Culture preservation

Culture preservation

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

2. CONTENTS Introduction Objectives of Culture Preservation Culture Preservation Center Techniques for the Preservation of Microbes Microorganisms are in Continues Metabolic active state Microorganisms are in Suspended Metabolic state Summary Conclusion References

3. 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.

17. 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

18. Storage using Liquid Nitrogen (Cryopreservation) In this method, the culture are rapidly frozen in liquid nitrogen at -196 o 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.

19. 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.

20. 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.

21. 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, 2 nd Ed. ASM Press Washington, D. C, P. No: 29-33.