ISOLATION OF PURE CULTURE

Microorganisms in nature are never found in pure populations. Different types of mixed microorganisms are normally present in soil, water, food, air and various parts of the human or animal body. To study the role played by a specific microorganisms in these environments, it is necessary to isolate the microorganisms in pure culture.

MIXED CULTURE: - A culture contains more than one species of microbes.

PURE CULTURE: - (A laboratory culture containing a single species of organism). A pure culture is usually derived from a mixed culture by transferring a sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample several times before inoculating the new medium.

It is extremely important to maintain isolated pure cultures for extended periods in a viable condition.

PURE CULTURE TECHNIQUES:- . The isolation of one kind of microorganism from a mixture of much different kind is called the pure culture technique.

THE FOLLOWING METHODS ARE USED TO ISOLATE PURE CULTURE.

METHODS OF ISOLATION OF PURE CULTURE (Types of pure culture technique)

STREAK PLATE METHOD: - Streak plate method is the most widely used method for isolation of cultures. The principle of this method is that, by streaking a dilution gradient is established across the face of the Petri plate where bacterial cells are deposited on the agar surface.

Streak plates are prepared by streaking a small amount of mixed culture over the surface at 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. The purpose of streaking is to thin out the inoculum (starter culture) successively so that microbes get separated.

A second steps may also be streaked from the same loop/needle without reinoculation. In the beginning of the streak, microbes are crowded and colonies develop closely but as the streaking proceeds cells gets separated as the needle contains less cells. Hence at the last streak, few and clearly separated colonies are developed. Transfer the well isolated colonies from streak plate to another new plate for isolation and purification (sub culturing).

POUR PLATE METHODS: - The main principle of this method is dilution of the inoculums in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium.

LOOP DILUTION TECHNIQUE:-

In this method, the mixed culture of bacteria is diluted directly in tubes of liquid (cooled) agar medium and mixed well. The medium is maintained in a liquid state at a temperature of 45° C to allow thorough distribution of the inoculums. In this method fixed amount of inoculums (generally 1 ml) from a sample is placed in the center of sterile Petri dish using a sterile pipette. The content of each tube are poured into separate Petri plates and then allowed to solidify. These plates are then incubated to develop bacterial colonies in both within the agar medium (subsurface colonies) and on the medium (surface colonies). A series of agar plates showing decreasing number of colonies resulting from the loop dilution technique.

SERIAL DILUTION TECHNIQUE:-

In this method the original inoculums may be diluted by using sterile water or a saline solution (by using pipette) so that the concentration of the microbes gradually becomes less.

A micro-organism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.

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.

E.g. A culture containing 10 ml of liquid medium, containing 1000 micro-organisms. i.e. 100 micro-organism/ml of the liquid medium. 1 ml of this medium is taken out and mixed with 9 ml of fresh sterile liquid medium then resulted medium have 100 micro-organisms in 10 ml or 10/ml. further 1 ml of this suspension is added to another 9 ml of fresh sterile liquid medium, each ml is now contained a single micro-organism. If this tube showed any microbial growth, there is a very high probability of that this growth has resulted from the introduction of a single micro-organism in the medium and represents the pure culture of that micro-organism.

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 liquid medium is maintained at 45°C temperature.

SPREAD PLATE METHOD: - The principle of this method involves using a sterilized spreader with a smooth surface made of metal or glass to apply a small amount of bacteria suspended in a solution over a plate that needs to be dry and at room temperature so that the agar can absorb the bacteria more readily.

In 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 count

ADVANTAGES OF SPREAD PLATE METHOD:

Ø It is a simple method.

Ø In this method, only surface colonies are formed.

Ø This method is also used for counting the microorganisms present in the inoculums.

Ø Microorganisms are not exposed to higher temperature.

MICROMANIPULATOR METHOD: A micromanipulator is a device which is used to physically interact with a sample under a microscope. It may typically consist of an input joystick, a mechanism for reducing the range of movement and an output section with the means of holding a micro tool to hold, inject, cut or otherwise manipulate the object as required. Micromanipulators are usually used in conjunction with microscope

Micromanipulator is devices that can pick up a single microbial cell from a colony of mixed culture. 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 on to a large drop of sterile medium on another cover slip. 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

Roll tube method: - 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 pre 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 being rotated by a motor Inoculation starts from the bottom and draws the loop gradually

Prepare ten-fold dilution and add 0.1 ml of diluted culture to molten agar cooled to 50°c poured in test tube. Now tilt the tube and roll so that medium is formed as a thin film around the wall of tube .Incubate and count the no of colonies on next day