To maintain pure culture for extended periods in viable conditions, without any genetic change is referred as preservation

1. PRESERVATIO
N METHODS
FOR PURE
CULTURE
Presented by:- Lokesh Patil
B Pharm 5th sem 3rd year

2. To maintain pure culture for
extended periods in viable
conditions, without any genetic
change is referred as
preservation
PRESERVATION

3. AIM OF PRESERVATION
The aim of preservation is to stop the cell
division at a particular stage i.e to stop
microbial growth or atleast lower the growth
rate.
Due to this toxic chemicals are not accumulated
hence viability of the microorganisms is not
affected
 To avoid contamination

4. 1. Periodic transfer of fresh
media
2. Oil overlay method
3. Storage at low temperature
4. Storage in sterile soil
5. Lyophilization or freeze
drying
METHODS OF
PRESERVATION

5. 1. Periodic transfer of fresh
media
Almost in all the microbiological laboratories, microorganisms are
preserved in agar slants, which after incubation is stored in the
refrigerators at low temperatures.
This agar slant media is continuously transferred to a fresh media in a
definite time interval.
The time interval may range from 1 day or 1 week or 6 months. This
procedure of transferring media is performed as long as preservation of
culture is required

6. 1. Periodic transfer of fresh
media

7. 2. Oil overlay method
This method was first used in 1947 by Buell & Weston, and is almost
similar to the periodic transfer method, however differs only in usage of
paraffin oil. The oil used in preservation must have specific gravity in
range of 0.865 to 0.890
The oil to be used should be sterilized by dry heat sterilization method in
hot air oven. The sterilized oil is poured over the culture containing
nutrient agar slant, upto 1 cm above the slant. The slant now is stored at
low temperature or room temperature

8. 2. Oil overlay method

9. 3. Storage at low
temperatures
This method is preferred for the short time preservation. In this method
refrigerators or cold rooms are used. If long period preservation is
required than subculturing of the culture is essential.
Use of liquid nitrogen is another long term preservation of culture. In this
method the cell remains viable for 10-30 years without changing their
property.

10. 3. Procedure
Dense suspension of microbes is prepared having a protective
agent ( glycerol or dimethyl sulphoxide), which is helpful in
prevention of cell damage due to formation of ice crystals.
The cell suspension is sealed into small vials and are kept for
freezing at -1500C. After this the vials are stored in liquid
nitrogen refrigerator (-1960C)

12. 4. Storage in sterile soil
Sterile soil method is preferred for spore forming microbes eg. Species of
Streptomyces, Aspergillus, Bacillus, Penicillium, etc. This method involves
storage of pure cultures of microbes in medium of sterile soil followed by
its preservation by refrigeration up to several months.

13. 4. Procedure
A suspension containing spores is prepared in a special
medium.
A mixture containing sand (78%), soil (20%), Calcium carbonate
(2%) is prepared and distributed into tubes. This mixture is
sterilized at a temperature of 1300C for 8-15 hours.
Prepared suspension is then added to the sterile mixture and
is allowed to grow for around 10 days.
Mixture containing tubes are inoculated under the vaccum in
desiccator. This is done to allow the evaporation of excess
water and then the tubes are sealed for preservation

15. 5. Lyophilisation
This method was first developed and used by Raper and Alexander of
National Research Laboratory, at Peoria. In this technique water is
removed from freezed product and is placed under vacumm, allowing the
ice to change directly from solid to vapour without passing through the
liquid phase, therefore the technique is known as freeze drying.

17. 6. Cryopreservation
Pure cultures are able to survive when stored for a long time with the aid
of cryopreservation. In this method, the microorganisms of culture are
rapidly frozen in liquid nitrogen -1960C. Some stabilizing agents ( Glycerol,
dimethyl sulphoxide) are also used for promotion of cell viability, survival
and prevention of cell damage due to formation of ice crystals

19. 7. Storage in silica gel
Silica gel powdered maintained at low temperatures can be used to store
both bacteria and yeast for upto 12-24 months. The fundamental principle
of this method is that quick desiccation at low temperature increases the
viability of the cells for longer time period.