Isolation and Preservation methods for Pure Culture

Mix Culture Media: A Culture media, which contain more than one kind of microorganism is called a mixed culture media.

Pure Culture: A pure culture is usually derived from a mixed culture by transferring a small sample into new.

_ Pure culture is defined as a laboratory culture containing a single species of organism.

Importance of Pure Culture:

a] Desired known species of microorganisms can grow once pure culture is isolated.

b] Pure culture can be correctly identified for accurate study and testing of microorganism in a clinical environment.

c] Experimenting with pure culture ensures the same results after repetition of experiment many times.

d] In pure culture, the spontaneous mutation rate is low.

Isolation: Isolation is the separating process of particular microorganism from the mixed culture.

Methods of isolation for pure culture:

1] Streak plate method

2] Pour plate method

3] Spread plate method

4] Serial dilution method

5] Special methods

     _Single Cell Isolation Methods

     _Enrichment Culture Method

Streak Plate Method:

The principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate where bacterial cells are deposited on the agar surface.

In this method the bacteria is isolated from mixed culture media by applying streak on the surface of culture media with the help of Inoculation loop.

Firstly inoculation loop is heated for sterilization, and then it streak on news sterile culture media.

Pour Plate Method: 

The main principle of this method is dilution of the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. 

In this method, 

The mixed culture of bacteria is diluted directly in the liquid agar medium tube (42-45°C) and mixed well. 

fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of sterile petri dish using a sterile pipette. 

The contents of each tube are poured into separate petri plates and then allowed to solidify. 

These plates are the incubated to develop bacterial colonies in both within the agar medium (subsurface colonies) and on the medium (surface colonies). 

Finally, isolated the colonies and are picked up by inoculation loop and streaked onto another petri plate to insure purity. 

Spread Plate Method:

The principle of this method involves using a sterilized spreader with a smooth surface made of metal or glass to apply a small amount of bacteria suspended in a solution over a plate that needs to be dry and at room temperature so that the agar can absorb the bacteria more readily. 

A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate.

In this Method,

a] A drop of diluted liquid from each tube is placed on the center of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod.

b] The medium is incubated for the colonies development on the agar medium plates.

c] The isolated colonies are picked up and transferred onto fresh medium to ensure purity

Serial Dilution Method: 

A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.

In this Method,

_Take 5 to 7 test tubes with field 9 ml of sterile distilled water.

_Now take 1 ml solution from original suspension and dilute it in 1st test tube.

_Then take 1 ml from 1st Test tube and dilute it in 2nd.

_Then take from 2nd and dilute it in 3rd.

_Repeat this process until all test tubes should be diluted.

_ After dilute all test tubes, take 1 ml of each test tube and pour into nutrient Agar plates.

_ As number of test tubes increases, the concentration of bacteria is decreases and the dilution is increases.

_ The method is used for actinomycetes bacteria [soil].