Isolation of Bacteria

Isolation of bacteria is a primary method to separate different groups of microorganisms. It is the method which helps us to differentiate different groups of organisms. It is the method, which allows us to discriminate different groups of bacteria on the basis of “Growth Pattern”.

Different bacteria grow differently on the different nutrient medium based on their growth requirements and the other factors like temperature, pH, oxygen availability etc. Isolation of bacteria is an important step for the identification and differentiation of the bacteria.

Content: Isolation of bacteria

  1. Definition
  2. Isolation Methods


Isolation of bacteria can define as the technique of separating one species of bacteria from the mixed culture of bacteria by different plating methods like pouring, spreading, streaking and serial dilution.

In simple words, isolation of bacteria can define as the method of obtaining a “Pure culture” which is devoid of any contamination and other related microorganisms.

Isolation Methods

There are several methods for the isolation of bacteria, which includes:


Pouring Method

Pouring is a simple method for the separation of bacteria. In the pouring method, the bacterial suspension which carries the huge bacterial population is taken.

pour plate method

By the help of pipette, take 1ml of a bacterial sample into the sterile Petri plate. For the growth of bacteria, there should be some nutrient source like carbon and nitrogen. Therefore, the most common Agar nutrient medium is firstly prepared and then added to the Petri plates containing the bacterial sample.

For the uniform distribution of the sample and the media, rotate the plates in the clockwise and anticlockwise direction. Before keeping the Petri plates in the incubator, allow the culture plates to solidify.

For the proper growth of bacteria, keep the culture plates in the incubator at the temperature of 35-37 degrees Celsius for the maximum period of 48 hours. After the incubation period of 24-48 hours, we can see the growth of bacterial colonies.

In the pouring method, the isolation of bacteria becomes difficult because of suspended growth of bacteria in the solid media. Some bacteria appear on the surface of the solid nutrient medium, and some appear under the surface of a solid nutrient medium.

In the pouring method, there is an overgrowth of bacterial colonies from which the isolation of pure culture is very difficult. This technique is least preferred for the separation of pure culture.

Spreading Method

Spreading method is again a very simple method to perform isolation of bacteria. It differs from the pour plate method, add the nutrient medium first before the addition of the bacterial sample.

spread plate method

The nutrient medium is added to the sterile Petri plates and then allowed to solidify. After the solidification of the nutrient media, add 1ml of the bacterial suspension over the surface of solid media.

For the uniform distribution of bacteria over the surface of solid media, take the spreader of T or L-shape to spread the bacterial suspension evenly. After that, incubate the culture plates for 35-37degrees Celsius for 24 to 48 hours.

We can see a number of bacterial colonies after the incubation period. In the spreading method, we can select the isolated colonies for the culturing of bacteria. For the isolation of pure culture, the spreading method is not very popular.

Streaking Method

Streaking method is very popular and the most widely used method for the isolation of pure culture. In this take the nutrient medium into the sterile Petri plates and allow it to solidify.

streaking method

After that take the inoculating loop and sterilize it on the flame until it gets red hot. Then, take the inoculum by the help of sterilized inoculating loop and streak over the solid nutrient media by keeping the plate close to the flame, to avoid contamination. After streaking of bacteria, incubate the culture plates for 24-48 hours at a temperature of 35-37degrees Celsius in the incubator.

In the streaking method, there is a limited population of bacteria, from which the isolation of pure culture is quite easier than the pour plate and the spread plate method. In this, one can separate the individual colony of bacteria for the isolation and culturing of bacteria.

Serial Dilution Method

This technique is much known for the isolation and culturing of bacteria. In the serial dilution method, take the bacterial suspension and dilute it serially in successive test tubes.

serial dilution method

By following the “Serial dilution” method, add 1ml of the sample to the neighbouring test tube sequentially in a series 10-1, 10-2, 10-3 and so on. After the sequential dilution of bacterial suspension, we can inoculate the bacterial culture by the above three methods, i.e. pouring, spreading and streaking.

It is very easy to isolate bacteria from less number of a bacterial population. In serial dilution, the more concentrated sample, i.e. 10-1 will produce the highest number of colonies. The more diluted sample, i.e. 10-4 will produce the least number of colonies.

So it should be clear to us that less diluted sample will contain more concentration of bacteria than that of water. And the more diluted sample will contain a high concentration of water than that of bacteria.

Therefore it is clear that the sample which contains low bacterial population will produce less number of colonies and vice versa. Select the colonies which show less growth for the isolation method.

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