In Microbiology laboratory, Culture methods are very crucial as it increases the chances of multiplication and identification of microorganism.
The following are various reasons for culturing microorganisms in the laboratory:
- Isolate bacteria in pure culture and identify the same by performing various tests.
- In order to maintain stock culture.
- To demonstrate biochemical, antigenic, and other phenotypic and genomic properties of the isolated colonies.
- Demonstrate susceptibility of the isolated bacteria to antibiotics, bacteriophages, bacteriocins, etc.
- Prepare antigens for various uses.
What Are Some Common Culture methods
Some common methods are used for culturing of bacteria. Some of these method includes
This is the most useful method for obtaining discrete colonies of the bacteria. It is done by streaking on the surface of a solid media plate using a platinum or nichrome loop of 2–4 mm diameter.
Here, a loopful of the inoculum is placed near the peripheral area of the plate. The inoculum is then spread with the loop to about one-fourth of the plate with close parallel strokes. From the primary inoculum, it is spread thinly over the plate by streaking with the loop in parallel lines. The loop is flamed and cooled in between the streaks to obtain isolated colonies.
The inoculated culture plate is incubated at a specific temperature overnight for demonstration of colonies.
Confluent growth occurs at the primary inoculum, but becomes progressively thinner, and well-separated colonies are demonstrated on the final streaks of the inoculum. Single isolated colonies obtained by this method are very useful to study various properties of bacteria. Streak culture is the most useful method for obtaining discrete colonies of the bacteria.
This method is useful to
- Carry out antibiotic susceptibility testing by disc diffusion method
- carry out bacteriophage typing
- produce a large amount of bacterial growth required for preparation of bacterial antigens and vaccines
The lawn culture provides a uniform layer of bacterial growth on a solid medium. It is carried out by flooding the surface of the solid media plate with a liquid culture or suspension of bacteria, pipetting off the excess inoculum, and finally incubating the plate overnight at a specific temperature.
Alternatively, the culture plate may be inoculated by a sterile swab soaked in liquid bacterial culture or suspension and incubating overnight for demonstration of the bacterial colonies.
The pour-plate culture is used to determine approximate number of viable organisms in liquids, such as water or urine. This method is used quantify bacteria in urine cultures and also to estimate the viable bacterial count in a suspension.
It is carried out in tubes, each containing 15 mL of molten agar.
- The molten agar in tubes is left to cool at 45° C in a water bath.
- The inoculum to be tested is then diluted in serial dilution. Then 1 mL each of diluted inoculum is added to each tube of molten agar and mixed well.
- The contents of tubes are poured into sterile Petri dishes and allowed to set. After overnight incubation of these Petri dishes at required temperature (mostly 37 °C).
- Colonies are found to be distributed throughout the depth of the medium, which can be counted using a colony counter
This method provides a pure growth of bacteria for carrying out slide agglutination and other diagnostic tests. It is carried out in tubes usually containing nutrient agar slopes.
This is prepared by stabbing the medium in tubes with a long, straight wire followed by incubating at 37°C.
It is frequently used for
- Maintaining stock cultures.
- Demonstration of gelatin liquefaction of bacteria.
- Demonstration of oxygen requirement of bacteria.
It is prepared in a liquid media enclosed in tubes, flasks, or bottles. The medium is inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes and incubating at 37°C, followed by subculture on to solid media for final identification.
A major disadvantage of using liquid culture is that it does not provide pure culture of the bacteria and also the bacterial growth does not exhibit special characteristic appearances but it is specifically used
- For blood culture and for sterility tests, where the concentration of bacteria is expected to be small. You can buy blood agar from Hardy Diagnostics at a very good prices.
- For culture of specimens containing antibiotics and other antibacterial substances, as these are rendered ineffective by dilutions in the medium
- When large yields of bacteria are required.
Source: Microbiology and Immunology Textbook of 2nd Edition (Subhash Chandra Parija)
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