Isolation and preservation of media
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The document discusses isolation and preservation methods for pure cultures in microbiology, emphasizing the importance of obtaining and maintaining pure cultures for accurate testing and research. It outlines various isolation techniques, such as streak plate, pour plate, spread plate, roll tube, and micromanipulator methods, along with their advantages and disadvantages. Additionally, it describes several preservation methods, including refrigeration, cryopreservation, soil storage, overlaying with mineral oil, and lyophilization, highlighting their benefits and limitations.
Isolation and preservation of media
- 1. ISOLATION & PRESERVATION METHODS FOR PURE CULTURE PREPARED BY- D.Mahendra,M.Pharm,(Ph.D),. 1
- 2. Pure Culture Technique 2 • 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 dispersethe individual cells across the medium surface or by thinning the sample many times before inoculating the new medium.
- 3. Why important ? Pure cultures are important in microbiology for the followingreasons- 3 1.Once purified, the isolated species can then be cultivated withthe knowledge that only the desired microorganism is beinggrown. 2. A pure culture can be correctly identified for accurate studyingand 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. o Pure culture spontaneous mutation rate is low o Pure culture clone is 99.999% identical
- 4. •Cultures composed of cells arising from a single progenitor •Progenitor is termed a CFU •Aseptic technique prevents contamination of sterile substances or objects •Common isolation techniques -Streak plate method -Pour plate method -Spread plate method -Roll tube method 4 ISOLATION TECHNIQUE OF PURE CULTURE
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- 6. 1.Streak platemethod microbial culture with an inoculating• Streakingis the processof spreadingthe needle on the surfaceof themedia. • Sterilizethe inoculating needlebyflameto make redhot andallowit to coolfor 30 seconds. • Thesampleisstreakedinsuchawaytoprovideseriesofdilution. • purpose-thinoutinnoculumtogetsepratecolonies. • subculturingcanbedonebystreakingwelisolatedcoloniesfromstreakplatetonewplate. 6
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- 8. 2. Pour plate method 8 • Thebacterial culture andliquid agarmedium aremixedtogether. • After mixing the medium,the medium containing the culture poured into sterilized petridishes( petriplates), allowed solidifying andthenincubated. • After incubation coloniesappear on the surface. DISADVANTAGES- 1.Microorganism trappedbeneath the surfaceof mediumhence surfaceas wel as subsurface coloniesaredevelopedwhichmakesthedifficuktiesincountingthebacterialcolony. 2. Tidiousand time consumingmethod,microbes are subjectedto heat shockbecause liquid mediummaintainedat45℃. 3. Unsuitable- Psychrophile
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- 10. 3. Spread plate method 10 agarmedium. Insteadit is mixed with • Thisisthe bestmethod to isolatethepurecolonies. • In this technique, the culture is not mixed with the normalsaline andserialydiluted. • 0.1 ml of sampletaken from diluted mixture, which is placed on the surfaceof the agarplate and spreadevenlyoverthe surfacebyusingLshaped glassrodcaledspreader. • Incubate theplates • After incubation, coloniesareobservedon the agar surface.
- 11. ADVANTAGES 1. It isasimplemethod. 2. In this method only surfacecoloniesare formed. 3. Micro-organismsarenot exposedto higher temperature. 11
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- 14. 4. Roll tube method. 14
- 15. 5. Micromanipulator method 15 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. the single cell of microbe sucked into micropipette and transfered to large amount of sterile medium. ADVANTAGES OF MICROMANIPULATOR METHOD- that the culturescomeThe advantages of this method are that one can be reasonably sure from a single cell and one can obtain strains with in thespecies. DISADVANTAGES- The disadvantages are that the equipment is expensive,its manipulation is very tedious, and it requires a skilledperson.
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- 17. PRESERVATION OF PURE CULTURE 17 T o maintain pure culture for extended periods in viable condition without any genetic change is referred as Preservation. During preservation most important factor is to stop microbial growth or at least lower the growth rate. D u e to this toxic chemicals are not accumulated and hence viability of microorganism is not affected.
- 18. Objectives of preservation 18 1. To maintain isolated pure culture for extended periods in a viable conditions. 2. To avoid contamination 3. To restrict Genetic Mutation
- 19. Why to Preserve Bacteria? 19 In nature there are only 1% bacteria which is pathogenic and harmfulto Animalia andPlantae. 99% of bacterial populations are of economicimportance for human beings and plants. In soil for nutrient uptake in food industry,in sewage treatment,inmedical industry. So the preservation of bacteria is one of the mostprofitable practice economically as well as environmentally.
- 20. 1. Academic purpose 2. Reserch Purpose 3. Biotechnology field 4. Fermentation Industry 20
- 21. Preservation methods of Bacteria 21 1. Periodic trnsfer to fresh medium 2. Storage at low temprature 3. Storage in sterile soil 4. Preservation by overlaying culture with mineral oil 5. Lyophillization or freeze drying
- 22. 1. Periodic transfer to fresh medium 22 • Strains can be maintained by periodically preparing a fresh interval at which the culture from the previous stock culture. • The culture medium, the storage temperature, and the time 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. • Many of the more common heterotrophs remain viable for several weeks or months on a medium like NutrientAgar. • 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.
- 23. 2. Storage at low temprature 1. REFRIGERATION 2. CRYOPRESERVATION 23
- 24. 1. REFRIGERATION 24 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) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continue slowly, nutrients are utilized and waste products released in medium. This results in finally the death of the micro
- 25. 2. CRYOPRESERVATION 25 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. In this method, the microorganisms of culture 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, however this method is expensive.
- 26. 3. Storage in sterile soil 26 Storing organisms in soil fall into twogroups; 1 sterile soil infested with small amount of inoculum,immediately dried and stored in refrigerator. 2 Soil infested with the organism,than incubatedallowing The organism to grow;thus the mycelium and propagative unitof second generation are preserved. The soil preservation method is useful for fungi,and by this method actinomycetes are maintained in soil for 4 to 5 years,and there are several bacterial spp which are also maintained in soi for several years.
- 27. 4.Preservation by overlaying culture with mineral oil 27 1. This is a simple and most economical method of maintaining pure cultures. 2. 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. 3. 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). ADVANTAGES 1. We can remove some of the growth under the oil with a transfer needle, inoculate a fresh medium, and still preserve the originalculture. 2. The simplicity of the method makes it attractive, but changes in the characteristics of a strain can stilloccur.
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- 29. 5. Lyophillization or freeze drying 29 • Freeze drying is a stabilizing process in which a substance is first frozen and then the quantity of the solvent is reduced, first by sublimation (primary drying stage) and then desorption (secondary drying stage) • Better preservation occurs with freeze-drying than with other methods because freeze-drying reduces the risk of intracellular ice crystallization that compromises viability • Removal of water from the specimen effectively prevents this damage • Lyophilization is greatest with gram-positivebacteria (spore formers) and decrese with gram -negative bactetia but viability can be maintained as long as 30 years.
- 30. • Large numbers of vials of dried microorganisms can be stored with limited space, and organisms can be easily transported long distances atroomtemperature 30 • The process combines freezing and dehydration- Organisms are initially frozen and then dried by lowering the atmospheric pressure with a vacuumapparatus • Specimens can be connected individually to the condenser (manifold method) or can be placed (in a chamber) where they are dehydrated in one largerairspace
- 32. StorageVials 1. Glassvials are used for allfreeze-driedspecimens vial is added of the outer 2. When freeze-drying is performed in a chamber,double glass vials areused 3. (chamber method) : an outer soft glass for protectionandpreservation of the dehydratedspecimen 4. Silica gel granules are placed in the bottom vial before the inner vial is inserted andcushionedwithcotton 5. Manifoldmethod- a single glass vial isused 6. storage vial must be sealed to maintain the vacuum and the dry atmosphericcondition CryoprotectiveAgents Two most commonly used agentsare Skim milk for chamberlyophilization,and sucrose for manifoldlyophilization 32
- 33. ADVANTAGES 33 Removal of wateratlow temperature Thermolabilematerialscanbe dried. Sterilitycanbemaintained. Reconstitutioniseasy
- 34. DISADVANTAGES 34 Many biological molecules are damaged by the stress associated with freezing, freeze- drying, orboth. E.g. the process of drying causes extensive damage to molds, protozoa, and most viruses Hence, these microorganisms can not be stored by thismethod The product is prone to oxidation, due to high porosity and large surface area. Therefore the product should be packed in vacuum or using inertgas Cost may be an issue, depending on theproduct