Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture.

OBJECTIVES

The aim of this study was to enumerate viable bacteria at different growth stages of a multispecies oral biofilm and to compare results obtained with the LIVE/DEAD BacLight Kit (Molecular Probes, Eugene, OR) with those from culturing and plate counting (colony-forming unit counts [CFUs]).

METHODS

The multispecies biofilm was grown from plaque bacteria on collagen-coated hydroxyapatite disks in brain-heart infusion broth for 3 weeks (phase І) with a weekly addition of new nutrients. This was followed by a 9-week nutrient-deprivation phase (phase ІІ); after which, the biofilm was reactived again by weekly additions of fresh BHI medium for 4 weeks (phase ІІІ). The number and proportion of live bacteria in biofilm was assessed by culturing and by confocal laser scanning microscopy using a LIVE/DEAD viability stain throughout the experiment.

RESULTS

The CFU counts dropped more than four logarithmic steps during phase ІІ. However, viability staining by LIVE/DEAD stain indicated only a 25% drop in viability. The CFU counts increased during phase III, but it took 4 weeks for them to return close to the original CFU numbers. Cell viability, as indicated by the staining, improved from 75% close to the original 100%.

CONCLUSIONS

Bacteria in the multispecies biofilm grown under nutrient deprivation became viable but nonculturable but could be brought back to a culturable state after reestablishing sufficient access to nutrients. The results indicate that viability staining better reflected true viability of the biofilm bacteria than culturing during the long starvation phase. The result of this study may have an impact on the interpretation of cultural studies on root canal microbiology/biofilms in vivo.