Effects of growth phase and antibiotics on quantitative DNA/RNA hybridization

Synthetic probes complementary to ribosomal RNA are increasingly used in the detection of bacteria. Many applications, however, require the quantitation of bacteria. We therefore tested the influence of growth phase and representative antibiotics (ampicillin, chloramphenicol and gentamycin) on the outcome of DNA/RNA filter hybridization using radiolabelled probes and a multisample digital autoradiograph for quantitative monitoring. Hybridization efficiency seemed influenced by the binding capacity of the membrane, availability of target molecules and physiological growth control. For chloramphenicol the absolute hybridization signal remained constant over the experimental period. Only a slight decrease was found in experiments with gentamycin whereas viable counts dropped 10,000-fold. For ampicillin a decrease in viable counts was paralleled by diminishing signal strengths. Relative signal strengths (counts per viable cell) increased in all experiments with antibiotics. In conclusion; (i) RNA probes seem to detect bacteria even after onset of antimicrobial therapy; (ii) DNA/RNA filter hybridization appears not suitable for accurate quantitation of bacteria.