Figure 10.

Efficacious trapping of volatiles. (A). Trapping. Air was pumped through tubing from an inoculated Pa10 (80 μL from a 10⁹/mL suspension), or from an uninoculated (control) 250 mL TSA plate, incubated for 72 h at 37 °C, bubbling through 25 mL of de-ionized water. The collected waters were then incubated in the double plate method with TSA plates inoculated with 10 μL of 5 × 10⁷/mL of conidial 10AF suspension for 24 h, and the growth was measured. “Interaction” refers to the water that had been exposed to Pa vapors, three asterisks = p < 0.001. (B). Trapped volatiles are inhibitory to Af metabolism as well as growth. The effects of the volatiles trapped in deionized water and control, described in the legend for Figure 10A, were studied for their effects on 10AF biofilm formation with the methodology detailed previously [40,43,44,45,46,52,53,54,58]. In this instance, parafilm was used to seal the 96-well plate, the plate lid was applied, and the plate was put in a plastic bag, with the aim of retaining, if necessary, the volatiles in the wells (in addition to those trapped in the water). “Interaction” refers to the water that had been exposed to Pa vapors, three asterisks = p < 0.001. (C). Comparison of de-ionized water trapping of volatiles by the double plate method and air pumping method. The water from 2 double plate experiments, trapping phase, was prepared from the incubation with the Pa10 on a TSA plate, as described in Figure 9A,B, and tested (“Double plate 1 + 2”). Two pumping methods were assayed for trapping of volatiles, one as shown in Figure 3b, the other as described in the text for the generation of Figure 10C (“Pump 1 + 2”). Controls were as previously described, the water was exposed to an uninoculated TSA plate. Growth of 10AF on a TSA plate was assayed (as detailed in the Figure 3a legend) after exposure in a double plate method to the trapped volatiles in water. The effects on growth from the water of the 2 double plate experiments were identical, and the results were pooled. The results from the water of the 2 pumping methods were identical, and their results were pooled. Comparisons are shown as a percent of the control. Three asterisks = p < 0.001 compare either of the 2 left bars to the control. One dagger signifies p < 0.05, comparing the 2 left bars. Pump methods of obtaining a gas phase containing volatiles from a Pa culture are modestly but significantly superior to aspiration via needle from double plates for obtaining the materials inhibitory to Af.