Pure Culture preservation Methods

Pure culture preservation of microbes are described in detain. Different short and long term preservation are explained in detail. Methods like Agar slant cultures (Sub culturing) & Refrigeration , Mineral Oil or Liquid Paraffin Method,Saline suspension storage, Drying in Vacuum, Storage at low temperatures (Cryopreservation) and Lyophilization (Freeze drying) are included.

1. Pure Culture Preservation Methods By Dr C R Meera Assistant Professor & HOD Department of Microbiology St. Mary’s College, Thrissur

2. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Why do we need culture preservation methods? Stock culture maintenance for laboratory classes, research works, or for other particular procedures in Microbiology Labs For maintenance of reference strains for taxonomic studies American Type Culture Collection (ATCC ) Microbial Type Culture Collection & Gene Bank (MTCC)

3. Objectives of Culture Preservation Maintenance of microorganisms for extended period of time in viable condition No contamination No lose of genetic and morphological characteristics. Preservation methods either stop or slow down the metabolism and multiplication of microbes. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India

4. Types of Culture Preservation Based on the period of storage, culture p reservation is divided into Short term methods and Long term preservation methods . Short term methods Long term methods Agar slant cultures ( Sub culturing) & Refrigeration Mineral Oil or Liquid Paraffin Method   Saline suspension storage   Drying in vacuum   Storage at low temperatures (Cryopreservation)   Lyophilization (Freeze drying ) Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India

5. Short Term Preservation Method Agar slant cultures (Sub-culturing) and Refrigeration Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Maintenance of pure culture by periodic aseptic sub-culturing to the suitable media- called stock cultures . Type of media , storage temperature and interval between sub-culturing depends on the bacterial species . Solid media is preferred as the toxic material produced by organisms diffuse into slant and thus away from microbial growth. Image Courtesy- ncbe.reading.ac.uk

6. Agar slant cultures (Sub-culturing) and Refrigeration Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Most heterotrophs can be easily maintained on nutrient agar. Nutrient agar slants in screw capped bottles are preferred as it prevent drying of the media. Disadvantage is that repeated sub-culturing leads to mutation in the strains. Prepared stock culture on getting sufficient growth are stored in cool dry place or refrigerator till use. Low temperature like 4 o C slow down the growth and metabolism of microorganisms- Nutrient use reduced Periodic sub-culturing is needed as bacterial waste products accumulate

7. Long Term Preservation Methods 1. Mineral Oil or Liquid Paraffin Method Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Agar slants in screw capped tubes are inoculated and incubated till good growth is obtained . Covered with sterile mineral oil or liquid paraffin to a depth of 1 cm above the slant. Paraffin layer prevents the dehydration of the medium and also produce anaerobic condition so that microorganisms remain in a dormant state . Can be used to preserve bacteria and fungi in viable condition at room temperature from months to years. Image Courtesy- youtube.com

8. Mineral Oil or Liquid Paraffin Method- Advantages Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India The unique advantage of the method is that sub-culturing can be done without affecting the stock culture . (Sub-culturing is done by taking loopful of growth under the oil from these slants, touching the loop to the glass surface of tube to drain off excess oil and then inoculating to fresh medium). Many species remain viable for longer periods under oil than in tubes without oil. Useful for strains susceptible to mutations on repeated sub-culturing Simple and economical method as equipment like dessicator , vaccum pump etc are not needed.

9. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Preservation Methods 2. Saline suspension storage Sodium chloride in high concentration is inhibitory to the microbes. 1 % salt solution (sub lethal concentration) is useful for culture preservation Bacteria are suspended in salt solutions in screw capped tubes to avoid evaporation . Tubes can be stored at room temperature and when required, can be sub-cultured to agar slants. Easy method for storing bacterial cultures for 2-3 years Particularly useful in labs with limited equipment facility. Image Courtesy- thomassci.com

10. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Preservation Methods 3. Drying in Vaccum Organisms are found to survive longer if they are dried in a desiccator than when air dried. Different methods are employed for drying in vacuum. Liquid drying or L –Drying - Organisms are suspended in liquid media and dried in a desiccator in vacuum over dehydrating agents such as H 2 SO 4 or P 2 O 5 ( Phosphorus pentoxide ) Image Courtesy- biocyclopedia.com

11. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 3. Drying in Vaccum L –Drying - P articularly useful for microbes which are sensitive to freeze drying as this method involves vacuum-drying of samples from the liquid state without freezing The microbial suspensions can be also dried on sterile coverslips, filter paper, in small test-tubes, or more conveniently in sterile ampoules which can subsequently be sealed off in vacuo Image Courtesy- tedpella.com

12. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 4. Freezing and Cryopreservation Glycerol suspension method Organisms suspended in nutrient broth containing 15% glycerol, frozen and stored at -29 o C (up to 2 years). Liquid Nitrogen Method Dense cell suspensions are prepared in the medium containing cryopreservative agents like Glycerol or Dimethyl sulfoxide (DMSO ). These agents prevent ice crystal formation during freezing process which may damage microbial cells. Image Courtesy- sciencephoto.com

13. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 4. Freezing and Cryopreservation Microbial suspension is sealed into small ampoules or vials and then frozen at controlled rate to -150 o C . Ampoules or vials stored in a liquid nitrogen refrigerator either by immersion in liquid nitrogen (-196°C) or by storage in a gas phase above liquid nitrogen (-150 o C). Useful for preservation of organisms that cannot withstand freeze drying . Cultures can be maintained for 10-30 years or more without any change in the characteristics of microbes . Disadvantage: Expensive method as liquid nitrogen has to be replenished at regular intervals to replace the loss due to evaporation.

14. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 5. Lyophilisation (Freeze drying ) Mainly useful to preserve organisms that would be killed by ordinary drying process. Dense microbial cell suspension is prepared in small vials and frozen at - 60 to - 78 o C. Vials are connected to high vacuum so that ice present in frozen culture undergo sublimation under vacuum . Sublimation is transition of substance directly from solid to gas state without passing through liquid state. This causes dehydration of bacteria with minimum damage to delicate cell structure.

15. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 5. Lyophilisation (Freeze drying ) Vials are then sealed under vaccum and stored in refrigerator. Many species of bacteria can be preserved by this method for more than 30 years in viable condition without any change in characteristics. Minimal storage place is required for lyophilised cultures as they are in powder form. Vials are also convenient to mail in sealed containers to other laboratories . Lyophilised cultures are revived by rehydrating the opened vials by adding liquid medium and then transferring it to the suitable media.

16. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India Long Term Methods 5. Lyophilisation (Freeze drying ) Image Courtesy- labmanager.com Image Courtesy- microbiologics.com

17. Pure culture preservation methods, Dr C R Meera, St.Mary’s College, TCR, Kerala, India