CONDITIONS FOR GROWTH OF AN OVINE CHLAMYDIA (BEDSONIA) IN CELL CULTURE

Abstract

When HEp2 cell cultures were inoculated with an ovine Chlamydia very-few inclusions were fonned when the pH of the nietliiim fell to below pH 7*0. Failure to form inclusions was not due to inactivation of organisms by add conditions during the adsorption period, but was apparently due to events subsequent to adsorption whereby inclusion development under acid conditions was inhibited. Although the ovine ChlamtjcHa was inactivated more rapidly at 37° than at 30°, nevertheless adsorption and penetration of organisms into IIEp2 cultures was more efficient at 37° than at 30". The rate of adsorption of organisms was dependent on volume, and the smaller the volume in which a particular do.se of organism.s was su.spended, the greater the number of inclasions formed. From the theory of Brownian motion it was possible to predict that the rate of adsorption on to surfaces by Chlamydia should be slower than that of viruses, because the size of Chlamydia limits tlieir diffusion. It was possible to calculate that no more than 40!£ of this Chlamydia suspension, in cell culture medium, might adsorb from a depth of 0-1 cm on to a surface in 18 hours at 37°. Under tliese conditions, experimentally, only 24S of the total Chlamydia particles in the suspension resulted in inclusions. Inclusion counts were Iiigher in HEp2 cells than in Vero cells. The inclasion counts in HEp2 cell cultures which had not been treated with DEAE-Dextran were increased by addition of 0-5% (v/v) foetal calf serum or bovine serum, or by 0-6-0-8X (w/v) bovine albumin fraction V to the medium in which the organisms were suspended. The counts decreased with higher concentrations of serum or albumin. In DEAE-Dextran-treated cultures inclusion counts were greatly depressed by the presence of bovine albumin fraction V or serum in the medium (except for a slight ri.st' in count with 0-5 (v/v) serum).

Susceptibility testing of Chlamydia trachomatis: from eggs to monoclonal antibodies

“…Although the microdilution plate has a smaller capacity than the vial, thus restricting the volume of inoculum, the significance of this is questionable because the most common inoculum volumes used currently for both methods range from 0.1 to 0.3 ml. An increased container volume per se does not improve isolation rates and may decrease the efficiency of adsorption onto the cells (11). The volume-to-cell surface ratio is a factor; the small elementary bodies must be centrifuged through the inoculum to come in contact with the cells, and an increased volume also increases the distance that they must travel.…”