Microbial Culture Preservation and its Methods

Various microbiological methods used for culture preservation in the labs

1. DAVANGERE UNIVERSITY SHIVAGANGOTHRI – 577 007 Department of Studies in Microbiology. Seminar Topic On : Microbial Culture Preservation. Presented By: DENNIS M. MONDAH MSc 3 RD SEMESTER Depart of Microbiology. Under The Guidance of : Dr. VINAY KUMAR Depart of Microbiology. Presented On: 31/01/2022.

2. Contents. Introduction Pure culture Objectives of preservation Methods of preservation Short term methods long term methods Summary Conclusion References

3. Introduction Microbial culture preservation is to maintain a pure culture for extended periods in a viable conditions, without any genetic change. The aim of culture preservation is to stop the cell division at a particular stage i.e. to stop microbial growth or at least lower the growth rate. Due to this toxic chemicals are not accumulated and hence viability of microorganisms is not affected.

4. Pure Culture. A   pure culture is a culture that is obtained from a single spore colony or cell. The pure culture contains only one single organism which can be used without worrying of contamination from other species. Preservation: - This is to maintain a pure culture for extended periods in a viable conditions, without any genetic change using different microbiological methods. The aim of preservation is to stop the cell division at a particular stage i.e. to stop microbial growth or at least lower the growth rate.

5. OBJECTIVES OF PRESERVATION The primary aim of culture preservation is to maintain the organism for extended periods in a viable conditions. To keep the culture isolate uncontaminated for a longer period to be used for educational , research, bioassay, industrial, or other purposes. variation or mutation, that is, to preserve the culture in a condition that is as close as possible as the original isolate.

6. Methods of Preservation. Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure culture free from contamination. There are two types of microbial culture preservation; Short term culture preservation. Long-term culture preservation.

7. Short-term Culture Preservation. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (sub-culturing ) to allow continuous growth and viability of microorganisms. Microbial cultures remain viable for several weeks or months on a medium like Nutrient agar . This is a simple method and any special apparatus are not required, however it is easy to recover the culture. The transfer has the disadvantage of failing to prevent changes in the characteristics of a strain due to development of variants and mutants and risk of contamination is also more in this process.

8. Short term methods of culture preservation include: Periodic transfer to fresh media. Preservation using saline suspension. Preservation by drying method. Preservation by refrigeration.

9. 1. Periodic transfer to fresh m edia . Strains can be maintained by periodically preparing a fresh culture from the previous stock culture. The culture medium, the storage temperature, and the time interval at which the transfers are made vary with the species and must be ascertained beforehand. The temperature and the 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.

10. 2. Storage in Saline suspension. Bacterial culture is preserved in 1% salt concentration in screw caped tubes to prevent evaporation. The tubes are stored in room temperature. Whenever needed the transfer is made on Agar Slant.

11. 3. Preservation by Drying method. Spores of some microbes which are sensitive to freeze- drying, can be preserved by drying from the liquid state rather than the frozen state.  Different procedures of drying methods are as follows: Paper disc: A thick suspension of bacteria is placed on sterile discs of thick absorbent paper, which are then dried over phosphorus pentoxide in a desiccation under vacuum. Gelatin disc: Drops of bacterial suspension in gelatin are placed on sterile plastic petri-plates and then dried off over P2O5 under vacuum. L-drying : Bacteria in small ampoules are dried from the liquid state using a vacuum pump and desiccant and a water bath to control the temperature. In this suspension of the organisms are dried under vacuum from the liquid state without freezing taking place . - Apart from the mentioned methods the organisms are also dried over Calcium Chloride in vacuum  and are stored in the refrigerator.

12. 4. Preservation by Refrigeration. Pure cultures can be successfully stored at 0- 4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) as the metabolic activities of the microorganisms are greatly slowed down but not stopped. Their growth continue slowly as nutrients are utilized and waste products released into the medium finally results in the death of the microbes after sometime.

13. Long term Preservation Methods. Long term microbial culture preservation methods include: Preservation by using liquid Paraffin/ Mineral oil Preservation by using of Glycerol Immersion in distilled water Preservation using sterile soil Lyophilization – Freeze drying Cryopreservation – Liquid Nitrogen

14. 1 . Preservation by using Paraffin. This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin is poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies with species). The advantage of this method is that we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture.

15. 2. Preservation using Glycerol. Bacteria can be frozen using 15% glycerol. The glycerol is diluted to 30% and an equal amount of glycerol and culture broth are mixed, dispensed into tubes, and then frozen at -10° C. When liquid bacteria cultures are added to a 50% glycerol solution, the glycerol infuses into the bacterial cells, making them structurally stable and allowing them to be stored safely. After mixing your samples, freeze them at −80 °C to ensure that they remain viable for as long as possible. They can be stored at -20 °C and transferred after 12–18 months.

16. 3. Immersion in Distilled Water. This is one of the another method in expensive and low-maintenance method for staining fungal culture so to immerse them in distilled water Fungi can be stored in this method at 20°c for up to 10-20 years depending upon the species For sporulating fungi; It involves inoculating agar slants of preferred media with fungal cultures and then incubating them at 25˚C for several weeks to induce sporulation. Sterile distilled water(6-7 ml) is added aseptically to the culture, and the surface of the culture is scraped gently with a pipette to produce a spore and mycelial slurry. This is kept in sterile glass vial at 25˚C and to retrieve a culture, 200-300µl of the suspension is removed from the vial and placed on fresh medium.

17. 4. Preservation Using Sterile Soil. It is mainly applied for the preservation of sporulating microorganisms such as Fusarium, Penicillium, Alternaria, Rhizopus etc. Soil storage involves inoculation of 1ml of spore suspension into soil (autoclaved twice) and incubating at room temperature for 5-10 days. The initial growth period allows the fungus to use the available moisture and gradually to become dormant. The bottles are then stored in refrigerator and the organisms can found viable after 70-80 years.

18. 5. Lyophilization Method. It is a vacuum sublimation technique that involves freeze drying the culture using products that are hygroscopic to protect them from moisture during storage. By freezing the cells in a medium that contains a lyoprotectant (usually sucrose) and then pulling the water out using vacuum(sublimation), the cells can be effectively preserved. Freezing must be very rapid, with the temperature lowered to well below 0˚C (as such -20˚C). Lyophilized cultures are stored in the dark at 4˚C in refrigerators. Many microbes preserved by this method can remain viable and unchanged in their characteristic for more than 20 years.

19. Advantages Only minimal storage space is required as hundreds of lyophilized cultures can be stored in a small area. Sterility can be maintained for a longer period of time. Disadvantage Handling and processing time increase Sterile diluent is needed upon reconstition. Equipment become costly and complex. Not all tools can be freeze dried. Slow process average cycle 42 - 47 Hours . High energy costs.

20. 6. Cryopreservation. Cryopreservation involves freezing cultures in liquid nitrogen at -196°C or in the gas phase above the liquid nitrogen at -150°C that helps in survival for long storage times. In this method, the microorganisms are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol or Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to formation of ice crystals and promote cell survival. This 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 to 30 years without undergoing change in their characteristics

21. Advantages Reduced risk of microbial contamination. Cultures can be preserved for longer periods The cultures do not undergo genetic mutations therefore maintaining the original characteristics. Disadvantages This method is expensive.

22. Summary. Microbial culture preservation is to maintain a pure culture for extended periods in a viable conditions, without any genetic change. The primary aim of culture preservation is to maintain the organism for extended periods in a viable conditions, uncontaminated to be used for educational, research, bioassay, industrial, or other purposes. There are two types of microbial culture preservation; Short term culture preservation include; Periodic transfer to fresh media . Preservation by refrigeration. 2. Long-term culture preservation include; - Preservation by using liquid Paraffin/ Mineral oil - Lyophilization – Freeze drying - Cryopreservation – Liquid Nitrogen

23. Conclusion. A  pure culture is a culture that is obtained from a single spore colony or cell . Microbial culture preservation aims to maintain a pure culture for extended periods in a viable conditions, without any genetic change using different microbiological methods . There are two types of microbial culture preservation; Short term culture preservation include; Periodic transfer to fresh media. Preservation by refrigeration. 2. Long-term culture preservation include; - Preservation by using liquid Paraffin/ Mineral oil - Lyophilization – Freeze drying - Cryopreservation – Liquid Nitrogen

24. References. Simione , F.P. and Brown, E.M. 1991. ATCC Preservation Methods: Freezing and Freeze Drying. American Type Culture Collection, Rockville, Maryland. Simione , F.P. 1992. Key issues relating to the genetic stability and preservation of cells and cell banks. J Parenter Sci Techno 46:226-32. De Paoli, P. 2005. Bio-banking in microbiology: From sample collection to epidemiology, diagnosis and research. FEMS Microbiology Reviews 29:897-910 Huba'lek , Z. 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology 46: 205-29. Moore, L.W. and Rene, V. 1975. Liquid nitrogen storage of phytopathogenic bacteria. Phytophathology 65:246-50.