Bacterial and Fungal Preservation Methods

Biopreservation is the process of preserving the integrity and functionality of cells.
Most bacteriological laboratories maintain stock cultures of microorganisms for educational, research, bioassay, industrial, or other purposes.
A wide variety of techniques are available for the preservation of bacteria and it may be difficult to choose a method for a particular strain, which not only assures survival, but which also makes certain that the genotype and hence the unique characteristics do not change.
The primary aim of culture preservation is to maintain the organism alive, uncontaminated, and without variation or mutation, that is, to preserve the culture in a condition that is as close as possible to the original isolate.

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Agar Slant Cultures

All microbiology laboratories preserve micro-organisms on agar slant.
The agar slants are inoculated and incubated until good growth appears.
They are then covered with sterile mineral oil to a depth of 1 cm above the tip of slant surface.
The slants are incubated for 24hr or more and are then stored in a refrigerator.
These cultures are periodically transferred to fresh media.
Transfers are made by removing a loop full of the growth, touching the loop to the glass surface to drain off excess oil, inoculating a fresh medium and then preserving the initial stock culture.
Time intervals at which the transfers are made which varies with the origin and condition of growth.
This is a simple and most economical method of preserving bacteria and fungi where they remain viable for several years at room temperature.

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 is a simple and most economical method of maintaining pure cultures of bacteria and fungi.
In this method, sterile liquid paraffin in 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 is preserved for several years.

Sodium chloride in high concentration is frequently an inhibitor of bacterial growth.
Bacteria are suspended in 1% salt solution (sublethal concentration in screw cap tubes to prevent evaporation).
The tubes are stored at room temperature. Whenever needed the transfer is made on agar slant.

Cryopreservation (i.e., freezing in liquid nitrogen at-196°C) helps 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, that prevent the formation of ice crystals and promote cell survival.

In this method, the culture is rapidly frozen at a very low temperature (around -70°C) and then dehydrated by vacuum.
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.
Freeze- drying method is the most frequently used technique by culture collection centres.

The Lyophilisation Process

In this process the microbial suspension is placed in small vials.
A thin film is frozen over the inside surface of the vial by rotating it in mixture of dry ice (solid carbon dioxide) and alcohol, or acetone at a temperature of −78^oC .
The vials are immediately connected to a high vacuum line. This dries the organism while still frozen.
Finally, the ampules are sealed off in a vacuum with small flame.
These cultures can be stored for several years at 40°C.
This method is also employed for preservation of toxins, sera, enzymes and other biological material.
To revive microbial cultures it is merely necessary to break open the vial aseptically, add a suitable stale medium, and after incubation make further transfers.
The process permits the maintenance of longer number of culture without variation in characteristics of the culture and greatly reduces the danger of contamination.

The organisms are dried over calcium chloride in the vacuum and are stored in the refrigerator.

http://www.biologydiscussion.com/micro-biology/preserving-microbial-cultures-top-5-methods/17821
http://www.biologydiscussion.com/microorganisms/culture-microorganisms/maintenance-and-preservation-of-pure-cultures-4-methods/55037
http://www.asmscience.org/content/book/10.1128/9781555817497.chap473
https://www.ncbi.nlm.nih.gov/pubmed/9629607
gc11.ac.in/wp-content/uploads/2017/02/preservation-of-cultures.ppt