Pure Culture:
A culture in which only one strain or clone present.
Top six methods used for obtaining pure culture of microorganisms.
I) Streak Plate Method
This method is most commonly used to isolate pure culture of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. the successive streaks " thin out " the inoculum sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the some loop/needle without re inoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited.
Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/needle and re streaked onto fresh media to ensure purity.
II) Pour Plate Method
This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium mentioned in the liquid state at a temperature of forty two degree Celsius. The bacteria and the melted medium are mixed well. The contents of each tube are poured onto separate petriplates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium and on the medium. These isolated colonies are then picked up by inoculation loop and streaked onto another petriplates to ensure purity.
III) Spread Plate Method
In this methods, the mixed culture or micro organisms is not diluted in the melted agar medium; it is rather diluted in a series of tubes containing sterile liquid, usually water or physiological saline. A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread evenly over the surface by means of sterilized bent-glass-rod. the medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well isolated colonies grow. This happens as a result of separation of individual microorganisms by spreads over the drops of diluted liquid on the medium of the plate.
The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.
IV) Serial Dilution Method
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. A microorganisms that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculation 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. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganism i,e 100 microorganism/ml of the liquid medium. If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganism in 10 ml or 10 microorganism/ml. If we add 1 ml of this suspension to another 9 ml of fresh sterile liquid medium, each ml now would contain a single microorganism. If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganisms in the medium and represents the pure culture of that microorganism.
V) Single Cell Isolation Methods
An individual cell of the required kind is picked out by this method from the mixed culture and permitted to grow.
i) Capillary Pipette Method
Several small drops of a suitably diluted culture medium are put on a sterile glass cover slip by a sterile pipette drown to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.
ii) Micro manipulator method
Micro manipulator have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjugation with a microscope to pick a single cell from a hanging drop preparation. The micro manipulator has micro pipette adjustment by means of which its micro pipette can be moved right and left, forward and backward and up and down. A series of hanging drops of a diluted culture are placed on a special sterile cover slip by a micro pipette. Now, a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micro pipette by gentle suction and then transferred to a large drop of medium on another sterile cover slip. When the number of cells increase in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yield a pure culture of this required microorganisms.
The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. This disadvantages are that the equivalent is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies.
VI) Enrichment Culture Method
Generally, it is used to isolate those microorganism which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixer culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and, specific condition of incubation favors the growth of desired micro organisms but, is unsuitable for the growth of other types of microorganisms.