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TECHNIQUES FOR MICROBE PRESERVATION

Microorganism are preserved in order to ensure optimal long term viability and their genetic stability. In laboratories, for research work and for clinical work in hospitals, it is not practical to grow microbes, whenever it is required. So, it is important to preserve the microbes before hand and use them, whenever necessary. Due to the advancement in technology, there are a lot of techniques by which, the microorganisms can be preserved. The process of microbe preservation is also widely known as Biopreservation. The primary aim of microbe preservation is to maintain the organisms alive and stable, without undergoing any variation or mutation, so as to maintain them as closely as possible to the initial isolate. Non-contamination of the microbes is also taken into consideration during these techniques.

Agar Slant Method

• 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 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 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.

Refrigeration

Working of Refrigerator

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, growth continues slowly and nutrients are utilised and waste products are released in the medium. This results in the death of the microbes after sometime.

Freezing

Freezing is a good way to preserve microbes. Generally, the colder the storage temperature, the longer the culture will retain viable cells.The problem faced by Bacteria (and other cells) stored in freezers is the ice crystals. Ice can cause damage to the cells by dehydration caused by localised increase 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 even rupture membranes. 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.

Freezer

A. Types of Freezers

• Laboratory freezers are those that can pull temperatures down to -20 to -40°C.  These are single stage systems (one compressor) and are 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.

• Ultra low freezers are two stage systems (two compressors each having a different refrigerant) which pull down to around -86°C.  Ultra low freezers are very prevalent, but space in them can sometimes be limited and competitive.  Ultra low 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 ultra low freezers substantially reduces chemical reactions within the culture. It is important to regularly monitor cultures to assess their level of viability.   

• Cryogenic freezers are very cold and rely on liquid nitrogen or specialised 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. Storing cells in cryogenic freezers is the most effective method for long-term storage.  The downside is the cost and potential vulnerability of stocks to power outages, mechanical failures, and failed deliveries 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. Both cryogenic freezers will cost several hundred dollars a month to operate.

Paraffin Method

Paraffin oil or liquid paraffin oil is obtained in the process of petroleum distillation. It is a colourless and odourless oil that is used for varied purposes.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 of culture and is 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. 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 there are chance for changes in the characteristics of a strain, to occur.

Saline Suspension

As the name suggests, saline in the sense, salt is used to make a suspension and preserve the microbes. Sodium chloride in high concentration is frequently an inhibitor of bacterial growth. Bacteria are suspended in 1% salt solution. It is the sublethal concentration in screw cap tubes, which prevents evaporation. The tubes are then stored at room temperature. Whenever needed the transfer is made on to an agar slant.

Cryopreservation

Freezers for cryopreservation

Cryopreservation is a process that preserves organelles, cells, tissues, or any other biological constructs by cooling the samples to very low temperatures. The responses of living cells to ice formation are of theoretical interest and practical relevance. Stem cells and other viable tissues, which have great potential for use in basic research as well as for many medical applications, cannot be stored with simple cooling or freezing for a long time because ice crystal formation, osmotic shock, and membrane damage during freezing and thawing will cause cell death. Cryopreserved cells or tissues possess some advantages for basic research and current and future clinical applications. With the constant availability of cryopreserved cells and tissues, extensive quality testing can be performed to determine the suitability of the cells or tissue for transplantation without the need to obtain fresh samples. The cryopreservation agents used are Dimethyl sulfoxide (DMSO), Ethylene Glycol, Glycerol and so on. These agents affects the rates of water transport, nucleation, and ice crystal growth.

Lyophilisation

It is called as Freeze-drying. 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. Lyophilised 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 centres.

A. Method

• 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°C .
• 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.

B. Precautions

• Heating the product to a very high temperature can cause melt-back or product collapse.
• Condenser overload caused by too much vapour hitting the condenser.
• Vapour choking – the vapour is produced at a rate faster than it can get through the vapour port; the port between the product chamber and the condenser, creating an increase in chamber pressure.

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