Isolation & preservation of culture of microorganism
- 1. Isolation & Preservation of Culture of microorganism DR. RACHANA CHOUDHARY ASSTT. PROF. DEPT. OF MICROBIOLOGY SHRI SHANKARACHARYA MAHAVIDYALAYA JUNWANI BHILAI
- 2. INTRODUCTION • Microorganisms are generally found in nature (air, soil and water) as mixed populations. Even the diseased parts of plants and animals contain a great number of microorganisms. • Study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture.
- 4. There are several methods of isolating the pure culture of bacteria, like, streak plate method, pour plate method, spread plate method and serial dilution. Why important ? 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. Testing/experimenting with a pure culture ensures that the same results can be achieved regardless of how many time the test is repeated.
- 5. COMMONMETHODSOF ISOLATIONOF PURE CULTURE The process of screening a pure culture by separating one type of microbes from a mixture is called Isolation. Common methods of isolation; I) Streak plate method II) Pour plate method • Serial Dilution technique III) Spread plate method
- 6. Special METHODSOF ISOLATIONOF PURECULTURE 1. Single cell Isolation methods (A) Capillary pipette method (B) Micromanipulator method 2. Enrichment Culture Method
- 7. (I)STREAKINGOR STREAKPLATETECHNIQUE • Streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.
- 9. VARIOUS METHOD OF STREAKING
- 10. II) Pour plate method • The bacterial culture and liquid agar medium are mixed together. • After mixing the medium, the medium containing the culture poured into sterilized petridishes (petriplates), allowed solidifying and then incubated. • After incubation colonies appear on the surface.
- 12. Disadvantages of Pour plate method • The microorganisms are trapped beneath the surface of medium when it solidifies. Hence, surface as well as subsurface colonies are developed and it is very difficult to isolate and count the subsurface colonies. • This method is tedious, time consuming and requires skill. • The microorganisms are subjected to hot shock because liquid medium is maintained at 45°C temperature. • Unsuitable for isolation of psychrophile bacteria.
- 13. PROCEDURE FOR SPREAD AND POUR PALTEMETHOD
- 14. (a) SERIAL DILUTION This method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.
- 15. • If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml. • If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. • If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.
- 16. III) Spread plate method
- 17. Advantages of spread plate method 1. It is a simple method. 2. In this method only surface colonies are formed. 3. Micro-organisms are not exposed to higher temperature.
- 18. SPECIAL METHODOF ISOLATIONOF PURE CULTURE Single cell isolation methods (A). Capillary pipette method: Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.
- 20. (B)-MICROMANIPULATOR METHOD Micromanipulators Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. ADVANTAGES OF MICROMANIPULATOR METHOD • The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. DISADVANTAGES • Disadvantages are that the equipment is expensive, • Its manipulation is very tedious, and it requires a skilled operator.
- 22. (2).Enrichment Culture Method: • Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture. • The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and, specific condition of incubation favours the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.
- 23. Maintenance and 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. 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. • The transfer is always subject to aseptic conditions to avoid contamination.
- 24. Objectives of preservation To maintain isolated pure cultures for extended periods (future use) in a viable conditions. To avoid the contamination To restrict genetic change(Mutation)
- 25. Application of Preservation • Academic Use: • Research Purpose: • Fermentation Industry: • Biotechnological Field: • 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. • 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. The transfer is always subject to aseptic conditions to avoid contamination. • Since repeated sub-culturing is time consuming, • It becomes difficult to maintain a large number of pure cultures successfully for a long time. • In addition, there is a risk of genetic changes as well as contamination. • Therefore, it is now being replaced by some modern methods that do not need frequent sub-culturing. • These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).
- 26. Methods of Preservation 1. Periodic transfer to fresh media (Sub culturing) 2. Storage in sterile soil 3. Storage at low temperature 4. Preservation by overlaying cultures with mineral oil 5. Freeze dying/Lyophilization
- 27. 1. Periodic transfer to fresh media • 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. • The transfer method has the disadvantage of failing to prevent changes in the characteristics of a strain due to the development of variants and mutants.
- 28. Advantages 1. It is a simple method, any special apparatus are not required. 2. Easy to recover the culture Disadvantage 1. Risk of contamination is more 2. It may be possible to change in genetic and biochemical characteristics
- 29. Storagein sterilesoil • It is mainly applied for the preservation of sporulating microorganisms (a single spore (endospore) within the cell). • Fusarium, Penicillium, Alternaria, Rhizopus, Bacillus, Aspergillus, Penicillium, etc. proved successful for store in sterile soil. • 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 at refrigerator. • Viability of organisms found around 70- 80 years.
- 30. Storageat lowtemperature • Culture medium can be successfully stored in refrigerators or cold rooms, when the temperature is maintained at 4˚C. • Another: liquid nitrogen can provide long term preservation of culture. In this method, dense suspension of microbes is prepared in a medium containing a protective agent(Glycerol or dimethyl sulfoxide) which prevent cell damage due to ice crystal formation. Suspension is sealed into small ampoules or vials and then frozen at - 150°c • At this temperature range the metabolic activities of microbes slows down greatly and only small quantity of nutrients will be utilized. • This method cannot be used for a very long time because toxic products get accumulated which can kill the microbes. • 10-30 Years without changing the characteristics.
- 31. Preservationby overlayingculturewith mineraloil or liquid paraffin storage • In this method sterile liquid paraffin is poured over the slant culture of microbes and stored upright at room temperature. • Where as cultures can also be maintained by covering agar slants by sterile mineral oil which is stored at room temperature or preferably at 0-5°C. • It limit the oxygen access that reduces the microorganism’s metabolism and growth, as well as to cell drying during preservation. • The preservation period for bacteria from the genera Azotobacter and Mycobacterium is from 7-10 years, for Bacillus it is 8-12 years. Adv: • Simple • Mainly used anaerobic microorganism • Cost effective method • Can preserve 10-15 years
- 32. 2. Lyophilization (Freeze-Drying) • Freeze-drying is a process where water and other solvents are removed from a frozen product via sublimation. • Sublimation occurs when a frozen liquid goes directly to a gaseous state without entering a liquid phase. • It is recommended using slow rates of cooling, as this will result in the formation of vertical ice crystal structures, thus allowing for more efficient water sublimation for the frozen product
- 34. Procedure • In this process, a dense cell suspension is placed in small vials and frozen at -60 to -70°C. • The vial are immediately connected to a high vacuum line. • The ice present in the frozen suspension evaporates (sublime) under the vacuum. • This results in dehydration of bacterial cell and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. • The vials are then sealed off under a vacuum and stored in the dark at 4°C in refrigerators.
- 35. Advantage of Lyophilization • Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area. • Remained viable for more than 30 years. • Frequent sub-culturing is not required • Maintained without contamination • Lyophilised strains remains genetically stable • Small vials can be sent conveniently through the mail to other microbiology laboratories when packaged in a special sealed mailing containers. • This method is employed for the preservaton of sera, toxin, enzymes and other biologicals.
- 36. CONCLUSION Pure culture maintenance technique is used for isolation & preservation of culture from different samples. These techniques are used for industrially important organisms. it is essential to check the quality of the preserved organisms stocks However preservation is essential as it reveals great importance in the field of science. Preservation helps in research purposes, industry as well as in Microbiological field.
- 37. REFERENCES • Textbook of Microbiology R. P. Singh • Textbook of Microbiology Dr. R.C. Dubey & Dr. D. K. Maheshwari. • Experiments in Microbiology, Plant Pathology& Biotechnology By K.R. Aneja. • www.google.com • Slidesharelink
- 38. Thank you