Chlamydia pneumoniae: Culture Methods

Abstract

After T’ang et al. in Peking first successfully cultivated trachoma organisms in chick embryo in 1957, egg culture became a standard method of isolation and growth of Chlamydia trachomatis [1]. The researchers’ success was owed to the use of streptomycin, but not penicillin, for control of contamination. Since egg culture is not only cumbersome for cultivation but also for purification of organisms from yolk sacs, finding a sensitive cell culture method was vigorously pursued. In 1965, Gordon and Quan described a method for isolation of trachoma agents in McCoy cell culture using a flat-bottomed culture vial [2]. To enhance the sensitivity, McCoy cells were irradiated with γ-radiation and centrifugation was applied during the absorption period after specimens were inoculated to culture vials. The procedure was simplified by Ripa and Mårdh, who replaced γ-radiation with the use of cycloheximide [3]. In their method non-irradiated McCoy cells were used. To enhance the sensitivity of McCoy cells to C. trachomatis growth, cycloheximide was added to culture medium for cultivating infected cells. Cycloheximide inhibits host cell protein synthesis but does not affect chlamydial metabolism; thus, it reduces the competition for nutrients between host cells and parasites and enhances the growth of chlamydial organisms. This cell culture method was quickly adapted, and replaced egg culture for isolation and growth of C. trachomatis. Other modified methods have appeared and been used, such as the use of DEAE-dextrantreated HeLa 229 cells [4]. However, the crucial factors are centrifugation and cycloheximide. The use of 96-well microtiter plates for isolation of C. trachomatis was first introduced by McComb and Puzniak in 1974 [5] and later popularized by Yoder et al. in 1981 for handling large volumes of clinical specimens [6].