Methods of Preservation of Pure Cultures (microbiology)

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METHODS OF PRESERVATION OF PURE CULTURES

After the isolation of any microorganism in pure culture, it is imperative to maintain the viability and purity of such microorganism by keeping the pure culture free from any contamination. To preserve microorganisms, it is necessary to reduce their metabolism up to its minimum due to which the processes that lead to ageing and death are slowed down and the microorganism can be maintained in its inactive state for several years. This process is known as ‘ preservation of pure cultures ’.  

There are several methods of preservation of pure cultures but some commonly used methods are discussed below:

1. Refrigeration

 Pure cultures can be stored successfully in refrigerators or cold rooms at 0-4 °C. 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 being utilized and waste products released in the medium, which finally results in death of the microbes after some time.

2. Paraffin method (storage in mineral oil)

This is a simple and most economical method of maintaining pure cultures of bacteria and fungi .In this method, sterile liquid paraffin or pre-sterilized mineral oil or medical paraffin (specific gravity 0.83-0.89) is poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic condition (reduced oxygen tension) and prevents dehydration of the medium. This condition helps microorganisms of pure cultures to remain in a dormant state and therefore, the culture is preserved for several years.

Procedure:

  1. Grow the culture in suitable medium in a culture tube till full growth is achieved.
  2. Sterilize the liquid paraffin in an oven for 1 hour at 180°C.
  3. Add sterile paraffin to a level of 1 cm above the highest point of agar slope.
  4. Plug the mouth of the tube with cotton plug or metal cap and store in refrigerator.

Revival of culture: The culture is revived by putting a loopful of the culture onto a fresh agar (plates) or broth (tubes) which are incubated till growth resumes.

3. Lyophilization (freeze-drying):

In this method, the culture is rapidly frozen at a very low temperature -70 °C and then dehydrated by vacuum. Under these conditions the cells are dehydrated and their metabolic activities are stopped, the microbes undergo dormant state and retain viability for years. These lyophilized pure cultures are then sealed and stored in the dark at 4°C in a refrigerator.

Procedure:

  1. Sterilize the ampoules in an oven for one hour at 180°C.
  2. Pour 0.5 ml of a broth culture into a sterilized ampoule.
  3. Push a cotton plug completely into ampoule. 4. Connect the ampoule to a hole             in the centrifuge head of freeze-drying apparatus.
  4. Centrifuge is continued for 5-10 minutes which results in rapid dehydration and freezing of the contents of the ampoules.
  5. Allow these for primary dehydration for 5 hours.
  6. The necks of the ampoules are drawn out and the ampoules are given secondary dehydration for 12 hours.
  7. Seal the ampoules under vacuum and store these at 4°C on the desk.
  8. Examine the ampoules for sealing using vacuum tester. Production of a blue   glow is indicative of sufficiently low pressure i.e. the proper sealing of the ampoules.
  9. Remove the cotton-wool plug, flame the ampoule neck.
  10. Add 0.2 ml of broth into the ampoule with a sterile pipette and keep it for rehydration for a few minutes.
  11. Using a sterile pipette, transfer the rehydrated culture to a broth tube
  12. Incubate the tube at 37°C for 24 hours. 14. From the culture, streak the agar plate aseptically.
  13. Incubate the plate at 37°C for 24 hours and study morphology of a given culture.

Revival of the culture: During opening of an ampoule, the ampoule neck is flamed, a stretch is made with a file around the ampoule, corresponding to the equator of the cotton-wool plug and open the ampoule by applying pressure at the stretch  mark.

4. Cryopreservation (storage in liquid nitrogen)

 This method is suitable for long term preservation of microorganisms that do not survive freeze drying. Some microorganisms are sensitive to the effects of cooling and warming. Such loss is reduced by the use of cryoprotectants such as glycerol and dimethyl sulphoxide (DMSO).In cryopreservation the microorganisms of a pure culture are rapidly frozen in liquid nitrogen at -196°C in presence of stabilizing agents (glycerol and DMSO) that prevents the formation of ice crystals which may kill frozen cells. By this method, some cultures retain viability for 10-30 years without ny alteration in their physiology or morphology.

Procedure:

  1. Grow the culture on a suitable medium in sterile Petri dish.
  2. Add 4 ml of 10 % glycerol (v/v) or 5 % DMSO on the growing culture plate.
  3. Scrap the culture with sterile pipette.
  4. Pour 0.5 ml of the cell suspension in cryo-tube (meant for storage).
  5. Transfer and store the culture in liquid nitrogen.
  6. Incubate the plates at 35°C for 24 hours until growth resumes.

Reviving of culture: The culture can be revived by keeping it at room temperature for 2-3 hours and thereafter transferred onto suitable media aseptically.

5. Storage at -70°C

The low temperature deep freezers are recommended for storage of certain microorganisms.

Procedure:

  1. Grow the cells under cryopreservation conditions.
  2. Transfer 0.5 ml suspension in small glass screw capped tube.
  3. Store the tubes at -70°C

6. Storage on glass beads at -60°C to -70°C

 Some microorganisms fail to show viable colonies due to repeated freezing and thawing. For such frozen bacterial cells with a cryoprotectants in glass beads is a reliable method. In this method, the individual glass bead is removed when needed without thawing the entire sample. It is mention worthy that various groups of microorganisms can be maintained by using beads of different colours. The animal pathogens are stored on red beads, plant pathogens on green, and microbes require special growth factors on blue. The alkalinity should be neutralized by using dilute HCI.

Procedure:

  1. Grow the culture on a suitable media.
  2. Prepare the broth medium recommended containing 15% glycerol and autoclave it.
  3. Suspend the culture in 10 ml of the above medium and remove the culture by scrapping to form a thick suspension.
  4. Pour 0.5 ml suspension in each bead containing screw capped vial and freeze the vial at -60°C to -70°C.
  5. Incubate the plates at desired temperature till growth resumes. Note: The glass beads are neutralized by dilute HCl, washing with tap water followed by thorough rinsing with de-ionized water is recommended. After drying, 20-30 beads in screw cap glass vial are added and used after autoclaving.

Revival of culture: The culture can be revived by removing the beads by rubbing over the surface of suitable solid medium with the support of a wire loop.

7. Storage in gelatin discs:

In this method, bacterial strain after growth on a suitable medium is suspended in molten nutrient gelatin. The drops of the molten gelatin are allowed to solidify on Petri dish and allowed to dry over a desiccant. The method is recommended for preservation of various species of Enterobacteriaceae,Staphylococcus etc.

Procedure:

  • Grow the culture on a suitable medium, add 3 ml gelatin containing pre-molten medium consisting of; Gelatin powder      10 g
    1. Meso-inositol          5g
    2. Nutrient powder    2.5g
    3. Deionised water     100ml
  • Pipette out 0.5 ml of bacterial suspension
  • Place 0.02 ml suspension on a Petri plate, cover it and keep at -20 °C until frozen. The drop will become opaque.
  • Transfer the plate to freezer dryer for dryness.
  • Take silica gel containing screw capped glass tube or vial and transfer the solid drop or gel into it, plug it tightly.
  • Store at 5°C to be revived and used as a pure culture.

8. Storage in soil

Various fungi like Rhizopus, Aspergillus, Fusarium, Alternaria, Penicillium, Melanospora, etc. can be stored in sterile soil for longer periods of time.

Procedure:

  • Grow the culture on a suitable medium until spores are formed.
  • Suspend the spores in 1 ml of sterile water.
  • Place the garden soil in a bottle, autoclave twice or thrice and pour sterile water containing spore suspension into it.
  • The bottle is left at room temperature to allow the culture to grow for 10 days.
  • Store the bottle with cap loose in refrigerator.

Revival of culture: The culture can be revived by spraying few soil particles on a suitable medium which is incubated at desired temperature to enhance growth of a given culture.

PROOF OF PURITY OF A GIVEN CULTURE

To ensure the purity of any given culture, it should obey the following criteria:
 
1. All the microorganisms should look alike microscopically and all the cells should stain in the same fashion.
2. When inoculated on agar based media, all the colonies formed should look alike in characteristics.
3. The biochemical characteristics of the isolated colonies should follow the same pattern.

 These are the methods of preservation of pure culture in microbiology.

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