Culture preservation

1. Davangere University
Department Of Microbiology
: Seminar Topic on :
PRESERVATION AND MAINTENANCE OF INDUSTRIAL
Presented By:
Rudramurthi C V
IV sem
Under the guidance of:
Dr.Virupakshaiah D B M
Department of microbiology

2. CONTENTS
1. Introduction
2. Objectives of Culture Preservation
3. Culture Preservation Center
4. Techniques for the Preservation of Microbes
• Microorganisms are in Continues Metabolic active state
• Microorganisms are in Suspended Metabolic state
5. Summary
6. Conclusion
7. References

3. 1. INTRODUCTION
• Pure culture is a culture obtained from a single spore or cell.
• Preservation can be defined as, maintenance of pure culture for extended
periods in a viable conditions without any genetic change.
• The aim of preservation is to stop the cell division at a particular stage.
• Due to this toxic chemicals are not accumulated and hence viability of
microorganisms is not affected.
• Streptomyces aureofaciens NRRL 2209 was the first microbe deposited in a
culture collection in support of a Microbially based patent application.

4. 2. OBJECTIVES OF CULTURE PRESERVATION
• To maintain isolated pure culture for extended periods in a
viable conditions.
• To avoid contamination.
• To restrict genetic change Or mutation.

5. 3. CULTURE PRESERVATION CENTER
• Culture collection centers that provide authentic examples of
organisms, often microorganisms or animal and plant cell cultures
that can be grown or maintained in a laboratory.
• Two different organizations that provide microbial cultures.
• MTCC : Microbial Type Culture Collection and Gene bank, India(1986)
• ATCC : The American Type Culture Collection, (1925)
• Although 27 culture collection centre in India are registered with
WDCM.

6. Microbial Type Culture Collection (IMTech, Chandigarh.
• National Fungal Culture Collection Of India (Pune)

7. 4. TECHNIQUES FOR THE PRESERVATION OF
MICROBES
• Microorganisms are in Continues metabolic active state.
i. Periodic transfer to Fresh media
ii. Preservation with Mineral oil
iii.Storage in Sterile Soil
iv. Storage in Saline suspension
• Microorganisms are in Suspended metabolic state.
i. Drying
ii. Lyophilization
iii. Storage in Silica gel
iv. Storage using Liquid Nitrogen (Cryopreservation)

8. • Continues metabolic active state preservation
technique
In this technique, Organisms preserved on general nutrient
medium by repeated sub culture. Here repeated Subculturing
is required due to depletion of drying of nutrient medium.

9. Periodic transfer to Fresh media:
• Strains can be maintained by periodically preparing a fresh culture
from previous stock culture.
• The culture medium, the storage temperature, and the time interval
at which the transfers are made vary with the species.
• The temperature and the type of medium chosen should support a
slow growth rather than rapid growth.
• Organisms remains viable for several weeks or month on NA medium
• But there is a probability of Occurance of mutation in the organism.

10. Overlaying Culture with Mineral oil
• Many pure cultures of bacteria and fungi can be preserved.
• Paraffin oil of Specific gravity 0.865 to 0.890 is generally
used.
• In this method, Sterile liquid Paraffin is poured over the
agar slant of culture to a depth of 1cm, above the tip of
surface.
• The layer of mineral oil ensure anaerobic conditions
and prevents dehydration of the medium.
• Some species can preserved satisfactory for 15-20 years by this
method.

11. Advantages:
• One can remove some of the growth under the oil with transfer
needle, inoculate a fresh medium, and still preserve the initial
culture.
• Cheap and easy method.
Disadvantages:
• Chances of air born contamination during subculture are more.
• Chances of mutation are more.

12. Storage in Sterile Soil
• This technique can be used to preserve the sporulating bacteria and
Fungi such as, Fusarium, Penicillium, Alternaria, and Rhizopus.
• Sterilization of soil and addition of minerals and nutrients to the
sterilized soil, then inoculation of 1ml of spore suspension into
sterilized soil and incubate at room temperature for 5 to 10 days.
• Initial growth period allows the fungus to use available moisture and
gradually became dormant .
• Then the bottles are stored in refrigerator and organisms can found
viable after 70 to 80 years.
• Cheap and convenient method.

13. Storage in Saline suspension
• The bacterial culture is preserved in 1% salt concentration in screw
capped tubes to prevent evaporation.
• The tubes are then stored in room temperature, whenever required
the transfer made on agar slant.

14. • Microorganisms are in Suspended metabolic state
Organisms preserved in suspended metabolic state by either
drying or storage at low temperature. Microbes are dried or
kept at low temperature carefully so that their revival is
possible.

15. Drying
• Spores of some microbes are sensitive to freeze drying can be
preserved by drying from the liquid state rather than the frozen
state.
• Different procedure of drying methods are:
• Paper disc
• Liquid drying
• Apart from this, the organisms are also dried over Calcium chloride
in vacuum and then stored in refrigerator

16. Lyophilization
• One of the best method for long term preservation of culture. It is generally
used to preserve Fungi, viruses, bacteria, enzymes, toxins and other
microbes.
• Lyophilization is a vacuum sublimation technique that involves freeze
drying the culture using products that protect them from moisture during
storage.
• Advantages: Less opportunity for changes in characteristics of oganisms
Culture once dried needs no further attention
Organisms can survive for period of 20 years or more
• Disadvantages: This is expensive and need expertise.

18. Storage in Silica gel
• The basic principle in this technique is quick desication at low
temperature, which allows the cell to remain viable for longer
period.
• Some of the species such as, Saccharomyces cerevisiae, Aspergillus
nidulans, Pseudomonas denitrificans, Escherichia coli can preserved
on anhydrous silica gel.
• This method is simple and mites free, Suitable for Oomycetous fungi.
• Repeated retrieval can result contamination, and this method is
suitable only for fungi

19. Storage using Liquid Nitrogen (Cryopreservation)
• In this method, the culture are rapidly frozen in liquid nitrogen at
-196o C in the presence of stabilising agents such as, Glycerol or
DMSO that prevents cell damage due to formation of ice crystals and
promote cell survival.
• Advantages: Many species has been successfully preserved that cannot
be preserved by Lyophilization. Species
can remain viable for 10-30 years
• Disadvantages: This method
is expensive.

20. 5. SUMMARY
Microbial culture preservation is to maintain a pure cure for extended
periods in a viable condition without any genetic change.
The primary aim of preservation is to maintain the organisms for
extended period in a viable condition, uncontaminated to be used for
industrial, bioassay, research or other purpose.
Microorganisms are in continues metabolic active state and
microorganisms are in suspended metabolic state are two techniques
for preservation of microbes.

21. 6. CONCLUSION
Maintenance of pure culture is used for isolation and preservation of
culture from different samples. These techniques are used for
industrially important organisms.
However preservation is essential as it reveals great important
in field of Science. Preservation helps in research purpose,
industrially as well as in microbiological field.

22. 7. REFERENCES
• Simione, F. P and Brown, E.M.1991. ATCC Preservation Methods:
Freezing and Freeze drying. American Type Culture Collection,
Rockville, Maryland.
• De Paoli, P. 2005. Bio-banking in microbiology: From sample
collection to epidemiology, diagnosis and research, FEMS
Microbiology Reviews. 29:897-910.
• JulianE.Davies and Arnold L. Demain, 1999. Manualof Industrial
Microbiology: Preservation of Industrial strains, 2nd Ed. ASM Press
Washington, D. C, P. No: 29-33.