Fig 1.
Sampling duration experiments for microorganisms collected on filters.
(a)–Comparison of 1×24 hour versus 3×8 hour parallel sampling. Two sets of sampling trains collected air at the same time and location for 24 hours. The first set sampled the air continuously for 24 hours on a single filter, while the other set’s filter was replaced every 8 hours. DNA extracted from the three 8-h filters was then pooled and the result is compared with the first set which was extracted alone. (b)–Testing loss of GFP-tagged bacteria during sampling. A set of filters sampled air at the same time and location for a total duration of 20 hours. Known quanta of GFP-tagged S. oneidensis cells were spiked onto the filters at staggered timepoints, exposing them to different durations of air sampling.
Fig 2.
Concentration approach during extraction.
The DNA from three filters is pooled during the spin-filter binding step of DNA extraction. The total DNA yield is then compared to the one filter extracted alone to investigate whether the expected 3-to-1 ratio is obtained.
Table 1.
Probe-and-primer sets for qPCR assays.
Fig 3.
DNA measurements of AHU filter samples for seven treatment pathways of sonication at varying temperature.
(a)—Total DNA yields (Qubit). (b)–Results from bacterial and fungal qPCR. N = 4 for each case. * denotes statistically significant difference to mean of DNA yield extracted with 65°C incubation.
Fig 4.
Improving DNA yield with additional heat and sonication lysis.
Additional sonication and thermal lysis show improved DNA yield for (a)—AHU filter samples and (b)–ambient air samples as measured by the Qubit fluorometer for total DNA (left bar, left axis) and by qPCR for bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA (N = 4).
Fig 5.
Comparison of two sampling approaches.
Comparison of a sampling approach utilizing a single filter continuously sampled for 24 h (grey bar) and a combined series of three filters, each operated for 8 h (black bar) expressed in terms of total DNA (left bar, left axis) measured by Qubit and in terms of bacterial (middle bar, right axis) and fungal (right bar, right axis) DNA measured by qPCR (N = 3).
Table 2.
qPCR results from a series of 20-h samples illustrating the decay of GFP-tagged Shewanella oneidensis cells.
Table 3.
qPCR cycle threshold value data for mid-concentration approach considering both 16S bacterial and 18S fungal probe-and-primer sets.