A pure culture theoretically contains a single bacterial species. There are a number of procedures available for the isolation of pure cultures from mixed populations.
A pure culture may be isolated by the use of special media with specific chemical or physical agents that allow the enrichment or selection of one organism over another.
Pure culture, in microbiology, a laboratory culture containing a single species of organism. A pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample manyfold before inoculating the new medium. Both methods separate the individual cells so that, when they multiply, each will form a discrete colony, which may then be used to inoculate more medium, with the assurance that only one type of organism will be present. Isolation of a pure culture may be enhanced by providing a mixed inoculum with a medium favouring the growth of one organism to the exclusion of others.
A major contribution to bacterial techniques was the development of methods using solid medium. For example LB medium or minimal medium for the cultivation of bacteria. Koch was convinced that microbes caused some diseases. However, to test this idea, he needed to isolate the causative agent. Almost all samples from diseased animals or any natural surface contained many different microbes and it was impossible to tell which one was the problem. A method was needed to separate these different bacteria. The most common method of isolation was to continually dilute a sample in liquid broth in hopes that at high enough dilution, only one type of microbe would be found. A problem with this method is that only the most populous microbe would be isolated, but that might not be one causing the disease. There were other technical problems as well with such a liquid-based system, so a solid medium would seem to provide distinct advantages. Koch had tried gelatin for these experiments with unsatisfactory results.
The differential and selective procedures will be utilized later in this course. Simpler methods for isolation of a pure culture include:
(i) spread plating on solid agar medium with a glass spreader and
(ii) streak plating with a loop.
The purpose of spread plating and streak plating is to isolate individual bacterial cells (colony-forming units) on a nutrient medium.
Asepsis can be defined as the absence of infectious microorganisms.
- Serial dilution
A serial dilution is the step-wise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M… Serial dilutions are used to accurately create highly-diluted solutions as well. A culture of microbes can be diluted in the same fashion. For a ten-fold dilution on a 1 mL scale, vials are filled with 900 microliters of water or media, and 100 microliters of the stock microbial solution are serially transferred, with thorough mixing after every dilution step. The dilution of microbes is very important to get to microbes diluted enough to count on a spread plate
Streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.The streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. This is dipped in an inoculum such as a broth or patient specimen containing many species of bacteria.The sample is spread across one quadrant of a petri dish containing a growth medium, usually an agar plate which has been sterilized in an autoclave. Choice of which growth medium is used depends on which microorganism is being cultured, or selected for. Growth media are usually forms of agar, a gelatinous substance derived from seaweed.
- Spread plate
Spread plates are simply microbes spread on a media plate. Microbes are in a solution, and can be diluted. They are then transferred to a petri dish with media specific for the growth of the microbe of interest. The solution is then spread uniformly through a number of possible means, the most popular is the use of sterile glass beads that are shook on top of the media, spreading the microbe-containing liquid evenly on the plate. Also common is the use of a bent-glass rod, often referred to as a hockey stick, due to its similar shape. The glass rod is sterilized and used to spread the microbe-containing liquid uniformly on the plate.
The purpose of this procedure is to distribute cells evenly so that well isolated individual colonies can be grown. These are then counted or used for further tests such as serial dilutions:
- Inoculate clinical specimen using a sterile spreader or alternative onto agar media. Gently spread bacteria over the entire culture medium surface backward and forward while rotating the plate. Avoid the spreader touching the edges of the agar plate
- Replace the lid and allow the plate to stand in an upright position (with the lid at the top) to dry for 10 to 20 minutes
- Incubate the spread agar plate at the appropriate temperature in an inverted position (with the lid at the bottom
Agar stab technique
The method is used to prepare stab cultures (to observe motility, or when inoculating certain types of solid medium) and to pick single colonies from a plate:
- Using aseptic technique pick a single well-isolated colony with a sterile inoculating stab needle and stab the needle several times through the agar to the bottom of the vial or tube
- Replace the cap and tighten loosely when incubating to allow gas exchange
- Incubate the stabbed agar plate/slant at the appropriate temperature