Isolation and preservation of pure Cultures.pptx

Detailed explaination of Isolation and preservation methods for pure cultures, various methods of isolation of microorganisms and preservation methods of cultures

1. UNIT I 1.3 Isolation and preservation methods for pure culture s Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

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3. 3 Pure Culture Technique Culture: Act of cultivating microorganisms or the microorganisms that are cultivated. Mixed culture: more than one microorganism. Pure culture: containing a single species of organism. A pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many times before inoculating the new medium. Importance Pure cultures are important in microbiology for the following reasons Once purified, the isolated species can then be cultivated with the knowledge that only the desired microorganism is being grown. A pure culture can be correctly identified for accurate studying and testing, and diagnosis in a clinical environment. 3. Testing experimenting with a pure culture ensures that the same results can be achieved regardless of how many time the test is repeated. Pure culture spontaneous mutation rate is low. Pure culture clone is 99.999% identical

4. 4 It is extremely important to maintain isolated pure culture for extended periods in viable conditions. Most microbiological laboratories usually maintain a large collection of pure culture as well as subculture of authentic species purchased from various cultural collection centers . American type culture collection ( ATCC) USA. National Collection of Industrial Bacteria. (NCIB) Scotland. National Collection Of Yeast Culture (NCYC) England. National Collection of Type Culture ( NCTC) England.

5. 5 Inoculating Loop - is used for transfer of bacteria. Nichrome wire is heated by red hot sterilization method There is no need of fume. The inoculating loop is made up from Platinum OR Nichrome wire.

6. 6 Methods of Isolation of Laboratory cultures Streak Plate method The streak plate method is a most widely used for the isolation of culture. There is no need of liquid media means nutrient broth. But there is a need of solid media because it's contain agar (nutrient agar ). Streak plate are prepared by a streaking a small amount of mixed culture over the surface of the solid medium in a Petri- plate with Platinum or Nichrome wire loop. The sample is streaked in a such away that to provide successive dilution. The purpose of streaking is to thin out the inoculum successively so that microbes get separated. A second plate may also be streaked from the same loop / needle without reinoculation. In the beginning of streak, microbes are crowded and colonies develop closely but as the streaking proceeds cell get separated as the needle contain less cell. Hence at the last streak ,few and clearly separated colonies are developed.

7. 7 Continues Streaking No overlapping of streaking zone. Loop is sterilized only once in beginning. Recommend for both culture. Discontinuous Streaking The streaking is discontinue. The loop is sterilized between carry out the inoculation form one zone to other zone. Rotate at 45° angle of each streaking

8. 8 2. Pour Plate Method Loop dilution Method Mixed culture is directly diluted in tubes of liquid agar medium. The medium is maintained in a liquid state of temperature 42 to 45 °c for distribution of the medium. Media is poured into Petri - plate to become Solidify. Incubate the properly for to allow the growth of microbes. Disadvantages The microorganisms are trapped beneath the surface of the medium when it's Solidifies. This method is tedious, time consuming, and requires skill. Microorganisms should able to with stand the temperature at 42 to 45 ° c . Surface colony of microorganisms difficult to transfer.

9. 9 The Media/Medium Diluted by using sterile medium or saline solution Mix 1 ml dilute sample. In 20 ml liquid Nutrient agar medium at 45 ° c Shake the Nutrient agar medium. Pour in Petri plate & Then Solidify and Incubate at 37° c Pour in Petri plate & Then Solidify and Incubate at 37 ° c B. Serial Dilution Method

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11. 11 3. Spread Plate Method In spread plate method the mixed culture is not Diluted in culture media. Microorganisms are spreaded over solidifying agar media with a sterile L - shaped glass rod spreader. Procedure Firstly prepare the Nutrient agar medium. It is Diluted in series of tubes containing sterile water or a saline solution. A sample is removed from each dilution tube ( 0.1 ml) and placed into the surface agar plated. The culture is spread by using a glass spreader on the surface of agar plate. The plates are incubated and then isolated colonies are observed after 24 hr, at 37 °c. Growth of Microorganisms is observed

12. 12 Spread plate method

13. 13 4. Micromanipulator Method The micromanipulator are device that can pick up a single microbial cell form colony of mixed culture. The micromanipulator are used in conjunction with microscope to pick up a single bacterial cell form hanging drop preparation. The single cell OR microbial cell is gently sucked into micropipette and transferred on to large drop of sterile medium on another cover slips

14. 14 5. Roll Tube Method Roll tube method is used for isolation of stringent anaerobes.

15. 15 Preservation of pure cultures  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 cultures free from contamination. Maintenance of pure cultures -methods   Periodic Transfer to Fresh Media Refrigeration Paraffin Method/ preservation by overlaying cultures with mineral oil Cryopreservation Lyophilization (Freeze-Drying) Preservation in sterile soil

16. 16 Periodic Transfer to Fresh Media   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 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 Disadvantage - failing to prevent changes in the characteristics of a strain due to the development of variants and mutants. Refrigeration   Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms Method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) Metabolic activities of the microorganisms are greatly slowed down but not stopped Growth continues slowly, nutrients are utilized and waste products released in medium

17. 17 Paraffin Method/ preservation by overlaying cultures with mineral oil   This is a simple and most economical method Sterile liquid paraffin in 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. Microorganisms remain in a dormant state The culture can be preserved form months to years Advantage - we can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the original culture. Disadvantage - changes in the characteristics of a strain can still occur.

18. 18 Lyophilization (Freeze-Drying)   Culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped The microbes go into a dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures are then sealed and stored in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers. Many species of bacteria preserved by this method have remained viable and unchanged in their characteristics for more than 30 years. https://www.youtube.com/watch?v=-INsuz3H1M0&t=197s

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20. 20 Cryopreservation   Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the liquid nitrogen at -150°C ) helps survival of pure cultures for long storage times. Microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C Stabilizing agents such as glycerol or Dimethyl Sulfoxide (DMSO) are added 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. M ost species can remain viable under these conditions for 10 to 30 years without undergoing change in their characteristics. H owever this method is expensive.

21. 21 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

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