Isolation and Preservation Methods for Pure Cultures

Isolation and Preservation Techniques for Pure Cultures

• It is unlikely to find pure cultures of microorganisms in nature, they are always mixed.
• Since the cultures are purified in laboratories where they are prepared and preserved for longer periods of time, they are obtained from different sources for study.
• There are several notable laboratories, such as National Chemical Laboratory, India Limited, National Collection of Type Cultures (NCTC), United Kingdom, and American Type Culture Collection (ATCC).

Pure Culture Techniques

Microorganisms in a pure culture are those belonging to a single species. Purification of microorganisms from a mixture using pure culture techniques is called purification. There are a number of important pure culture techniques, which include,

• Streak plate method
• Micromanipulator method
• Spread plate method
• Pour plate method

1. Loop dilution technique
2. Serial dilution technique
3. Roll tube method

Streak Plate Method

• The most common method is the isolation of pure bacteria.
• The mixed culture is streaked across the agar medium with the aid of an inoculation loop or needle tip.
• A thin layer of inoculum is applied, and subsequent streaks separate the microorganisms.
• A colony is allowed to grow on these plates.
• Striking establishes a dilution gradient across the face of a Petri plate when bacteria are deposited on agar surfaces.
• Confluent growth takes place in the part of the medium that contains the fewest bacterial cells because of this dilution gradient.
• When each colony is a clone of a pure culture, the microbial cell it is made up of is the source of the colony.
• To ensure purity, isolated colonies are isolated and picked up with sterile inoculating loops/needles before being spread onto fresh media.

Micromanipulator Method

• Single cells are isolated using this technique.
• There are micromanipulators that enable one to remove a single cell from a mixture of cells.
• With the help of the instrument, a single bacterial cell (usually from a hanging drop preparation) can be taken out of the fluid.
• Micrometer adjustments enable the micropipette of the micromanipulator to be positioned right, left, forward, and backward.
• In order to place drops of diluted culture on sterile coverslips, a series of hanging drops is placed by a micropipette.
• An investigation is now conducted on a hanging drop containing only one microorganism cell.
• Gently suction is used to draw the cells into the micropipette, and then the drop of sterile medium is placed on a sterile coverslip.
• As the number of cells within the drop increases as a result of multiplication, it is transferred to a culture tube containing suitable medium.
• The obtained pure culture consists of the pathogen in question.
• A benefit of this method is that it is reasonable sure that the cultures are obtained from a single cell, and it is possible to obtain strains from a single species of organism.
• This equipment is expensive, it requires a lot of manual manipulation, and it is difficult to use without a skilled operator.
• The reason why this technique is only used for highly specialized studies is due to this reason.

Spread Plate Method

• The medium used in this method is sterile, typically water or physiological saline, not melted agar. This method differs from the pour plate method in that there is no dilution in melted agar.
• Using a sterilized bend-glass-rod, one drop of such diluted liquid from each tube is mixed with a sterilized bend-glass-rod, and spread evenly over the agar plate.
• On the agar medium plates, colonies can grow well-isolated on some plates, while others can grow on less-isolated plates.
• A diluted drop of liquid is spread over the medium to separate individual microorganisms.
• Each colony is then removed from the plate and transferred to new medium to ensure purity.

Pour Plate Method

• Agar medium is melted and mixed with diluted samples before plating.
• In agar plates, the main purpose is to provide a thorough distribution of bacteria throughout the medium by diluting the inoculum in successive tubes of liquefied agar.
• Mixed cultures of bacteria are directly diluted in liquid agar tubes at 42-45°C, in the presence of melted agar medium.
• The melted medium is thoroughly mixed with the bacteria.
• Incubate the tubes' contents separately in different Petri plates after they have solidified.
• The development of bacterial colonies is characterized by both subsurface and surface colonies forming within the agar medium.
• A loop is then used to pick up the isolated colonies and streak them onto another Petri plate so they will remain pure.

Serial Dilution Technique

• A sterile water or saline solution is used to dilute the original inoculum.
• Incubate the dilutions in sterile petri dishes, mix them with liquid nutrient agar at 45°C, and then pour them into, allow to solidify, and then incubate.

Roll Tube Method

• Using this method, obligate anaerobes can be isolated.
• Inert gas or CO2 is pumped through the test tubes to remove oxygen.
• Using nutrient agar, melt two ml at 50°C, add one ml to a test tube, and stir.
• To set the agar film on the sides of test tubes, inoculum is added, and tubes are rolled under flowing water.
• After incubation, colonies form.

Disadvantage of Pour Plate Technique
It is difficult to separate and count colonies that form under the surface of the ground.
Using heat.
Unsuitable for psychrophile bacteria isolation.
Requires expertise and takes time.

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