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Saturday, 16 March 2013

Preserving Microbial Cultures


When once microorganisms are cultured in the laboratory, they are usually sub cultured on agar plates or slants at regular intervals to maintain viability.
Some of the microbes routinely used for experiments should always be available for any immediate experimentation. In order to have a ready availability of cultures, the microbial cultures should be maintained properly and in viable conditions.
Most microbiological laboratories maintain a large collection of strains, frequently referred to as ‘stock culture collection.’

Various methods are:

1.       Periodic  transfer to fresh media
2.       Preservation by overlying with mineral oil
3.       Lyophilization/freeze-drying
4.       Storage at low temperature
5.       Sordelli’s method of preservation of cultures
6.       Preservation at -40 degree centigrade in glycerin
7.       Preservation in soil
8.       Paraffin method
9.       Use of refrigerator or cold room storage
10.   Preservation of soil cultures

Periodic transfer to fresh media:


·         Strains can be maintained by periodically preparing a fresh stock culture from the previous stock cultures.
·         The culture medium i.e.; storage temperature and the time intervals at which the transfers are made vary with the species and must be ascertained beforehand.
·         The temperature and type of medium chosen should support  a slow rather than a rapid rate of growth so that the time interval between transfers can be as long as possible.
·         Many of the more common heterotrophs remain viable for several weeks or months on a medium like nutrient agar.
Disadvantages:  Changes in characteristics of a strain due to development or variants in mutants.

Preservation by overlying cultures with mineral oil:



·         Many bacteria can be successfully preserved by covering the growth on agar slant with sterile mineral oil.
·         The oil must cover the slant completely. Therefore,  to ensure this, the oil should be about ½ inch above the tip of the slanted surface.
·         Maintenance of viability under this treatment varies with species.
·         This method of maintenance has the unique advantage that one can remove some of the growth under the oil with a transfer needle, inoculate into a fresh medium and inoculate into a fresh medium and still preserve the original culture.
Disadvantages: Change in characteristics of strain.

Lyophilization or freeze-drying:

·         Many bacteria die if cultures are allowed to become dry, although spore-formers can remain viable for many years.
·         However, freeze-drying can satisfactorily preserve many kinds of bacteria that would be killed by ordinary drying.
·         In this process, a dense cell suspension is placed in small vials and frozen at -60 to -78 degree centigrade.
·         The vials are then connected to a high-vacuum line.
·         The ice present in the frozen suspension sublimes under the vacuum i.e.; evaporates without first going through a liquid water phase.
·         This results in dehydration of the bacteria with a minimum damage to delicate cell structures.
·         The vials are then sealed off under a vacuum and stored in a refrigerator.
·         Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years.
·         Lyophilized cultures are revived by opening the vials, adding liquid medium and transferring the rehydrated cultures to a suitable growth medium.
Advantages:
1.       Only minimal storage space is required.
2.       Hundreds of lyophilized cultures can be stored in a small area.
3.       Small vials can be sent conveniently through mail to other microbiology laboratories when packaged in special sealed mailing containers.
Storage at low temperature (liquid nitrogen):
·         In this procedure cells are prepared as a dense suspension in a medium containing a cryoprotective agent such as glycerol or dimethyl sulfoxide (DMSO), which prevents cell damage due to ice crystal formation during the subsequent steps.
·         The cell suspension is sealed into small ampoules or vials and then frozen at a controlled rate to -150 degree centigrade.
·         The ampoules or vials are then stored in a liquid nitrogen refrigerator, either by immersion in liquid nitrogen or by storage in the gas phase above the liquid nitrogen            -150 degree centigrade.
·         The liquid nitrogen method has been successful with many species that cannot be preserved by Lyophilization and most species can remain viable under these conditions for 10-30 years or more without undergoing change in their characteristics.
Disadvantages: This method is relatively expensive, since the liquid nitrogen in the refrigerator must be replenished at regular intervals to replace the loss due to evaporation.

Sordelli’s method of preservation:

·         This is a method of preservation simpler than freeze drying, but as reliable as latter.
·         This method can be safely used when the samples to be preserved are small in quantity.
·         In this method, the culture should be preserved or incubated on solid medium for the required period.
·         The inoculum is emulsified in a loopful of horse serum and is deposited on the inner wall of the small tube, which is inserted into another large tube.
·         A small quantity of phosphorus pentoxide is placed at the bottom or the outer tube with the help of glass rod.
·         The inner tube must be placed in such a way that it is held over the bottom of the outer tube but not directly touching the chemical placed at the bottom.
·         The outer tube is then connected to a vacuum pump and after the air is removed, the outer tube is sealed.
·         This tube containing the culture can be stored at room temperature away from light.
 

Preservation at -40 degree centigrade in glycerol:

·         Cultures can be preserved for a number of years in glycerol at a temperature of -40 degree centigrade in a freezer.
·         In this method, about 2ml of glycerol solution is added onto the agar slant culture.
·         The culture can be emulsified by shaking the culture and emulsion is transferred to ampoules with each ampoule having 5ml of culture.
·         These ampoules are placed in a mixture of industrial methylated spirit and carbon dioxide and are frozen rapidly at -70 degree centigrade.
·         The ampoules are then removed and placed directly in a deep freeze at -40 degree centigrade.
·         For utilization of stock cultures, the ampoules are kept in a water bath at 45 degree centigrade for about a few seconds and then used.

                      Preservation in soil:

·         Soil borne bacteria and fungi can be stored in their natural habitat i.e.; soil.
·         About 5gms of soil sample is autoclaved at 15lb pressure for 30 minutes and inoculated with 1ml of aqueous suspension of cells/spores.
·         The microorganisms are allowed to grow for 10 days and the soil culture thus obtained is stored in refrigerator.
·         Microorganisms tend to undergo less variation in soil than in agar.

                      Paraffin Method:

·         Simple and cost effective method.
·         Used for preserving cultures of bacteria and fungi for several years at room temperature.
·         In this method sterile liquid paraffin is poured over the slant of microbes and stored upright at room temperature.
·         The layer of paraffin prevents dehydration of the medium and ensures anaerobic conditions.
·         As a result, the microbes remain in dormant condition.
·         It has been seen that in some instances bacteria remained viable for even up to 15-20 years.

Cold Room Storage:

·         Live cultures on a culture medium can be successfully stored in refrigerator or cold rooms. When the temperature is maintained at 4 degree centigrade.
·         At this temperature range the metabolic activities of microbes slow down greatly.
·         As a result, bacterial metabolism will be very slow and only less quantity of nutrients will be utilized.
Disadvantages: Cannot be used for long time storage because not only the nutrients get utilized but also waste products get accumulate, killing the microbes.

Preservation of Soil Cultures:

·         A sample of soil is passed through a sieve of 2mm and collected in sufficiently large test tubes.
·         1% solution of dextrose is added to the tubes and mixed thoroughly.
·         These cultures are then kept in a boiling water for about 15 min and then autoclaved for 1 hour at 13 pound pressure for 2 successive days.
·         Pure culture of soil microbes can be kept in this medium and preserved for a number of months under refrigerator.



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