Isolation of bacteria

Isolation of bacteria

In natural environment(soil,air,water,food,sewage,body surfaces) microorganisms exist in mixed population.It is only in very rare situations that they occur as a single kind of bacterium i.e a bacterial species.To study the cultural,morphological and immunological characteristics of a species,normally from in its habit,ie it must be isolated in pure culture.A culture which contains more than one kind of microorganisms is called a mixed culture and only one kind of organism is called pure culture.However before attempting isolation,it is often helpful to use a selective method first.

The microbial population in our environment is large and complex. Many different microbial species normally inhabit various parts of our bodies such as the oral cavity, the intestinal tract, and the skin. These microbes may be present in extremely large numbers. For Eg: a single sneeze may disperse from 10000to 100000 bacteria. One gramme of faeces may contain 10^11 bacteria . Infertile garden soil,microorganisms may number several billion per gramme include many species of bacteria, fungi,algae and protozoa. A study of the microorganism in these habits requires knowledge of the specific microbes present . the, in turn, requires unravelling the complex mixed population into the culture of separate distinct species .

Selective methods favour the growth of the desired species while discouraging or even the other organism present in the mixed culture . chemical, physical or biological methods are used in order to achieve selection of a particular kind of bacterium . a pure culture of the microorganism that forms discrete colonies on solid media may be most simply obtained by one of the modifications of the plating method.Is viable organism give rise through growth to a colony from which subculture can be readily made.There are several different methods of getting pure culture from a mixed culture.The three most frequently used method are :

1. Streak plate method
2. Pour-plate method
3. Spread-plate method

In all these methods,the techniques involved thinning out the organism so that the individual species can be selected from the others

• Streak plate method:

The streak plate method is the most economical and practical method of obtaining discrete colonies.In this method,a sterilised loop is dipped into a suitably diluted suspension of the organism which is then streaked on the surface of an already solidified agar plate to make a serious a parallel,not overlying streaks.This manipulation”Thins out “the bacteria on the agar surface so that some individual bacteria are separated from each other.When streaking is properly performed,the bacterial cell will be sufficiently far apart in some areas of the plate.To ensure that the colony developing from one cell will not merge with that growing from another streaking is made in different ways:

1. Quadrant streak method
2. Radiant streak method
3. T streak streaks method
4. Continuous streak method

• Pour plate method:

In both pour plate and streak plate method,the mixed culture is first diluted a to provide only a few cells per millilitres before being used to inoculate media.Since the no of bacteria in the specimen is known beforehand,a series of dilution is made so that at least one of the dilution will contain the suitably sparse concentration of cells.In the pour plate method, a measured amount of sample is mixed with a large volume of sterile water or physiological saline called diluent or diluent blank.Dilution is usually made in multiples of ten.A single dilution is calculated as

Dilution=Vol.of sample or solute/Total vol. of sample and diluent

Serial dilution is then prepared by transporting a known volume of the dilution to second dilution blank and so on.Once diluted,The specific volume of diluted sample from various dilution is added to sterile Petri plate to which molten and cooled(temperature of 45-50c)suitable agar medium is added for thorough distribution of the inoculum.The sample and medium are homogeneously mixed before solidification and incubated and a series of agar plate showing decreasing no of colonies are counted ,averaged and total no of organism per ml of given sample is calculated as;

No of organism per ml=No of colonies/Inoculation size x Dilution

Advantages

1. Somewhat less skill is required to get good isolation
2. Isolation, as well as enumeration, can be done

Disadvantages

1. Some organisms are trapped beneath the surface of medium when it gets and therefore both surface and sub-surface colonies developed
2. The organisms being isolated most be able to withstand the temporary exposure to 45c of liquid organisms medium,unsuitable for isolating psychrophiles.However, some microaerophiles and some species may be benefited.

Loop dilution method

This method consists of diluting one or more loopful of organisms with tubes of liquefied media serially in such a manner that one of the plates poured will have an optimum number of organisms per dilutions.

Spread plate method

Spread plate method aid in the isolation as well as enumeration of organisms in a mixed culture . usually, dilution is done in series of tubes containing a sterile liquid , water of physiological salines . measured amount of diluted sample is placed onto the surface of the agar plate and spread evenly over the surface by means of a sterile , bent glass rod .volums greater than 0.1 ml are rarely used because an excess liquid is not soaked in the media and may cause the colonies to coalesce as they form making counting difficult. The number of colonies formed after incubation is counted and total no.of organisms are determined as;

No.of bacteria=No of colonies×dilution factor/Inoculum size(0.1)

If the column size is 0.1 then number of bacteria;

No. of bacteria= no.of colonies/0.1×dilution

Advantages:

1. Isolation as well as Enumeration.
2. The organism does not require to withstand the temperature of liquid agar, Thus this technique may be performed in a quantitative manner to enumerate strict psychrophiles.
3. Strict aerobes are favoured while microaerophiles tend to grow slower.

Disadvantages:

1. The problem of spreaders as well as crowding of colonies that makes the enumeration difficult.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.