Common Methods of isolation of pure culture

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General methods of pure culture isolation

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Common Methods of isolation of pure culture Pure culture of microorganisms that form discrete colonies on solid media e.g. yeasts most bacteria many other microfungi and unicellular microalgae may be most commonly obtained by plating methods such as streak plate method pour plate method and spread plate method. But the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method. Streak Plate Method This method is used most commonly to isolate pure cultures 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 inoculums sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. 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 restreaked onto fresh media to ensure purity. 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 maintained in the liquid state at a temperature of 42-45°C agar solidifies below 42°C. The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates allowed to solidify and then incubated. When bacterial colonies develop one finds that isolated colonies develop both within the agar medium subsurface colonies and on the medium surface colonies. These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity. Pour plate method has certain disadvantages as follows: i the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies and ii the microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms. However the pour plate method in addition to its use in isolating pure cultures is also used for determining the number of viable bacterial cells present in a culture.

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Pour Plate Method 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. Spread Plate Method In this method the mixed culture of microorganisms is not diluted in the melted agar medium unlike the pour plate method 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 centre of an agar plate and spread evenly over the surface by means of a 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 spreading over the drop of diluted liquid on the medium of the plate. Serial Dilution Method As stated earlier this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum 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 1000 microorganisms i.e. 100 microorganisms/ml of the liquid medium. Serial dilution method

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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 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium each ml would now 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 microorganism in the medium and represents the pure culture of that microorganism. Special Methods of Isolation on of Pure Culture 1. Single Cell Isolation methods An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow. The following two methods are in use. i Capillary pipette method Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn 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 Micromanipulator method Micromanipulators have been built which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell particularly bacterial cell from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette 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 coverslip by a micropipette. Now a hanging drop is searched which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism. 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. The disadvantages are that the equipment 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. 2. Enrichment Culture Method Generally it is used to isolate those microorganisms which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed 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

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growth of desired microorganisms but is unsuitable for the growth of other types of microorganisms. Proof of Purity of Cultures Assuming that one has isolated a pure culture how does one establish that it is pure A pure culture is one in which the cells are all of one kind i.e. demonstrate "likeness". Hence the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows: 1.The microorganisms look alike microscopically and stain in the same fashion. 2. When plated all the colonies formed look alike. 3. Streaks stabs etc. are uniform. 4. Several isolated colonies perform identically i.e. ferment the same sugars and so on.

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