Technique accumulation of Cultures — one of the most powerful methods available to the microbiologist. To highlight certain microorganisms from natural sources can be used almost infinite number of combinations of various external factors, both chemical and physical. The methods allow the desired enrichment to allocate certain types of microbes, using their specific needs. These methods can be infinitely varied in order to isolate and study not previously described organisms capable of growing in the conditions created by the researcher. Here, we briefly describe a number of methods of preparation, which can be used to isolate natural sources of the basic physiological groups of microorganisms, mainly bacteria.

Cumulative environment hemogeterotrofov

Terms and nutrients necessary length of accumulation in various synthetic medium hemogeterotrofov shown in the tab below and in the table below shows in detail the composition of each of these environments.

For the accumulation of organisms performing fermentation important chemical nature of the organic substrate. To the substrate could ferment, it should not be too oxidized nor too recovered. Lovely substrates for fermentation are sugar, but can be fermented and many other organic compounds roughly the same level of oxidation. Incubation enrichment cultures should be performed under anaerobic conditions — not only because some organisms derive energy by fermentation, are obligate anaerobes, but also to prevent competition from aerobic forms. As a source of nitrate nitrogen can not be used, since in this case be able to grow and denitrifying bacteria. If accumulating organisms form acid, and themselves are sensitive to it, it can be added to the medium calcium carbonate.

The synthetic media under anaerobic conditions it is also possible to carry out the accumulation of three distinct physiological groups of bacteria hemogeterotrofnyh for which terminal oxidants in the respiratory metabolism instead serve 02 other inorganic compounds. In these cases, should not use such readily fermentable organic substrates like sugar. For denitrifying bacteria as terminal oxidant nitrate added to the medium, and as a carbon source and anergy — acetic acid, butyric acid or alcohol. Sulfate-reducing bacteria are added to relatively high concentrations of sulfate, because for them it is a terminal-specific oxidizing agent; best sources of carbon and energy is lactic or malic acid, but acetic. For reducing carbonate (methane-) bacteria as oxidizer need C02, and the carbon and energy source can be, for example, formic kislota7. It should also be noted that for the accumulation of bacteria reducing sulphates and carbonates, preferably used as the nitrogen source, ammonia, since the addition of nitrate would favor the development of microorganisms, reducing nitrates. For accumulation of bacteria reducing carbonates and nitrates, should be reduced to a minimum sulphate concentration in the medium, to prevent enhanced growth of sulfate-reducing bacteria.

Obviously, for the accumulation of microorganisms, receiving energy from aerobic respiration, aerobic conditions are needed. Organic nutrient in this case can be both fermentable and non-fermentable substrates. However, if a fermentable substrate, the need abundant and constant aeration culture, as the depletion of oxygen in the environment as a result of the accumulation of anaerobic respiration favors. Easily fermentable compounds are generally stimulate the growth of facultative anaerobes. Therefore, for inclusion into the collection medium glucose or any other sugar, both aerobic and anaerobic conditions grow mainly Enterobacter species.

Using a non-fermentable substrates usually leads to the accumulation of obligate aerobic, most members of the genus Pseudomonas. For example, including as the sole organic agent sodium benzoate (1 g / l) may be prepared oxidizing benzoate strains Pseudomonas putida. If the only source, both carbon and nitrogen will asparagine (2 g / l), the culture will be dominated by oxidizing asparagine or strains of related species him.

In synthetic media for storing aerobes as nitrogen source typically use an ammonium salt. If you do not add nitrogen compounds, we can distinguish the culture of nitrogen-fixing bacteria of the genus of aerobic Azotobacter. Azotobacter Types can be used as the sole organic nutrient great many different substrates, including alcohol, butyric and benzoic acid, and glucose.

Cumulative protection chemoautotrophic and photosynthetic organisms

To accumulate chemoautotrophic and photoautotrophic organisms from the environment to exclude organic compounds, as well as the sole carbon source to use C02 or bicarbonate.

Photosynthetic shapes need light, and culture chemoautotrophs need to cultivate in the dark to prevent the development of photosynthetic microorganisms. During the incubation, cultures must maintain either aerobic or anaerobic conditions depending on whether these organisms require oxygen. Exceptions to this rule are the algae as they in themselves produce oxygen metabolism, in fact all the same whether we incubate the culture algae accumulation in aerobic or anaerobic conditions.

Medium intended for enrichment cultures and the cultivation of photosynthetic organisms should contain sodium, as it is known that this element is necessary to photosynthetic bacteria.

The environment for the accumulation of non-sulfur purple bacteria should contain a suitable organic substrate, and sometimes bicarbonate. This substrate should not be easy to ferment; typically use acetic, butyric, or malic acid. If the carbon substrate (as, for example, oleic acid) is recovered to a greater degree than the material of the cell, it is necessary to add bicarbonate to the environment have been accompanied by the consumption of C02 photosynthesis. When using substrates such as malic acid, which is formed by the metabolism of the C02, the addition of bicarbonate is not required. Since fotogeterotrofnye bacteria require different growth factors, it is usually in the environment for their accumulation of a small amount of yeast extract.

Using sophisticated media

Some enrichment cultures of bacteria having highly complex nutrient requirements, to obtain a defined medium is not possible. Nevertheless, sometimes such organisms can be isolated from natural sources, using specially designed complex medium. Examples include lactic acid bacteria. They are characterized by high resistance to lactic acid, which they themselves form the fermentation of sugar. For accumulation of lactic acid bacteria use a weakly buffered medium containing glucose and some rich source of growth factors (for example, 20 g glucose and 10 g yeast extract to 1 liter of medium). After inoculation, which is desirable to produce a natural material containing a lot of lactic acid bacteria (for example, pieces of vegetables, raw milk, waste water), incubated under anaerobic conditions. Usually first developing bacteria such as Enterobacter and Escherichia. However gradually accumulates in the medium of lactic acid, and the conditions are becoming less and less beneficial to the bacteria, while the lactic acid bacteria continue to grow. Finally, the environment becomes acidic so that the lactic acid bacteria predominate, and most other organisms die.

Another example of a complex environment, quite selective for a particular group of organisms — an environment designed for the accumulation of bacteria propionovokis-mated. These bacteria during fermentation form propionic acid, acetic acid and CO2. Although they are easy to ferment glucose, they can not compete in glucose-containing medium with Enterobacter audio, audio with lactic acid bacteria have been growing rather slowly and do not tolerate acidic conditions. However, propionic acid bacteria can also be fermented to lactic acid, which is not a suitable substrate for most other organisms, carrying out fermentation. This ability and use for their savings. To this end, the neutral medium containing 20 g of sodium lactate and 10 g of yeast extract in 1 liter was inoculated with some natural material containing propionic bacteria, and incubated at 30 ° C under anaerobic conditions. The best inoculum for such crops is Swiss cheese, since it is mainly due to the maturation of the activity of propionic acid bacteria.

Complex environment can be successfully used for the selective cultivation of acetic acid bacteria. These bacteria are well adapted to the environment with high concentrations of alcohol. In addition, other bacteria, they are less sensitive to the acetic acid which are formed from the alcohol in the breath. For their accumulation of a complex medium containing alcohol, inoculated with the appropriate material and incubated under aerobic conditions. Good sources of inoculum may be fruits, flowers and unpasteurized (taken directly from the barrel) beer. It is possible to use a medium containing 40 ml of alcohol and 10 g of yeast extract per 1 L, pH, adjusted to 6.0. Beer and hard cider are also excellent memory mediums, since these drinks are similar to the natural environment in which to grow well acetic acid bacteria. Culture should have a large surface of contact with air, but the inoculated medium is generally not heavily aerated as many acetic acid bacteria grow best in the film they form on the surface.

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