Introduction
In nature, microorganisms exist as mixed populations. Soil, water, food, air and even the human body contain many different microbial species living together. To study the role and characteristics of a single species, it must be separated and maintained as a pure culture. Pure culture techniques help isolate only one type of microorganism from a mixture.
Microbiological laboratories maintain large collections of pure cultures that are used for teaching, research and industrial applications. These cultures may be obtained from national and international culture collection centres such as ATCC (USA), NCIB (Scotland), NCYC (England), NCTC (England) and NCL (India).
What is a Pure Culture?
A pure culture contains only one species of microorganism. The process of separating a single species from a mixed population is called isolation. Several laboratory methods are used to achieve this.
Major Methods Used for Isolation of Bacteria
- Streak plate method
- Pour plate method
- Loop dilution technique
- Serial dilution technique
- Spread plate method
- Micromanipulator method
- Roll tube method
1. Streak Plate Method
The streak plate method is the most common and effective technique for isolating pure cultures. A small amount of mixed culture is streaked over the surface of a solid agar plate using a sterile wire loop.
The purpose of streaking is to spread out the microbial cells so that individual cells get separated. As streaking continues, the loop carries fewer cells, allowing isolated colonies to appear in later streak areas.
Well-separated colonies from the final streak are transferred to fresh plates for complete purification.
2. Pour Plate Method
In this method, the mixed inoculum is diluted directly in cooled molten agar (around 45°C). The inoculated agar is poured into sterile Petri plates, allowed to solidify and incubated.
a) Loop Dilution Technique
The culture is diluted repeatedly by transferring small drops of inoculum into several tubes of molten agar. Each tube is poured into plates to obtain isolated colonies.
b) Serial Dilution Technique
Here, the inoculum is diluted by adding it stepwise into tubes of sterile water or saline. A measured volume (usually 1 ml) from each dilution is mixed with molten agar and poured into plates. Colony counts multiplied by the dilution factor help estimate bacterial numbers.
Disadvantages of Pour Plate Method
- Microorganisms may become trapped inside the agar, forming subsurface colonies.
- It is more tedious and time-consuming than streaking.
- Bacteria are briefly exposed to heat (45°C), which may damage sensitive species.
- Not suitable for psychrophilic bacteria.
3. Spread Plate Method
In this method, the mixed sample is diluted in sterile water or saline. A small amount (0.1 ml) from each dilution is spread evenly onto the surface of a solid agar plate using a sterile glass spreader.
Advantages
- Simple and easy to perform
- Produces only surface colonies
- Useful for counting viable microorganisms
- No heat exposure to organisms
4. Micromanipulator Method
This method isolates a single microbial cell from a mixed culture. A micromanipulator connected to a microscope is used to pick up one cell using a micropipette. The single cell is transferred onto sterile medium to develop into a pure culture.
This method gives very high accuracy but requires skill and precision.
5. Roll Tube Method
The roll tube method is specially used for isolating strict anaerobic bacteria. A culture tube coated with pre-reduced agar and flushed with oxygen-free nitrogen is used.
During inoculation, the tube is rotated while the inoculating loop touches the agar. The process starts from the bottom and moves upward. After inoculation, the tube is sealed and incubated anaerobically.
Advantages
- Provides well-isolated colonies of anaerobes
- Prevents exposure to oxygen
Key Points
- Pure cultures are essential for identification, research and industrial use.
- Streak plate method is the most commonly used isolation technique.
- Pour plate and spread plate methods help in counting microorganisms.
- Micromanipulator and roll tube methods are used for special cases like single-cell isolation and strict anaerobes.
Detailed Notes:
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