Since microorganisms are very small dimensions, in the study of individuals can obtain only limited information about their properties. Therefore, usually microbiologists study population consisting of millions and billions of individuals. Such a population or culture obtained by cultivating microorganisms in a more or less specific conditions. The culture containing microorganisms only one type, called the net, and a culture that contains more than one kind of microorganisms — mixed. If the culture grows only two kinds of microorganisms, and their special support in association with each other, so called two-culture.
Thus, the basis of microbiological methods comprise two procedures: selection, ie. E. The isolation of specific micro-organism found in nature mixed populations, and cultivation — growing microbial population in an artificial (culture) in the laboratory environment. What would view any work microbiologist, it will always use these two procedures. They are basically the same in the study of viruses, bacteria, fungi, algae, protozoa and even very small invertebrates. Moreover, in recent years, similar methods have been used when working with cell lines or tissues of higher plants and animals (tissue culture). In spite of all the biological diversity of organisms, which deals microbiology, its unity as a science is caused by the fact that it always used the basic methods.
One and the same strain can not be recovered a second time from the same source at a different time because any population genetic diversity of microorganisms is too large, as well as the genetic variability of the microorganisms. Therefore, the term «wild-type» refers to all of the other variations of the cells of this type or any other strain of this type, but not included in the collection of micro-organisms (ie not having the description and name).
Methods of obtaining pure CULTURES
Microorganisms are ubiquitous, so getting a pure culture is not limited to, to highlight this type of mixed natural microbial populations: it is necessary to maintain even a dedicated body and its offspring in such artificial conditions that would preclude contamination of the culture of other species. For the development of micro-organisms do not need a lot of space; therefore it is possible to create the necessary conditions in vitro, the flask or Petri dish. These three types of vessels are most commonly used for culturing microorganisms. The culture vessel must first be sterilized (m. E. It must be destroyed all life), and then, after it entered test organisms are protected from contamination from the outside. The main source of pollution — air, which always contain microbes. The lid covering the petri dish, is specifically designed to prevent microbes from entering the air. Test tubes and flasks with the same purpose plug caps. Typically, this is done using a plug of cotton, but is now often used, particularly for tubes, metal caps and plastic screw-cap.
The outer surface of the culture vessel, of course, also be a source of contamination; when the tube or flask open to add to it or take a sample of the material, can get into foreign organisms. To avoid this, immediately after the tube is removed, and again just before the closing of the neck of the container it is burned in a flame.
Typically, the inoculum (microbial material used for seeding or inoculation, the medium in the culture vessel) is made on a metal wire or loop, that before the use of fast sterilized in the flame. Liquid culture also transferred via pipette. For this the end of it, which is taken in the mouth with cotton wool plug, pipette wrapped in paper and placed in a glass or metal pencil case and sterilized. Paper and pencil case prevents contamination of both internal and external surface of the pipette until their use.
To further reduce the possibility of accidental contamination, can be passaged in culture desktop box or a small closed room, which receives a specially treated air with a reduced content of microorganisms.
Isolation of pure cultures by plating on plates
Pure cultures of microorganisms forming on solid media, individual colonies (t. E. The majority of bacteria, yeast, filamentous fungi and many single-celled algae), the easiest way to select, using a modification of the method of seeding in the cup. This method is based on the fact that individual organisms are immobilized on the surface or in the depth of the culture medium supplemented with agar or some other gelling agent. The growth of each of viable organisms leads to the formation of a separate colony from which it is easy to make reseeding to the new environment.
Typically, most often for this purpose use the crop on the cup touch. The sterilized curved wire is dipped in the diluted slurry adequately microorganisms and then subjected to a number of its non-contiguous parallel strokes across the surface already solidified agar layer in a cup. Each subsequent stroke is a gradual dilution of inoculum so that even if the first touches and give missteps growth, something along the following strokes isolated colonies grow. Isolation can be carried out also by the drilling depth. To serial dilutions of the inoculum is mixed with molten, but cooled agar medium and then the samples were poured into a sterile petri dish, where they are frozen, the resulting colonies formed in agar depth.
Special problems arise when allocating method seeding on plates of strictly anaerobic bacteria. If the contact with oxygen is not immediately lead to the death of the studied organisms, it is possible to sow in the cup as usual but then incubate them in a closed container, from which the absorption by chemical or incineration of combustible material removed oxygen. To select more oxygen sensitive anaerobes better, however, to use a modification of the method of deep planting, known as culture, divorced stirring. In this case, the test tube is inoculated and cooled with molten agar, the tube contents were stirred and approximately one-tenth of it is transferred to the next tube with agar. The contents of the tube mixed well and used to inoculate a third tube and so on. After 6-10 serial dilution tubes is rapidly cooled and sealed by applying to the surface layer of sterile petrolatum and paraffin, which prevents air penetration in the column of agar. In such cultures, colonies grow in agar, and as a result they are not as easy to control the transfer. To this was removed with a sterile needle and petrolatum wax layer and column agar gently blown from the tube into a sterile petri dish by passing the gas containing no oxygen through a capillary pipette lowered between the wall of the tube and agar. To be able to study and carry colony column sterile agar is cut with a knife on wheels.
When using a suitable technique, then the separation of bacteria from a natural population often mixed in the first series of plates or cultures diluted stirring, grow well separated from each other colonies. Is it possible to taking material from a colony and sowed them a suitable environment, assume that a net culture? Often they do it, but isolated cultures may be far from clear. Micro-organisms vary considerably in their needs, and, therefore, there is no single environment and a single set of conditions that would ensure the growth of all existing in the natural populations of the species. Probably only a very small part of them would be able to form colonies on each given one environment. Therefore, in the first cup at each visible colony might have to be a thousand cells of other microorganisms, which were also applied to the agar surface, but did not give a macroscopically visible growth, although they may still be viable. It is likely that some of them will also be captured when moving culture. For pure culture should never take a sample from the first cup. The cell suspension obtained from a single colony in the first cup should dissipate streaked into a new cup. If all colonies in the second cup will appear identical, then an isolated colony well can already be used to obtain a pure culture.
Isolation of pure culture anaerobic bacteria breeding method. It is shown that a whole lot of test tubes with dilutions. In more densely sown tubes (right) confluent growth is observed, and in the latter two tubes of the series of dilutions (from left) shows well separated from each other colonies. After agar stiffened each tube slipped mixture petrolatum and paraffin, in order to prevent penetration of atmospheric oxygen suppressing the growth of anaerobic bacteria. Wednesday red pH indicator, with growth comes acidification and the indicator changes its color to yellow.
Not all microorganisms capable of growing on solid media is required to form the individual colonies. Some «bacteria that move using flagella (eg, Proteus and Pseudotnonas), can quickly spread through a slightly damp surface svezherazlitoy medium bowl. This can be avoided by using a medium with well-dried surface on which cells can not move. Spirochetes and those organisms which are moved by sliding (myxobacteria, tsianobakterii1) can move along the surface of the agar gel or through it, even if the surface to dry. The ability of these organisms to movement helps to clean them, as they may themselves be separated from other species immobilized on agar. Thanks to this is often possible to clean up, allowing the cells to move around and making repeated subcultures from the front edge of the migrating population.
Sometimes the allocation of natural populations of organisms useful to include in the environment a substance that selectively inhibits the growth of certain bacteria. Especially effective certain antibiotics due to their biological specificity. Bacteria differ greatly in their sensitivity to penicillin; Therefore, low concentration can be popolzovavshis penicillin to inhibit growth of the bacteria present in the initial population. At higher concentrations of penicillin is usually toxic to prokarioticheskih2, but not for eukarioticheskih3 organisms. Therefore, it is very useful for the purification of cultures of protozoa, fungi and eukaryotic algae contaminated with bacteria. In contrast, prokaryotes are insensitive to polyene antibiotics (such as nystatin), which are usually toxic to eukaryotes. The addition of such antibiotics in the environment can sometimes be used advantageously for the purification of bacterial cultures, strongly contaminated by fungi or amoebae. To facilitate the release of micro-organisms can use, and many other substances with selective toxicity.