Introduction:
Microorganisms in nature are never found in pure cultures, they are always mixed.
Hence to study them, the pure cultures are obtained from various laboratories where they are prepared and preserved for longer durations.
The notable laboratories are National Chemical Laboratory, India Ltd., American Type Culture Collection (ATCC) USA, National Collection of Type Cultures (NCTC) England etc.
Pure Culture Techniques:
A pure culture is a population of only one specific species of a microorganism.
The isolation of this only one kind of microorganism from a mixture is called “Pure Culture Techniques”.
The important pure culture techniques are listed as follows,
Streak Plate Method.
Pour Plate Method.
Loop Dilution Technique.
Serial Dilution Technique.
Spread Plate Method.
Micromanipulator Method.
Roll Tube Method.
Streak Plate Method:
Most commonly used method 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 inoculum sufficiently and the micro-organisms are separated from each other.
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 loops/needle and re-streaked 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.
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 an inoculation loop and streaked onto another Petri plate to insure purity.
Serial Dilution Technique:
In this technique the original inoculum is diluted with sterile water or saline solution.
The dilutions are then mixed with liquid nutrient agar @ 45°C and poured in sterile petri dishes allowed to solidify and then incubated.
Disadvantages of Pour Plate Technique:
Formation of subsurface colonies, which are difficult to separate and count.
Use of heat.
Not useful for isolating psychrophile bacterias.
Time consuming and requires skilled persons.
Spread Plate Method:
In this method, the mixed culture or 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 center of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod then medium is 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.
The isolated colonies are picked up and transferred onto fresh medium to ensure purity.
In contrast to the 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.
Micromanipulator Method:
This is a single cell isolation technique.
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 within 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.
Roll Tube Method:
This technique is used for isolation of obligate anaerobes.
The test tubes are made oxygen free by flushing other inert gas or CO2.
Nutrient agar is melted at 50°C, two ml is taken out and added in test tube.
Inoculum is added and test tubes are rolled under water flow to set the agar film on sides of the test tube.
Incubated to get the colonies.
