Pure Culture Techniques: A pure culture consists of a population of only one species of microorganisms. The isolation of one kind of microorganism from a mixture of many different kinds is called the pure culture technique. The methods widely used for the isolation of microorganisms are as follows:
Pure Culture Techniques
- Streak plate method
- Pour plate method
- Loop dilution technique
- Serial dilution technique
- Spread plate method
- Micromanipulator method
- Roll tube method.
(i) Streak plate method:
The streak plate method is the most widely used method for the isolation of cultures. Streak plates are prepared by streaking a small amount of mixed culture over the surface of the solid medium in a Petri plate with a platinum or nichrome wire loop. The sample is streaked in such a way as to provide successive dilution (Fig.1).
The purpose of streaking is to thin out the inoculum (starter culture) successively so that microbes get separated. A second plate may also be streaked from the same loop/needle without inoculation. At the beginning of the streak, microbes are crowded and colonies develop closely but as the streaking proceeds cells get separated as the needle contains fewer cells. Hence at the last streak, few and separated colonies are developed. Transfer the well-isolated colonies from the streak plate to another new plate for isolation and purification (sub-culturing).
(ii) Pour plate method:
In this method, the mixed culture is diluted directly in tubes of liquid (cooled) agar medium (Fig.2). The medium is maintained in a liquid state at a temperature of 45 oC to allow thorough distribution of the inoculum. The inoculated medium is transferred into Petri plates, allowed to solidify, and incubated. A series of agar plates showing the decreasing number of colonies resulting from the loop dilution technique is shown in Figure.2.
In the serial dilution technique, the original inoculum may be diluted by using sterile water or a saline solution (by using a pipette) so that the concentration of the microbes gradually becomes less. Mix 1 ml dilute sample in a 20 ml liquid nutrient agar medium at 45 oC. Shake the liquid nutrient agar medium and pour in a sterile Petri plate, solidify and incubate it. The plate gets well-isolated colonies and then counts the total number of colonies in the sample, and multiplies the number of colonies by the dilution factor.
Disadvantages of pour plate techniques:
- The microorganisms are trapped beneath the surface of the medium when it solidifies. Hence, surface, as well as subsurface colonies, are developed and it is very difficult to isolate and count the subsurface colonies.
- This method is tedious, time-consuming, and requires skill.
- The microorganisms are subjected to heat shock because the liquid medium is maintained at 45 oC temperature.
- This method is unsuitable for isolating psychrophile bacteria.
(iii) Spread plate method:
In the spread plate technique, the mixed culture is not diluted in the culture medium. It is diluted in a series of tubes containing sterile water or a saline solution. A sample is removed from each dilution tube (0.1 ml) and placed onto the surface of an agar plate. The culture is spread by using a glass spreader on the surface of the agar plate. The plates are incubated and the isolated colonies are observed after 24 hours and counted (Fig.3).
Advantages of the spread plate method:
- It is a simple method.
- In this method, only surface colonies are formed.
- This method is also used for counting the microorganisms present in the inoculum.
- Microorganisms are not exposed to a higher temperature.
(iv) Micromanipulator method:
Micromanipulators are devices that can pick up a single microbial cell from a colony of mixed cultures. Micromanipulators are used in conjunction with microscopes to pick up a single bacterial cell from hanging drop preparations. The single microbial cell is gently sucked into the micropipette and transferred onto a large drop of the sterile medium on another coverslip. The micromanipulator method makes one reasonably sure of the pure culture coming from a single cell. The device however has to be used with skill and precision.
(v) Roll tube method:
The roll tube method is used for the isolation of stringent anaerobes. A stoppered anaerobic culture tube is used for isolation which has been coated with a reduced agar medium containing oxygen-free nitrogen. When the stopper is removed the tube is kept anaerobic by continuously flushing it with oxygen-free CO2 from a gas cannula. Inoculation is done with a transfer loop held against the agar surface as the tube is rotated by a motor. Inoculation starts from the bottom and draws the loop gradually upward (Fig.4). After inoculation the tube is restoppered and incubated anaerobically to get well-isolated colonies.
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