What are the different techniques of obtaining pure culture?
Posted January 30, 2024
Answer
The different techniques for obtaining pure culture include the streak plate method, the pour plate method, the spread plate method, serial dilution, single cell isolation methods, and enrichment culture method.
- The pour plate method is a microbiological procedure used to isolate and quantity viable microorganisms in a liquid sample. This involves adding the sample into molten agar before it solidifies, allowing growth within the agar medium. This method creates evenly dispersed colonies within the solid medium when a suitable dilution of the sample is plated.
- The streak plate method involves streaking a mixed microbial sample in a specific pattern (e.g. quadrant streaking) onto an agar plate. As the streaked plate is incubated, individual cells are spread out and form isolated colonies to be quantified.
- The spread plate method involves spreading a small volume of diluted microbial sample over the surface of an agar plate using a sterile spreading bent-glass-rod. For all 3 of these methods, after obtaining isolated colonies on an agar plate, an inoculation loop is used to pick up individual colonies. These colonies are then streaked onto a new petri plate for purification.
- Serial dilutions involve diluting a sample in a series of consecutive dilutions, each with decreasing microbial concentration. A small volume of each dilution is then plated using spread or pour plate methods. The dilution that results in well-isolated colonies is selected for obtaining a pure culture.
- Enrichment culture is used to isolate microorganisms that exist in low numbers or have slower growth rates compared to other species in a mixed culture. This method involves creating a suitable environment by adjusting the nutrients and altering the incubation conditions in the culture medium. By adjusting the medium's composition and incubation features, it encourages the growth of the targeted microorganisms while inhibiting the growth of unwanted species.
- Single isolation methods consist of the capillary pipette method and the micromanipulator method.
- The capillary pipette method involves placing multiple small drops of a properly diluted culture onto a sterile glass coverslip using a pipette with a fine capillary tip. Each drop is examined under a microscope until a drop containing just one microorganism is identified. Once this drop is found, it is carefully extracted using a sterile capillary pipette and transferred to fresh growth medium. This isolated microorganism within the drop then begins to proliferate, leading to the development of a pure culture made up solely of that particular microorganism.
- Micromanipulators are tools used alongside microscopes to select individual cells from mixed cultures. By making micrometer adjustments, the micropipette attached to the micromanipulator can be precisely moved in various directions. In the process, a sequence of hanging drops, created from a diluted culture using a micropipette, is arranged on a specialized sterile coverslip. The goal is to locate a hanging drop containing only a single cell of the microorganism. Once identified, this single cell is drawn into the micropipette using gentle suction and then transferred to a larger drop of sterile growth medium on another sterile coverslip. As this cell begins to multiply and the number of cells in the drop increases, the contents of the drop are moved to a culture tube containing appropriate growth medium. This process ultimately results in the development of a pure culture consisting only of the desired microorganism.
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