Food Protection Trends - November/December 2020 - 395

EZ-10HC-PUR, EZ Reach Sponge, World Bioproducts,
Libertyville, IL, with a 1.5- by 3-in. [3.8- by 7.6-cm] sponge)
before inoculating the surface. The swabbing procedure was
as follows, with swabbing locations shown in Fig. 1b. First, as
much of the broth as possible was squeezed out of the swab.
Starting at the top left, swabs were swiped within the template
area from left to right, right to left, ending at the bottom right
(as many swipes as needed). The swab was flipped over and
the surface was reswabbed in the same manner starting at the
bottom right, from bottom to top, top to bottom, ending in
the top left (as many swipes as needed). This was repeated
two more times in both diagonal directions. The handle of the
swab was twisted off, and the swab was returned to the bag
containing the neutralizing broth.
After the negative-control surface was swabbed (Fig. 1b;
Neg S1d), the positive-control surface and S1d (Fig. 1b) were
inoculated with one of the microbial suspensions (prepared
as described above). For both the positive control surface and
the S1d (Fig. 1b), a 12- by 12-in (1 ft2) inoculation area was
designated, and 25 spots of 20 µL each of the inoculum (500
µL total inoculum volume) was placed over each 1-ft2 area.
The inoculum was allowed to dry before wiping experiments
took place. Drying of the inoculum ranged from 45 min to
2 h, was dependent on the surrogate, and was confirmed
by visual inspection. The entire 1-ft2 inoculated area of the
positive control surface was swabbed as described above.
The surface wiping procedure is diagrammed in Fig. 1a.
A standard terry cloth bar rag (catalog B00KKRCS2Q, All
In Safety, Bloomfield, NJ), 16 in. (40.6 cm) by 19 in. (48.3
cm), was folded in half and then into thirds, resulting in a
50-in2 (322.6-cm2) wiping area. The folded towel was placed
into a bucket containing one of three solutions: (i) PBS (2
L), used as a no disinfectant control; (ii) Oasis 146 (Ecolab,
Saint Paul, MN), prepared per manufacturer's instructions
(QAC; 2 L hard water + 7.8 mL concentrated disinfectant;
final target disinfectant concentration of 400 ppm), used
as a representative "clean" disinfection solution; or (iii)
QAC prepared per manufacturer's instructions (described
in (2)), with an additional 5% soil load prepared according
to an ASTM standard (1), used as a representative "dirty"
disinfection solution. The cloth was submerged in the bucket
solution and used to manually mix the bucket's contents with
a gloved hand by swirling the contents in a circular motion.
After thorough wetting, the cloth was squeezed out by hand
and was then used to wipe in a back-and-forth motion from
S1d to surface 1-clean (S1c). Wiping started at the top left
corner of S1d (where the dried inoculum was located), and
the cloth was used to wipe top left to top right, right to left,
left to right, ending in the bottom right corner of S1c with a
total of eight swipes. Wiping was then repeated starting back
in the top left to bottom left, bottom to top, top to bottom,
ending in the top right with a total of eight swipes.
The folded cloth was then placed back into the bucket of
solution, squeezed out, and used to wipe surface 2 (S2) in

the same back and forth motion described above (Fig. 1a),
making sure to use the same area of the cloth surface for
wiping. The cloth was then immediately used to wipe surface
3 (S3; Fig. 1a), before being placed back into the bucket with
solution and squeezed out before wiping surface 4 (S4; Fig.
1a) in the same back and forth motion described above.
A 1-ft2 swab sample was taken from all surfaces (Fig.
1b) using the swabbing procedure described above for the
negative control surface. Microorganisms were eluted from
the swabs by squeezing the swab in the neutralizing buffer
120 times between two fingers before enumeration by plating
serial dilutions of the neutralizing broth in PBS on selective
medium with incubation as described above in enumeration
methods for each microorganism. Microorganism counts
from the 1-ft2 sampling areas were adjusted to reflect number
of microorganisms present on the entire 6-ft2 surface.
Bucket solution sampling
Bucket solution was sampled immediately after wiping S1,
S3, and S4 by drawing 1mL from the bucket using a pipette
(Fig. 1a). The sample was transferred to a 15-mL conical tube
containing 9 mL of D/E neutralizing broth. Samples were
processed for enumeration of microorganisms as described
above. In all cases, no difference was seen in the counts from
the three 1-mL solution samples (data not shown). Results
were calculated to reflect the total number of organisms
remaining in the entire 2-L volume of the bucket solution by
averaging the counts from the three 1-mL solution samples
and multiplying by 2,000.
Statistical analysis
All experiments were independently replicated in
triplicate on separate days. Results are presented as mean
total CFU or PFU per 6 ft2 surface ± standard deviation
and as a percentage of the total CFU or PFU transferred
to, and remaining in, the bucket solution for liquids (Excel,
Microsoft Corporation, Redmond, WA).
RESULTS AND DISCUSSION
In food service establishments, schools, and many other
settings, the use of the cloth and bucket method for cleaning
environmental surfaces is common. Because little is known
about the potential for these practices to spread pathogenic
bacteria, viruses, and spores, the aim of this study was to
characterize the spread of representative gram-positive and
gram-negative vegetative bacteria, a spore, and a surrogate virus
from laminate surface to laminate surface using a scaled-up "real
life" experimental design. In addition, we sought to characterize
the concentrations of those organisms transferred to, and
remaining in, the bucket solutions after wiping events had been
carried out because residual organisms could be the source of
potential ongoing recontamination in real-world settings.
To replicate the initial contamination source, S1d was
inoculated with the test microorganism; after which, a

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