Materials and methods

We tested 4 collection methods: (1) cotton swabs, (2) replicate organism detection and counting (RODAC) agar plates, (3) sponge sticks (Romer Labs, Newark, DE) using manual extraction and (4) sponge sticks using extraction with the Seward Stomacher (Seward, Davie, FL). Test surfaces included stainless-steel and laminate squares. We tested the following organisms: Klebsiella pneumoniae ATCC 700603, Staphylococcus aureus ATCC 43300, and Clostridioides difficile ATCC 9689. ⁵ We performed 9 replicates for each surface–method–organism combination.

Dilutions containing ∼10,000 CFU per milliliter (CFU/mL) of bacteria were made for each batch of testing. A viable colony count was obtained for each dilution by performing plate counts in triplicate. Colony counts were then averaged and used to calculate the estimated CFU/mL for each test dilution.

Known quantities of the test dilution were drawn into a pipette tip then dispensed as tiny droplets across each test surface. Applying known quantities allowed for the calculation of the estimated number of bacteria placed onto each test surface. Inoculated surfaces were allowed to air dry inside a biological safety cabinet ∼1 hour prior to sampling.

For Klebsiella pneumoniae and Staphylococcus aureus testing, Dey-Engley neutralizing agar was used for all methods. For Clostridioides difficile testing, Clostridium difficile selective agar containing 7% horse blood, sodium taurocholate, and lysozyme was used for all methods.

Swab samples were collected by rubbing a swab moistened with Remel DE-neutralizing broth over each 15.5-inch² test surface, followed by a dry swab. Swabs were rotated as sampling was performed. Both swab tips were broken into a tube containing 1 mL phosphate-buffered saline. Tubes were mixed using a vortex, then an aliquot of each was plated to the appropriate agar plate. Each RODAC agar plate was pressed onto a 3.875-inch² round test surface and was held in place for 30 seconds. Sponge-stick samples were collected by rubbing a sponge premoistened with DE-neutralizing broth over each 15.5-inch² test surface. Each sponge head was ejected into a sterile bag containing phosphate-buffered saline. For manual agitation, each bag was kneaded by hand for 1 minute. For the stomacher method, bags were processed at 260 rpm for 1 minute. The contents of each bag were poured into individual tubes then centrifuged at 3,500 rpm for 15 minutes. The supernatant was removed and discarded from each tube. The remaining sample was mixed and measured, then an aliquot was plated to the appropriate agar plate.

The K. pneumoniae and S. aureus test plates were incubated at 35°C for 24 hours and 48 hours, respectively. C. difficile test plates were sealed inside anaerobic jars immediately after inoculation then were incubated at 35°C for 48 hours. After incubation, a digital colony-counting device was used to enumerate the colonies growing on each of the agar plates. Figure 1 illustrates steps used to assess the different sampling methods. Colony-forming units recovered per square inch (CFU/inch²) were calculated for each replicate, and the average was obtained for each set of 9 replicates. For each category, 95% confidence intervals (CI) were calculated using the average and standard error of the replicate counts.

Fig. 1.

Illustration of steps used for assessing the effectiveness of 4 sampling methods. Photographs illustrating various stages of study: (A) applying bacterial dilution to test surface; (B) colonies of Staphylococcus aureus growing on agar plates from samples obtained by swabs; (C) collecting surface sample using sponge stick; and (D) colonies of Clostridioides difficile growing on a RODAC agar plate.