using 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 dormant state and retain viability for years.
Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators.
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 special sealed mailing containers.
Lyophilized cultures can be revived by opening the vials, adding liquid medium, and transferring the rehydrated culture to a suitable growth medium.
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.