In preparing synthetic medium microbiologists are often faced with the fact that the sterilization process precipitate. This is especially true if the environment contains relatively high concentrations of phosphate. Precipitates due to the fact that complexes form insoluble phosphates with some cations, particularly calcium and iron. Usually it does not affect the nutritional value of the medium, but the precipitate may impede observation of the development of microbes or quantify their growth. Precipitate formation can be avoided by separately sterilized concentrated solutions of calcium and iron, and then add them to the already sterilized and cooled environment. This difficulty can also be avoided by adding a small amount of medium substance, which forms with these metals soluble complex (chelate) and thereby prevents the formation of an insoluble complex with phosphate. Most often, this is done using ethylenediaminetetraacetic acid (EDTA) at a concentration of about 0.01%.
Maintaining aerobic conditions
For the life of obligate aerobes require oxygen. Aerobic microorganisms grow well on the surface of agar plates or in a thin layer of the liquid medium. In liquid cultures unstirred growth normally occurs only at the surface and in depth, anaerobic conditions are created, and the growth is not possible there. Therefore, to obtain large populations in liquid culture medium must be aerated. With this purpose in the laboratory using various devices for shaking which aerated medium, continuously mixing it. Applied also another method of aeration, which is that in the culture of all time, passed a sterile air stream. To increase the contact surface between the gas and the liquid, air can be supplied through a porous «spray», from which it leaves very small bubbles.
Methods of cultivation obligate anaerobes
Many strictly anaerobic microorganisms are very sensitive to molecular oxygen and rapidly die in contact with it. Therefore, contact with air such crops should be minimized or even eliminated. Furthermore, the growth of some strict anaerobes can start only in environments with low redox potential (about 150 mV or even lower). Therefore, when growing many anaerobes must first be added to the culture medium of a reducing agent such as cysteine, thioglycolic acid, sodium ascorbate or Na2S. Prepare such an environment should be at its storage and use to exclude access of air. During the growth of this culture can be fed into the vessel a gas containing no oxygen: C02 or N2.
Culture strict anaerobes in liquid media are usually grown in test tubes or flasks, brim-filled environment and closed with a rubber stopper or screw the plastic cover. When using solid media cultures can avoid contact with the air in several ways. Organisms that can tolerate short-term contact with the air, can be isolated by streaking on plates, which are then placed in a hermetically closed anaerobic desiccator. Then oxygen is removed, to which the air is evacuated and filled desiccator some inert gas (e.g., N2) or is placed into a substance absorbing O2. Sometimes combine both of these methods of oxygen removal. To select more oxygen sensitive anaerobes better to use the method of agar dilution and immediately after sowing seal the tubes. In a variant of this method uses a rotating tube, in which the molten agar is distributed along the walls of a thin layer. The tube is filled with a gas mixture containing no oxygen, and then sealed with a rubber stopper.
Supply crops with carbon dioxide
When cultured photoautotrophs and chemoautotrophs is often necessary to ensure a sufficient amount of carbon dioxide cells. Culture can grow through C02 coming into the culture medium from the air by diffusion, but the concentration of CO2 in the atmosphere is extremely low (0.03% in the open air, and only slightly higher in enclosed spaces), so for autotrophic carbon dioxide is often the factor limiting growth . To create the best conditions, a culture saturated air artificially enriched with CO2 (to a concentration of 1-5%). As discussed above reasons, it complicates the task of monitoring pH, and therefore have to modify the buffer system environment. When cultured autotrophs capable of growing under anaerobic conditions in closed containers (e.g., magenta and green sulfur bacteria), the requirement can be satisfied at C02, by adding the medium NaNSOz. Soluble carbonates can not be used in environments that are in contact with the air because the rapid exit of CO2 in the atmosphere leads to a strong alkalized medium.
For the cultivation of phototrophic microorganisms (algae, photosynthetic bacteria) need light. Provide adequate lighting at the same time, and the desired temperature — the task is difficult. When cultured non-photosynthetic organisms to maintain the proper temperature thermostat is used. However, most commercially available thermostats are not equipped with internal lighting system, and can not be used in the cultivation of phototrophic organisms.
Exposing a culture of daylight, you can get pretty bad and non-controlled lighting. This should avoid direct sunlight, since the intensity may be excessive and culture can be so very hot that its growth will be impossible. Many phototrophic microorganisms tolerate continuous lighting; in these conditions, they grow much faster, so better to use artificial lighting. When this value is large spectral composition of light. In some ways more convenient fluorescent lamps, since they give relatively little heat, which facilitates the maintenance of the desired temperature. However, in the long wavelength region of the visible spectrum and the near infrared radiation of such lamps is weaker compared to sunlight. These lamps may be used in the cultivation of algae and cyanobacteria, photosynthesis occurs at wavelengths less than 700 nm, but they do not allow or provide very little light at wavelengths from 750 nm to 1000 nm, which is necessary for photosynthesis purple and green bacteria. For these groups of photosynthetic bacteria as a source of artificial light only acceptable lamp filament, so that if needed the high intensity lighting, there arise problems with heat removal. The easiest way in this case be placed in the culture vessels illuminated side glass or plastic water bath, the temperature of which may be stabilized, creating water circulation. You can also put together a culture of incandescent bulbs in a box and keep it at the proper temperature by ventilation or cooling device.