Preservation And Storage Of Microorganisms

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Preservation Of Microbial Cultures

Culture handling and storage are very essential in Microbiology. Pure cultures, of microorganisms can be easily contaminated, undergo genetic changes or even die off if not properly handled. Scientists preserve one microorganism or-the other for various reasons.

Research scientists trying to characterize microorganisms and identify them need to produce reliable subcultures for further tests on their isolates. In industrial laboratories there is need to maintain stable cultures of the strains used for industrial fermentations. Even in strain improvement programmes of industries, it is essential to be able to produce subcultures which are reliable and this means that a system of culture handling and preservation must be built into the strain improvement programme. Technical laboratories also maintain a library of microbial strains that gave typical reactions. These cultures serve as positive and negative controls for these reactions in those laboratories.

Some organisms are also difficult to isolate and purify or may be isolated from extreme environments, the loss of such cultures may often mean that they are irreplaceable in such laboratories. Even those that can be reisolated, the time, information and cost of such reisolation can be reduced if the culture is preserved.

Various methods are available for preservation and storage of microbial cultures. The method an individual may choose will depend on the number of cultures to be preserved the climate, time and the facilities available. With some of the methods it is possible to regain the organisms in the condition in which they were at the time of isolation. Short preservation may involve subculturing every two to four days. This is particularly necessary for organism that are poorly viable and die off in a few days after the completion of their growth. Some species such as Streptobacillus and Neisseria gonorrhea etc are difficult to preserve irrespective of the temperature of storage. They must be subcultured if there is need to preserve them.

Methods Of Preserving Microbial Cultures

1. Periodic Transfer:

This involves periodic transfer from the stock to fresh medium. The period varies according to the viability of the organism. For organisms like Neisseria gonorrheae the transfer could be from two to four days while for others like Staphylococci, Streptoccoci they can remain viable for months thereby increasing the time of transfer. The variable involved in periodic transfer include transfer frequency, medium used and holding temperature. These factors can lead to increased mutation rates.

2. Glycerol:

This method is effective for stworing aerobic microorganism especially fungi.

3. Storage in Minimal Medium, distilled water or water agar:

These involves washing cultures and storing them in these media. These cultures can be viable for 3 to 5 months or more.

4. Freezing in growth media:

This method is often used for very short term storage. It is not a reliable method and can , result to damage of microbial structures.

Drying Cultures:

Most delicate bacteria can be preserved for long periods by different drying methods.

1. Dry Agar Slants

  1. Grow cultures on nutrient medium in test tubes or bottles, use agar gel with added nutrients (mineral salts plus a carbohydrate source, or process of vegetable or plant material). A weak decoction of potato carrot extract and agar may be used.
  2. Store at room temperature preferably in a wooden cupboard to protect culture from dust or in a refrigerator or cold room at 5 – 8°C in a deep freeze at -20°%.
  3. Transfer to fresh medium every 6 months.

Drying by Stamp’s Method

  1. Prepare a small amount of 10% gelatin in nutrient broth.
  2. Add 0.25% ascorbic acid.
  3. Add a thick suspension of the organism.
  4. Immerse paper sheets in wax.
  5. Drain and allow to set.
  6. Drop the suspension of the organism on the waxed surface.
  7. Store the discs in screw capped bottles in & refrigerator.

Note: Petri dishes smeared with a 20% solution of silicon in light petroleum and dried in a hot air oven can be used instead of waxed paper.

Drying by Rayson’s Method

  1. Prepare 10% serum broth or gestation broth
  2. Add a suspension of the organism
  3. Cut discs of about 10mm diameter from cellophane
  4. Sterilize in petri dishes v. Drop broth containing organism on disc
  5. Dry in a vacuum desiccator over phosphorous pentoxide
  6. Store in screw capped bottles in a refrigerator

Note: To recover the organism, use a sterile forceps, pick one Disc and place it into a tube of broth and incubate.

Preservation Without Drying

Some bacteria which grow readily can survive for several months in semi solid nutrient agar (0.5-1%). The coliforms and members of genus Staphylococcus are examples of the bacteria which survive for several months on semi solid agar.

Note: Members of the salmonella and shigella group survive well for a year or more on Dorset egg slant cultures in screw capped bottles kept at room temperature in the dark, organisms which do not grow readily usually require more frequent attention

Freeze Drying (Lyophilization)

This involves suspending bacteria in a mixture of one part of 30% glucose in nutrient broth and three parts of commercial horse serum and freezing and drying by evaporation under vacuum. This method preserves the bacteria as well as yeasts, Cryptococci, Nocardia and Streptomyces indefinitely.

Freezing

Many microorganisms remain viable for long periods when frozen.

Preservation In Silica Gel

This method of preservation is used mainly for fungal spores.

Note: Survival of spores is for up to eleven years depending on species of microorganisms. When required a few crystals are scattered on an agar plate for organisms to grow.

Preservation in Water

1. Grow young cultures of the microorganisms in agar

2. Cut from the edge of the young fungal cultures in agar

3.° Place cut agar containing organism in sterile distilled ( water in screw cap bottles

4. Screw down caps

5. Store preparation at room temperature

Note: Good revivals for several years have been obtained for many fungi.

Preservation of Viruses (plant viruses)

Advances in science of virology are continually rendering earlier virus descriptions incomplete. The description of new viruses require direct comparison with already known ones. Preservation is therefore prerequisite to identification. Several methods are available for the preservation of viruses.

Many viruses are directly detectable in crude sap preparations of dry plant materials by serology and electron microscopy.

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