Figure 9.

Biofilm forming isolates delay the onset of a viable, but non-culturable state. (A) F. tularensis LVS (grey), a biofilm forming population (black) and a purified constitutive biofilm forming isolate (white) derived from wild-type LVS were streaked onto chocolate agar and incubated at 37°C. At the time indicated, CDM was inoculated and incubated shaking at 37°C for 24 h. The OD₆₀₀ was obtained as a measurement of bacterial growth. Samples were diluted as required to maintain the linear range of measurement. Graphed data show the average of at least 3 independent experiments with the standard error of the mean. (B) An aliquot of the bacteria present from the 24h broth culture was assayed for viability using Live/DEAD stain followed by fluorescent microscopy. In these representative images, bacteria with an intact membrane (live) stain green while those with an impaired membrane (dead) are indicated by red staining. LVS exposed to 70% EtOH for 15 minutes was stained and imaged alongside samples to serve as a control.