Cholesterol requirement of mycoplasmas

Cholesterol requirement for growth of mycoplasmas was tested in a serum-free medium supplemented with albumin, l-arginine, palmitic acid, and various concentrations of cholesterol dissolved in Tween 80. In cases in which Tween 80 was shown to inhibit growth, the test medium was supplemented with cholesterol dissolved in ethanol. Of the 31 species examined, all but Mycoplasma laidlawii, M. granularum, and Mycoplasma species strain S-743 exhibited a growth response to cholesterol. No requirement for cholesterol could be shown with the stable L-phase variants of Streptobacillus moniliformis and Proteus species. The results provide experimental support for the view that the large majority of the established Mycoplasma species require cholesterol for growth.     

Formaldehyde fixation of cells does not greatly reduce the ability to amplify cellular DNA

Formaldehyde fixation of cells is routinely used to study DNA-protein interactions in vivo. In these studies, DNA is often analyzed using a polymerase chain reaction technique. Although it is known that formaldehyde can damage DNA, no studies have been performed so far to compare the efficiency of DNA amplification between normal and fixed cells. Here we show that formaldehyde fixation results in a 15% to 20% reduction in the ability to amplify cellular DNA. The loss of amplifiability is independent of the length of the amplification region and the degree to which DNA is compacted on packaging into chromatin.     

Cholesterol uptake is dependent on membrane fluidity in mycoplasmas

The transfer of elaidate-enriched Acholeplasma laidlawii cells in culture from 37°C to 4°C virtually arrested exogenous cholesterol incorporation into the cell membrane. Cholesterol uptake continued, though at a slower rate, in oleate-enriched A. laidlawii cells undergoing similar temperature shift-down. It is concluded that the incorporation of exogenous cholesterol into the cell membrane of living mycoplasmas is rapid when the membrane lipid bilayer is in the liquid-crystalline state and very slow when the lipid bilayer is in the gel state.