Agar overlay plaquing technique for Pseudomonas aeruginosa in HeLa monolayers

The solid agar overlay procedure used for viral plaquing was adapted to the study of Pseudomonas aeruginosa plaques in HeLa monolayers. After adsorption of the organism to the HeLa monolayers, an overlay consisting of 10% newborn calf serum (NBCS), 90% Eagles basal medium (EBM), and 1.5% agar containing neutral red was added. Plaques developed after 24 hr of incubation at 37 C which were indistinguishable from viral lesions. Optimal conditions for plaques included: serum in the adsorption fluid as well as the overlay medium; 1-hr adsorption time; healthy HeLa monolayers; and the presence of EBM. The formation of plaques instead of colonies in agar was due to the inhibitory effect of NBCS or EBM. By omitting both of these components from the overlay, colonies appeared in place of plaques. Adult bovine serum acted in a similar bacterial colony-suppressing fashion, but fetal or agamma calf serum did not. Furthermore, NBCS or adult bovine serum when incorporated in the overlay medium instead of fetal or agamma calf serum displayed a plaque-suppressing effect by reducing the number and size of plaques formed. The findings lend further evidence to support the hypothesis that P. aeruginosa may produce plaques from a protected intracellular site within HeLa cells.