A Method for the Rapid Enumeration of Mycoplasma Species growing in Broth Culture

Abstract

Summary: A method is outlined whereby Mycoplasma species growing in broth culture can be rapidly counted, using the relationship between the number of organisms and the mean lactate dehydrogenase (LDH) content of each organism. The uses of this technique are discussed.

Studies of Lactate Dehydrogenase of Mycoplasma mycoides var. mycoides

“…Quantitative yields of LDH were about 5 x I O -~ i.u./cell for the T, vaccine strain, a value higher than that ( 4 1 x 1 0 -l~ i.u./cell) obtained by Windsor & Boarer (1972). However, their work was confined to cultures up to 72 h. We found that LDH increased up to 96 h, although cell numbers were almost constant after 72 h. After 96 h, rapid decreases in both cell counts and LDH yields occurred.…”

Simple staining procedure permits rapid counting of mycoplasma colonies

“…There are numerous reports of attempts to facilitate quantitation of broth-grown or agar-grown organisms (1,2,4,11,12,16,17). For example, Low and Eaton (10) used a dilution of the Dienes stain as an aid in determining CFU.…”

Quantitation of bacillus subtilis L-form growth parameters in batch culture.

“…Calf thymus DNA (Sigma Chemical Co.) was used as standard. Lactate dehydrogenase (LDH; EC 1.1.1.27) activity was measured by the method of Windsor and Boarer (7) on samples of culture that were subjected to four cycles of freeze-thawing to lyse the L-forms. One unit of activity corresponded to the oxidation of 1 ,uM of NADH per min at 250C.…”

“…Apparently, LDH activity in Sig-1 was less than the level found in Mycoplasma sp. (7), because the LDH assays produced the least correlation with other measurements. OD, CFU, and DNA measurements produced similar calculated generation times, but total DNA established the end of exponential phase with greatest accuracy.…”