Isolation of pure culture

Isolation of pure cultures method

A culture that contains only one kind of microorganism is called the pure culture or axenic culture ,in contrast to culture containing more than one kind of microorganism is called mixed culture or contaminated culture. Pure cultures are isolated in the microbiological laboratories to study the following

• colony characteristics

• biochemical properties morphology

• Staining reaction

Two steps are needed for obtaining a pure culture

1. isolation it is a separation of a particular microbe from a mixed population that exist in nature.

2. cultivation it is the growth of microbial population in artificial environment i.e culture media under lab conditions

History of origin of pure culture techniques

In 1872, a microbiologist named as Schratter had observed the growth of different source of bacteria in isolated mass of various colours on slices of decaying potato with his microscope. He saw that all the microorganism in any one colony were always exactly the same it was obvious that by cultivating microbes on solid nutrient media it was possible to obtain isolated colonies of any single kind each a pure culture

in the earlier days gelatin was used as solidifying agent, later come the use of Agar Agar which had many advantages over gelatin . In order to prevent contamination of pure cultures by dust ,a student in Robert courts laboratory RJ Petri suggested the use of circular shallow dishes with covering glass covers ,these are widely used today as Petri plates

For isolation of pure culture solid Agar medium is always used this is because

• It can trapped the individual cells or spores in place of their origin.

• isolated a separate colonies can be obtained on the liquid medium, it is not possible to keep the colonies separated from each other

Methods for obtaining pure culture

There are the following methods which are used for obtaining the pure cultures some of the as follows

1. Streak plate method

It is the most practical method of obtaining discrete colony at the end of pure culture. It was developed by two bacteriologist Loeffler and Gaffkey in the lab of Robert Koch's

In this method a sterilised loop or inoculation needle is dipped into a suitable delute suspension of Organism. This is then streaked on the surface of solid media contained in a petri plate . In this way, a series of parallel non overlapping streaks are made. The aim of this exercises is to obtain colonies of microorganism that are pure i.e growth derived from a single cell or spore . After streaking those plates are incubated in inverted position at a temperature that favours the growth of the desired microbe.

The plates are then examined for the growth of microbes. A confluent growth is seen where the initial streak is made and at the end of the streak ,we find discreet colony . A well isolated colony selected from each plate and is subculture this gives pure culture.

2. Pour plate method

The forerunners of the present pour plate method was developed in the lab of the famous bacteriologist Robert Koch . Now-a-days the original method is used with some modifications

In this technique ,successive dilution of the inoculum are added into sterile petri plate to which is poured the melted and cold ( 42 -45 degree Celsius) agar medium , then thoroughly mixed by rotating the petri plates which is then allowed to cool and solidify. Plates are then incubated at the desired temperature and isolated colonies are sub cultured

3. Spread plate method

the spread plate technique is used for the separation of mixed population of microorganism ,so that individual colonies can be associated in this technique. Microbial suspension is spread over the solidify agar medium with the real glass spreader ,while the petri plates are rotated on a turnable table. The theory behind this technique is that as the particulate is move at the same stage, single cell will be deposited with the spreader on the agar surface . Some of these cells will be separated from each other by a distance ,sufficient to allow the growth of colonies then develop to the free from each other.

4. Enrich method

The principle behind this is too enrich the desired microbe by providing all the favourable condition which induce its maximum growth.Examples are

1. For cellulose decomposers cellulose or filter paper is added to the medium . Thus bacterial cell like Cellulomonas can grow on this medium and can be isolated.

2. For isolation of bacteria which hydrolyse casein ,skim milk is added to the medium

3. For isolation of lactose fermenting bacteria ,the medium is enriched by sodium lactate as an energy source

4. For isolation of anaerobic microbes ,oxygen is eliminated from environment and anaerobic conditions are provided . The medium is enriched with suitable things and the desired microbe is isolated. This method requires an indepth knowledge about a microbe and its environmental requirement

5. Repressive method

This method is of two types

• Using selective media In this method desirable microbes are prevented from growing by the addition of certain chemicals to the basic medium . These chemicals do not interfere in the growth of desired microbe but repress the growth of all other microbes . Some of the example of such isolations are

• when isolation of bacterial species is desired, fungal colonies can be suppressed by adding some antibiotic in the medium. One such antibiotic which is widely used is actidione (cyclohexaneamide). It inhibits protein synthesis in fungi and therefore suppress the fungal growth . In this way we can get a pure culture of bacteria.

• For isolating nitrogen fixing microbes, nitrogen sources omitted from the medium for example Mannitol Agar medium is used to isolate Azotobacter

• For isolation of fungi ,Rose Bengal is added to the medium, this inhibits bacterial growth

• Heating the broth culture which is contaminated, at 85 degree Celsius for 5-10 minutes or more kills all non spore formers . Such suspension if inoculated will give a pure culture of endospore forming bacteria.

• Addition of very high concentration of salt suppress many bacteria and helps in the isolation of Staphylococcus

Differential method

There are many differential method media which allow the growth of most of the microbes but help us to identify the desired colony. In the media ,some dyes or reagents or chemicals are added which help us to identify desired colony in a mixed culture . Once identified, these can be easily subculture and isolated.

Example,

• For isolating Lactobacillus, differential medium is prepared by adding lactose as carbon source and pH sensitive dye BCP (bromocresol purple), lactic acid bacteria fermented lactose and produce lactic acid and so the dye changes from purple to yellow ,in the area surrounding such colonies. The colonies are then isolated

• For isolation of bacteria

Another differential medium EMB agar( eosine methylene agar) is used to isolate bacteria from sewage water . Generally coliforms are isolated from sewage. E.coli give the brilliant green colony on this medium which is anucleated and show metallic shine . Similarly the colonies of Enterobacter on this medium are gummy pink in colour and generally nucleated.

• For isolation of Staphylococcus

A dye phenol red is added to the medium because of bacterial fermentation acid is produced and the medium shows changes in colour from red to yellow around the bacterial colony.

Sub culturing or picking off

After the isolation of pure culture i.e isolation of well separated discrete colonies ,the next step is sub culturing . Some of the microbial cells from the isolated colonies are transferred to a slant or agar plate. Sub culturing is done with a sterilised inoculation needle These isolated culture are kept for further examination and used . Each of these new culture represents the growth of a single species and then called a pure culture . Subculture term is used to describe the procedure of transferring the microbes from their parent growth source to a fresh medium or from one medium to another.

Picking off is the term which is used instead of sub culturing when the transfer is done from a solid medium to liquid medium example from Agar to broth.