Pour plate method has certain disadvantages as follows: These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. The bacteria and the melted medium are mixed well. 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☌ (agar solidifies below 42☌). 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. This method involves plating of diluted samples mixed with melted agar medium (Fig. Such isolated colonies are picked up separately using sterile inoculating loop/needle and re-streaked onto fresh media to ensure purity. Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture.
Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited.
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