Techniques of Bacterial preservation various techniques

1. PRESERVATION OF BACTERIAL
CULTURE
BY:
B.Navaneethakrishnan
1ST MSc Applied Microbiology
Sacred Heart College[Autonomous]
Tirupattur
TO:
Dr.N.Karthik.
Department of Microbiology
Sacred Heart College[Autonomous]
Tirupattur.

2. PRESERVATION:
It is a technique to maintain pure culture for extended periods in viable
conditions without any changes known as preservation.

3. OBJECTIVES OF PRESERVATION:
• To maintain isolated pure cultures for extended periods in a viable
condition.
• To avoid contamination.
• To restrict the genetic changes.[mutation]

4. METHODS OF PRESERVATION:
• SHORT TERM METHODS.
• LONG TERM METHODS.

5. SHORT TERM METODS:
. Periodic transfer to fresh media.
• Preservation of bacteria using glycerol.
• Storage by drying method.
• Storage by refrigeration.

6. PERIODIC TRANSFER TO FRESH MEDIA:
• Culture can be maintained by periodically.
• It is an advantageous as it is a simple method. Special apparatus not
require.
• Many more microbes remain viable for several weeks and months.
• The transfer has the disadvantages of failing to prevent changes in
characteristic strain.

7. PRESERVATION OF BACTERIA USING
GLYCEROL:
• Bacteria can be frozen using 15% glycerol.
• The glycerol is diluted to 30% and equal amt of glycerol.
• The viability of organism varied such as E- coli, D- pneumonia etc for 5
months.
• N- meningitides for 6 weeks and H- influnzae for 4 months.

8. STORAGE BY DRYING METHOD:
• Spores of some microbes is sensitive to freze dying can be preserved by
drying from liquid state.
• Different procedures of drying methods are follows:
• gelatin disc.
• L.drying.
• Paper disc.
• Apart from the mentioned methods the organisms are also dried over calcium chloride. [Cacl].
• At such condition the organism survive for longer period than the air dried cultures.

9. STORAGE BY REFRIGERATION:
• Culture medium can be sucessfuly stored in refrigerators or cold rooms.
• This temperature range metabolic activity of microbes is slowsdown. Small quantity
of nutrients is require.
• The method cannot be used for very long time because toxic products get
accumulated which can kill microbes.

10. LONG TERM METHODS:
• Mineral oil or liquid paraffin storage.
• Storage in saline water.
• Immersion in distilled water.
• Storage in sterile soil.
• Lyophilization. [freeze – drying].
• Cryopreservation.

11. MINERAL OIL OR LIQUID PARAFFIN
STORAGE:
• In this method sterile liquid paraffin poured over the slant culture of
microbes. And stored in room temperature.
• Cultures also maintained by covering agar slants by sterile mineral oil
which stored in room temperature at 0-5 degree celcius.
• It limit the oxygen access that reduces microbes metabolism and
growth.
• The preservation period for bacteria from the genera Azatobacter sp
and Mycobacterium sp is 7 – 10 yeas for Bacillus is 8-12 years.

12. STORAGE IN SALINE SUSPENSION:
• Bacterial culture is preserved in 1% salt concentration in screw caped
tubes to prevent evaporation.
• Tubes are stored in room temperature.
• Whenever needed transfer is made on agar slant.

13. IMMERSION IN DISTILLED WATER:
• It is another inexpensive method and low maintenance method for
storing culture is to immerse in distilled water.
• Organism can be stored in this method at 20 degree celcius.
• It can be survived up to 2-10 years depending upon the species.

14. STORAGE IN STERILE SOIL:
• It is a technique mainly used preservation of sporulating microbes.
• Eg – Fusarium sp, penicillium sp, Alternaria sp, etc.
• Soil storage involves inoculation of 1ml of spore suspension into soil.
• The initial growth period allows the microbes gradually become
dormant.
• The bottles are stored in refrigerator.
• Viability of organisms found around 70-80 years.

15. LYOPHILIZATION [FREEZE- DRYING]
• It is a vaccum sublimation technique.
• Freeze drying products are hygroscopic and must be protected from
moisture during storage.
• By freezing the cells ina medium that contain a lyoprptectant pulling
the liquid using vaccum.
• Many microbes preserved by this method have remained viable un
changed characteristics more than 20 years.
• It very advantageous only minimal storage space required to preserve.

16. CRYOPRESERVATION:
• It is one of freezing technique helps survival of pure cultures for long
storage time.
• In this method the microbe culture can be rapidly frozen in liquid
nitrogen at 150 degree celcius. In the
• This method species can remain viable for 10-30 years without
undergoing change in their characteristics.

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