1. Streak Plate Method
This technique is most typically used to isolate pure cultures of bacteria. A tiny quantity of mixed culture is spread over the surface of the agar medium using the tip of an inoculation loop/needle (Figure 1). The repeated streaks adequately “thin out” the inoculum and separate the microorganisms.
It is typically best to streak out a second plate using the same loop/needle without reinoculation. These plates are incubated to facilitate colony development. The basic idea of this approach is that streaking creates a dilution gradient across the face of the Petri plate when bacterial cells are deposited on the agar surface.
Confluent growth does not occur in the region of the medium where few bacterial cells are deposited due to this dilution gradient. Each colony is most likely the progeny of a single microbial cell, indicating a pure culture clone. To assure purity, such isolated colonies are picked up individually using a sterile inoculating loop/needle and re-streaked onto new medium.
2. Pour Plate Method
Plates containing diluted samples combined with melted agar media are used in this procedure. The basic idea is to dilute the inoculum in consecutive tubes containing liquefied agar media in order to ensure that the bacterial cells are evenly distributed throughout the medium.
The mixed culture of bacteria is diluted immediately in tubes containing melting agar medium that is kept liquid at 42-45°C (agar hardens below 42°C). The bacteria and the melted media are well combined.
Each tube’s contents are placed onto a separate Petri plate, allowed to harden, and then incubated. When bacterial colonies form, isolated colonies form both inside the agar media (subsurface colonies) and on the medium (surface colonies). To ensure purity, these isolated colonies are scooped up by the inoculation loop and smeared onto another Petri plate.
3. Spread Plate Method
In this method, unlike the pour plate method, the mixed culture or microorganisms are not diluted in the melted agar medium. Instead, they are diluted in a series of tubes with sterile liquid, usually water or physiological saline.
A drop of the diluted liquid from each tube is put in the middle of an agar plate, and a sterilised bent-glass rod is used to spread it out evenly over the surface. Now, the medium is being kept warm.
When the colonies grow on the agar medium plates, it turns out that some plates have colonies that are well separated from each other. This happens because the drop of diluted liquid spread out on the plate separates the microorganisms into their own groups.
To make sure the colonies are pure, they are picked up and put on new medium. In this method, unlike the pour plate method, only surface colonies form, and the microorganisms don’t have to be able to handle the heat of the melted agar medium.
4. Serial Dilution Method
As was already said, this method is often used to get pure cultures of microorganisms that can’t be grown on solid media and only grow in liquid media. A series of dilutions can be used to separate a pure form of a microorganism that is the main one in a mixed culture. The inoculum is diluted over and over again in a sterile liquid medium, and small amounts of each dilution are added to a large number of tubes of sterile liquid medium.
The goal of this dilution is to put a microbial suspension into a series of tubes that is so weak that only one microbe grows in some of the tubes. For simplicity, let’s say we have a culture with 10 ml of liquid medium and 1,000 microorganisms, which means there are 100 microorganisms per ml of liquid medium.
If we take 1 ml of this medium and mix it with 9 ml of fresh, sterile liquid medium, we would have 100 microorganisms in 10 ml, or 10 microorganisms/ml. If we added 1 ml of this suspension to 9 ml of fresh, sterile liquid medium, each ml would now have a single microorganism. If there is microbial growth in this tube, it is very likely that a single microorganism was added to the medium and that this growth is the pure culture of that microorganism.
5. Single Cell Isolation Methods
An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow.
i) Capillary pipette method: Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillars pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.
Figure 5: Capillary pipette method.
ii) Micromanipulator method: 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.
6. Enrichment Culture Method
Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture.
The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and, specific condition of incubation favours the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.