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Isolation, preservation and detection of industrially useful microorganisms is a time consuming and very expensive process. Therefore, it is essential to keep the isolated organisms in a viable condition so that it retains the desirable characters and it can be used whenever required for industrial production. This is done by storing it by creating certain special environmental conditions by which it remains in a viable condition but in an inactive state.
This phenomenon is called as preservation of culture. The preservation of culture should be done in such a way that it eliminates the genetic changes, prevents contamination and retains the viability.
Though there are several methods or techniques of preservation of industrially useful organisms, a description of only some important methods are given below:
(1) Repeated Sub-Culturing:
This is the most common, simplest and routine method of preservation of microorganisms. Selected microorganisms are initially grown on agar slants. After sufficient growth has taken place, they are transferred to fresh medium before they lose their viability. The appropriate time period for such transfer ranges from a week to few months (generally four to eight months).
Though an organism may be kept viable by this method but there is a probability of occurrence of mutations in the organism, which may lead to strain degeneration and subsequent uselessness of the organism for commercial usage. That is why it is less frequently used for preserving microorganism.
Advantages:
1. This method is cheap,
2. Needs no special equipment,
3. Recommended for small collection centers, and
4. Retrieval easy
Disadvantages:
1. Change in physiological and genetical characters, and
2. Time consuming.
(2) Storage under Liquid Nitrogen:
This method is also called as cryogenic storage method, because a cryoprotective agent in the form of 10% glycerol is used. Industrially useful microorganisms are stored under very low temperature ranging from 150°C – 196°C. In this method ranging, low temperatures are created by employing liquid nitrogen. Metabolic activities of microorganisms are reduced considerably at this low temperature. This method is generally employed for the preservation of fungi, bacteriophages, viruses, algae, yeasts, animal and plant cells, and tissue cultures.
This technique involves growing the desired microorganism in sufficient quantity either in the form of cells or spores or fragments of fungal mycelium. The grown up culture is suspended in 10% glycerol. The suspension is then introduced in to small ampoules at the rate of approximately 0.5 ml each. The ampoules are usually made up of borosilicate glass.
The ampoules containing culture suspension are frozen and sealed hermitically. Freezing is done either by directly dipping the ampoules into the liquid medium or hanging the ampoules initially over the column of liquid nitrogen for some time and finally dipping into the liquid.
The frozen ampoules are then dipped one above the other on small aluminum containers at the rate of six ampoules per can. The cans are then packed in aluminum boxes, 20 per each box. The perforations allow free flow of liquid nitrogen. There may be loss of viability in few cells during freezing process but there is virtually no loss of viability during storage phase.
Advantages:
1. Viable cultures may be preserved for many years by this method, especially those cultures which do not withstand preservation by freeze drying.
2. Though the equipment is costly, the process is economical.
3. The cultures remain viable under these conditions for 10-30 years without undergoing any change in their characteristics.
Disadvantages:
1. Evaporation of liquid nitrogen and replacement of lost liquid nitrogen regularly and periodically. If this is not done the apparatus will fail due to which there will be loss of valuable cultures, and
2. The method is relatively expensive.
(3) Employment of Dried Cultures:
This technique has been used extensively for the storage of fungi and actinomycetes particularly for sporulating mycelial organisms. Moist soil is generally used as a preservating medium. Moist soil is first sterilized and then inoculated with a desired culture and incubated for several days to allow some growth to occur. The soil with growing organism is dried at room temperature for a period of two weeks. The dried soil is then stored in a dry atmosphere or in a refrigerator.
Silica gel and porcelain beads may be used alternatively for soil. It is possible to preserve a culture for more than 20 years. Pridham and his associates reported that out of 1800 actinomycetes preserved by this method about 50% were viable, after 20 years storage.
(4) Lyophilization:
It is one of the best methods for long-term preservation of microorganisms. It is generally used for the preservation of fungi, viruses, bacteria, enzymes, toxins, sera and other microorganisms. It is a convenient method for the preservation of large number of cultures.
Lyophilization, which is also called as freeze drying involves freezing of a culture followed by its drying under vacuum which results in the temporary inhibition of metabolic activities of microorganisms.
The technique consists of the following stages:
1. The organism is allowed to grow to the maximum stationary phase on a suitable sterilized medium.
2. The cells are suspended in a protective medium like milk, serum or sodium glutamate.
3. A few drops of suspension are transferred to a glass ampoule.
4. The ampoules are then frozen by immersing into a freezing mixture of dry ice and alcohol at 78°C and are subjected to high vacuum until evaporation takes place completely (Fig. 2.16a).
5. The ampoules are then sealed and stored in a refrigerator (Fig. 2.16b).
6. The method of revival vary from laboratory to laboratory. Generally, during revival process the ampoules are decaped under sterile conditions and the dried pellets consisting of cells of the culture are transferred to a suitable liquid medium and are allowed to dissolve in order to make a suspension of cells. Then the cells are streaked on to agar plates. Sometimes there may be a need to undertake repeated sub-culturing for getting a culture exhibiting all characters. Cells of a lyophilized culture may remain viable for 10 years or more.
Advantages:
1. Culture once dried needs no further attention,
2. It needs very cheap storage equipment like refrigerator, and
3. It is easy to transport freeze-dried ampoules to far off places in large numbers in relatively small boxes.
1. This is expensive and needs expertise.
(5) With Mineral Oil:
This is one of the cheap and easy methods of preservation. Many microbes can be successfully preserved for longer time.
In this method tubes with sterile agar slants are inoculated with a given culture. The tubes are incubated till sufficient growth of the given microbe takes place. The grown up culture is covered with a suitable mineral oil to a depth of about 1 cm above the top of the slanted surface using sterile technique. Thus, over laid cultures can be stored at room temperature or preferably at low temperature by about 15°C.
Paraffin oil of specific gravity 0.865 to 0.890 is generally used in this method. The oil is sterilized either in McCartney bottles for 15 minutes at 103.41 × 103 Nm-2 pressure or in an autoclave at 103.41 × 103 Nm-2 pressure for 2 hours and then dried in an oven at 170°C for 1-2 hours, before it is used. Maintenance of viability of a culture under this treatment largely varies with the species and generally ranges from 10-20 years.
Advantages:
This method of maintenance has the unique advantage that you can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium and still original culture can be preserved. It is easy to control mites problem.
1. Chances of air-borne contamination during sub-culturing are more,
2. Chances of mutations are more, and
3. Retarded growth or inability to sporulate on retrieval.
(6) Storage in Soil:
Spore suspension in sterile water is poured into a culture bottle containing twice autoclaved loam soil (20% moisture). Fungal growth is allowed for few days and then stored with loose caps in a refrigerator. Fungi like Khizopus, Altemaria, Aspergillus, Penicillium and Fusarium which have long viability and stability can be maintained by this method.
1. Cheap and convenient,
2. Free from mites infestation, and
3. Though some variations may occur but the cultures are stable and survive upto 10 years.
(7) Silica Gel Storage:
Mc Cortney bottle is filled partly with medium grain non-indicating silica gel and sterilized by dry heat. The bottles are kept in a tray of water to the depth above the level of the gel. The water is frozen by placing the tray in a deep freeze with the temperature 17°C to 102°C. Spore suspensions are prepared in sterilized and cooled 5% skimmed milk and added to silica gel crystals in the tray of frozen water using Pasteur pipette and wetted three quarters to avoid over saturation.
The gel bottles are left in the ice bath for about 20 minutes until the ice around them have melted a little. The crystals are agitated to ensure thorough dispersion of the suspension. Bottles are dried with the caps loose for 10-14 days at 25°C until the silica gel crystals separate. Bottles are reversed down from tightly and stored over indicator silica gel in air tight container at 4°C. The indicator gel requires replacement once or twice in a year.
1. This method is simple and mites free.
2. Suitable for oomycetous fungi.
3. Stability in some cultures like Neurospora and Aspergillus is more.
1. Repeated retrieval can result contamination, and
2. Suitable only for fungi.