Posted 10/22/2014 2:05 PM (GMT 0)
Here is something that I found in a Mycoplasma book:
Mycoplasmas, or more correctly mollicutes, the bacterial class which incorporates all the degenerate, wall-less bacteria, including mycoplasmas, acholeplasmas, ureaplasmas, spiroplasmas, entomoplasmas, mesoplasmas, phytoplasmas and the recently reclassified eperythrozoans and haemobartonellas, are characterized by their small genome size (0.58– 1.38 Mbp), a low G+C content (23– 40 mol%) of the genome and a permanent lack of a cell wall.
Over 200 species have so far been described. According to the recent taxonomy of prokaryotes, the mollicutes belong to the phylum Firmicutes, which contains the Gram-positive bacteria and also comprises the Bacilli and Clostridia, from which the mollicutes have derived by a process of degenerative evolution.
The difficulty of culturing mycoplasmas in vitro is a major obstacle to research and laboratory diagnosis of these fastidious organisms, and it is highly likely that many more mycoplasmas exist in nature but have not yet been isolated, despite great efforts over many years (Razin et al., 1998) including the introduction of PCR; in addition, many isolated mycoplasmas still grow very poorly even on the best mycoplasma medium (Razin, 1994).
The limited capability of mycoplasmas to synthesize macromolecules essential for growth reflects their evolutionary development, which has resulted in the small size of the mycoplasma genome. To overcome these deficiencies, complex media are used for their cultivation. The medium is usually based on beef heart infusion, peptone, yeast extract and serum with various supplements (Razin, 1991). Mycoplasmas are completely dependent on the host for exogenous fatty acids and require amino acids, nucleic acid precursors, lipid precursor molecules and vitamins. The medium must contain sterol such as cholesterol, which may be replaced by other sterols such as cholestanol or ergosteroll (Rodwell and Mitchell, 1979).
Glycerol oxidation is very important for the synthesis of glycophospholipid and glycerides, which is consequently important for lipid synthesis. Glucose is the main source of energy in fermentative mycoplasmas, as well as a source of carbon for the synthesis of other sugars and polysaccharides. The fermentative mycoplasmas can also use maltose, trehalose, starch and glycogen (Razin and Freundt, 1984); pyruvate can replace glucose in non-fermentative mycoplasmas such as Mycoplasma bovis and M. agalactiae for energy production (Miles et al., 1988). Peptones provide the media with different polypeptides, di-peptides and amino acids (Miles, 1992).
A novel medium, called TSB-1, that is devoid of ruminant peptone and which may improve isolation of animal mycoplasmas from tissues and increase growth yields for antigen and vaccine production, has been reported (Khan et al., 2005; Patel et al., 2008); the use of vegetable peptones also reduces the risk of contamination of vaccines with agents causing the transmissible spongiform encephalopathies. Different types of animal sera (calf, horse, porcine) are used at 5– 20% as a source of essential lipids. Other nutrients are provided by the sera, including sugars, urea and inorganic ions. There is a considerable difference in the nutritional properties of different animal sera, which depends on their lipid concentration. Animal sera are usually inactivated by heating in a water bath at 56°C for 30 min to reduce the complement component of the serum, which can cause cell lysis. Continual efforts have been made to replace the serum component with albumin, fatty acids and cholesterol supplemented with serum albumin to neutralize free fatty acid toxicity (Razin, 1978), but few of these efforts have been successful.
Beef heart infusion and yeast extract provide a variety of nutrients, including nucleotides, vitamins and mineral salts. Fresh yeast extracts are superior to commercial dehydrated extracts because they contain labile components which are destroyed during commercial processing. The addition of organic components including DNA and NADH (a coenzyme present in animal tissue and yeast extracts) may enhance the growth of different types of mycoplasmas by lowering the oxidation– reduction potential of the media and making them more suitable for the growth of anaerobic or microaerophilic organisms (Miles, 1992). Energy sources are provided through the inclusion of glucose, pyruvate, arginine or urea.
Mycoplasmas lack a cell wall and therefore they are more susceptible to cell lysis in hypo-osmotic media than other cell-walled bacteria, so they need sodium chloride to increase medium tonicity. They also require an osmotic pressure of 7– 14 atmospheres for optimal growth. For most mycoplasmas growth is best at pH 7– 8 (Rodwell and Mitchell, 1979) and typical media have a pH of 7.6. The growth of mycoplasmas is sensitive to any change in pH; a decrease in pH to less than 6.5 due to sugar fermentation causes a limit to growth and consequently leads to cell death; an increase in pH above 8.0 may also lead to cell death.
Mycoplasma media should be well buffered because of the narrow range of pH values for mycoplasma growth. The buffers mostly used are phosphate buffer and N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Miles, 1992). Oxygen plays an important role in mycoplasma growth. The need for oxygen depends on the strain; some mycoplasmas prefer to grow in anaerobic conditions while others prefer microaerophilic conditions. Gentle aeration may increase growth rates, which may be due to the fact that this increases the rate of oxidation and thus the production of ATP during the metabolism of glucose or other carbohydrate (Miles and Agbanyim, 1998), while excessive aeration may reduce the culture viability. Although the numerous nutritional requirements of mollicutes dictate the need for complex growth media, the notion that the richer the medium the better may be wrong. It appears that the lack of growth of mycoplasmas in a rich medium is, in some cases, not the result of the lack of specific nutrients but rather is due to the presence of a component toxic to mycoplasma.
The growth inhibitors found in the complex media are mostly components of the peptone and yeast extract. While DNA amplification techniques are being used with ever-increasing frequency for the detection and identification of mycoplasmas, the isolation of a mycoplasma by conventional techniques is still required by most national and international authorities, particularly where diseases of great importance are concerned, such as contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP). Furthermore, live mycoplasmas are required in antibiotic sensitivity tests, molecular typing, vaccines and for use as antigens in diagnostic procedures. Consequently, the use of a high-yielding, and ideally selective, medium is still essential.