Methods of seeding on plates usually give satisfactory results in separation of bacteria and fungi, since the overwhelming majority of these organisms grow well in solid media. However, there can not be successfully cultivated on solid media, some bacterial species with larger cells; Many protozoa and algae can also grow only in a liquid medium. Although in recent years for the virus isolation techniques used seeding cup, many viruses are much easier to distinguish from the liquid medium. Of course, there never get pure culture as viruses — obligate intracellular parasites. In this case, the objective is to obtain a two-culture consisting of a certain virus and its biological host.
The easiest way to obtain pure cultures in liquid media — is a method of dilution. The inoculum was serially diluted with sterile medium and aliquots of each dilution are inoculated with a large number of tubes with a medium. The purpose of this operation — a series of tubes inoculated is so diluted suspension of bacteria that make the likelihood in a test tube, even one individual is sufficiently low (about 0.05). If so diluted inoculum is sown a lot of tubes, then the theory of probability can be calculated that 95% of all planted tubes will not get any cell. In one animal enters the 4.8% test tubes, two — 0.12% in test tubes, and 0.002% of the inoculated test tubes would be three specimens. Therefore, if the tube 6 is then grown, it is likely that growth caused penetrating into only one organism. The probability of this is 0.048 / (0.048 + 0.0012 + 0.00002) = 0.975.
The probability that the increase is due to hit in a single tube body very rapidly with increase in the average number of organisms (cells) in the inoculum. Therefore, it is important that the selection was made out of that series of tubes where the vast majority of cases, no growth is detected.
Dilution method, however, has one major drawback. It can be used to select only those organisms which predominate in a mixed population of microbes. They can not successfully use the allocation for larger organisms, unable to grow on solid medium (eg, protozoa or algae), as in natural populations of bacteria are usually much superior to them in numbers. This limits the applicability of the method of dilution.
If you can not use the dilution method or the method of sowing on cups, there remains only one thing — to resort to controlled release under the microscope of a mixed population of a single cell or organism. This procedure is called an isolated single cell. Technical difficulty it is inversely proportional to the size to the body. This method is relatively simple, if a microorganism has large cells, for example, when working with algae or protozoa, but use it to isolate the bacteria much more difficult.
In the case of larger organisms need to capture a single individual in a thin capillary pipette, and then rinse it several times in a relatively large volumes of sterile medium to remove the impurity of small microbes. These operations are performed manually by controlling them visually at a relatively low magnification, e.g., using a dissecting microscope.
If secreted by the body is so small that it is difficult to see with an increase of less than 100, then the capillary can not be, because at high magnifications can not be sufficiently finely manipulated manually. In this case, use a special mechanical device — micromanipulator and specially made with very thin glass instruments. Purpose micromanipulator is to reduce the amplitude of the movements made by hand; This allows a small workspace (microdrop) under constant observation through a microscope (h500-1000) to carry out very small and precisely controlled movement of instruments.
Heavyset selection — to get a pure culture. Sometimes, however, this is not possible, or so difficult that almost does not make sense. In such cases the alternative is to achieve a lesser degree of cleaning and to receive the two-culture comprising only two kinds of microorganisms. As already noted, the use of two-component cultures — the only possible method of maintaining the virus since it obligate parasites cellular organisms. Obligate intracellular parasitism is also typical for some groups of microorganisms having cell organization. In all these cases, the two-culture — the best attainable version of cultivation under controlled laboratory conditions.
Many simply that under natural conditions feed on smaller organisms, too, the easiest way to maintain in the laboratory in the form of a two cultures together with their smaller victim. This is the case, for example, ciliates, amoebas and slime molds. Such associations, «apparently never obligate, as a careful study of the power of some of these groups have shown that they can be grown in pure cultures. However, the nutritional needs of the simplest are often extremely complex and, therefore, the preparation of media to keep them pure cultures — a difficult and laborious. Bicomponent culture quite suitable for maintaining the microorganisms, and for many experimental purposes.
Two-culture was prepared in two stages. You must first obtain a pure culture of the organism supply (in the case of the host obligate intracellular parasites or victims in the case of protozoa). Then, by using any of the numerous methods (plating on solid media plates in the presence of the supply body, producing dilution in liquid medium or isolating individual cells) or isolate the parasite and predator introduce it into pure culture of the supply body.
To successfully maintain a binary culture requires considerable skill, as it is important that remained more or less stable biological balance between the two components. The medium employed should ensure the growth of the supply of the body, sufficient to meet the needs of the parasite or predator, but at the same time should not be too rich, or the development of the supply of the body can suppress the growth of the body associated with it, or lead to the formation of harmful products of metabolism for him.