Department of Biotechnology

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E-Module on

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Techniques of culturing IIM

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• Resource Person- Dr. Jitender Kumar
• HOD, Biotechnology

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• Techniques of culturing IIM

Isolation and Preservation of Micro-organisms

Isolation of microbial pure culture

• Micro-organisms are generally found in nature (air, soil and water) as mixed population.
• To study the specific role played by a specific micro-organism in its environment, one must isolate the same in pure culture.
• The two major steps of obtaining a pure culture are as follows:
• The culture has to be diluted until the various individual micro-organisms are separated far apart on agar surface that after incubation they form visible colonies isolated from the colonies of other micro-organisms.
• An isolated colony has to be aseptically picked off the isolation plate.

Pure culture/mixed culture

• Pure culture
• Originate from one bacterial strain.
• All the colonies look the same.
• Mixed culture
• Originate from many bacteria strains.
• Colonies have different shape/ sizes.

Pure Colony

Mixed Colony

Points to be taken under consideration during the streaking

• The inoculation loop has to be sterilized properly before the inoculation of the colonies from the agar plate.
• Every time the loop is sterilized by the heat it must be cooled before incubating the next colony.
• There are several methods of isolation of pure culture of bacteria, like, streak plate method, pour plate method, spread plate method, and serial dilution.

Why it is important to have Pure Culture?

• Once purified, the isolated species can then be cultivated with the knowledge that only desired micro-organism 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 to how many time the test is repeated.
• Pure culture spontaneous mutation rate is low.
• Pure culture clone is 99.999% identical.

Common Methods of Isolation of Pure Culture

• The process of screening a pure culture by separating one type of microbes from a mixture is called isolation.
• Some common isolation methods are:
• Streak Plate Method
• Pour Plate Method
• Spread Plate Method
• Serial dilution methods

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Streaking/ Streak Plate Method

• In this method the tip of a fine structure wire loop called inoculation needle consist of a wooden or glass handle with a nichrome wire the end of which is bend to form a loop is used to transfer microbes from culture.
• The straight wires are similar to wire loop except they do not have loop. These are used to transfer culture in colony formed on solid culture medium.
• In such case, the colony from solid medium is streaked on the surface of nutrient agar medium in a sterile petri dish.

This techniques consist of the following steps

1. Hold the broth culture containing tube in left hand and shake it.
2. Sterilize the wire loop of the incubation needle on burner flame.
3. Remove the cotton plug of the broth culture tube by little finger or right hand.
4. Flame the mouth of the test tube immediately.
5. Insert the wire loop to form a thin film and replace the cotton plug.
6. The thin film in the loop is streaked in either zig-zag manner by removing the loop backwards and forwards firmly. Care should be taken that loop should not be firmly pressed against the agar surface.
7. Incubate the petri dish in incubator at the required temperature.
8. Growth of the bacteria will be visible (after an overnight incubation) on the streaked marks.

Pour Plate Method

• A bacterial culture and liquid agar medium are mixed together.
• After mixing the medium, the medium containing the culture poured into sterilized petri dishes, allowed solidifying and then incubated.
• After incubation colonies appear on the surface of agar plates.

Disadvantages of pour plate method

1. The micro-organisms are trapped beneath the surface of medium when solidifies. Hence, surface as well as subsurface colonies are developed and it is very difficult to isolate and count subsurface colonies.
2. This method is tedious, time consuming and requires skill.
3. The micro-organisms are subjected to hot shock because liquid medium is maintained at 45 degree Celsius temperature,
4. This method is unsuitable for isolation of psychrophile bacteria.

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Procedure for Spread and Pour Plate Method

Serial Dilution

• This method is commonly used to obtain pure cultures of those micro-organisms that have not yet been successfully cultivated on solid media and grow only in liquid media.
• Micro-organisms that predominates in a mixed culture can be isolated in pure form by a series of dilution.
• 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.

• If we add 1 ml of this suspension to another 9 ml of fresh sterile liquid medium, each ml would now contain a single micro-organism.
• If this tube shows any microbial growth, there is very high probability that this growth has resulted from the introduction of a single micro-organism in the medium and represent the pure culture of that micro-organism.

Spread Plate Method

• This is the best method to isolate the pure colonies.
• In this technique, the culture is not mixed with the agar medium. Instead it is mixed with normal saline and serially diluted.
• 0.1 ml of sample taken from diluted mixture, which is replaced on the surface of the agar plate and spread evenly over the surface by using L shaped glass rod called Spreader.
• Incubate the plates.
• After incubation, colonies are observed on the agar surface.
• Advantages of Pour Plate Method:
• It is a simple method.
• In this method surface colonies are formed.
• Micro-organisms are not exposed to higher temperature.

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Maintenance and Preservation of Pure Cultures

• Once a micro-organism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the micro-organisms 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 micro-organisms.
• The transfer is always subject to aseptic conditions to avoid contamination.

Objectives of Preservation

• To maintain pure cultures for extended periods (future use) in a viable conditions.
• To avoid the contamination.
• To restrict genetic change (mutation).

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Methods of preservation of microbial cultures

• Low temperature storages
• Lyophilization
• Storage as frozen culture
• Subculturing
• Storage in glycerine stab
• Storage in sterile soil

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References

• General Microbiology by Stainier
• Biotechnology: Expanding Horizon – B.D. Singh (Kalyani Publication)
• Biophysical and Biochemical Technology – Wilson and Walker (Cambridge University Press)
• Principle of Gene Manipulation and Genomics – Primrose (Blackwell Publication)
• General Microbiology by Stainier
• Biotechnology: Expanding Horizon – B.D. Singh (Kalyani Publication)
• Biophysical and Biochemical Technology – Wilson and Walker (Cambridge University Press)
• Principle of Gene Manipulation and Genomics – Primrose (Blackwell Publication)

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