Food Protection Trends - March/April 2021 - 197

+ YE and was incubated again at the same temperature and
duration. Each strain suspension was harvested by centrifugal
force (Rotor FA-45-24-11, model 5424, Eppendorf North
America, Hauppauge, NY) at 6,000 rpm (3,548 × g for 88
mm rotor) for 15 min and was washed with phosphatebuffered saline (VWR International, Radnor, PA) to remove
sloughed cell components, excreted secondary metabolites,
and growth mediums. Individually subcultured and harvested
strains were then composited to prepare inocula of fourstrain mixtures for each pathogen.
Bacterial inoculation
Formerly used stainless steel coupons (type 304, #2b
finish, [1.2 by 5 by 0.3 cm]), measured using a digital slide
gauge (Marathon Watch Company Ltd., Richmond Hill,
Ontario, Canada), were washed and rinsed with detergent,
water, 99% acetone (Fisher Scientific, Fair Lawn, NJ), and
70% ethanol (Fisher Scientific), to eliminate any residue, and
were then sterilized by autoclaving (11, 31). Dry, sterilized
coupons were spot inoculated with 100 μL of wild-type,
10-fold diluted bacterial suspension (target inoculation level
of 7.5 log CFU/cm2), and another set of sterilized coupons
was inoculated with 100 μL of the pressure-stressed suspension (target inoculation level of 7.5 log CFU/cm2). The
coupons were stored aseptically for 1 h at ambient temperature under a biosafety cabinet for attachment of cells onto the
surface of coupons. The inoculated coupons were aseptically
transferred into 15-mL sterile Eppendorf conical tubes
containing 4 mL of sterile organic milk. This arrangement
was done to ensure that coupons were half-submerged,
ensuring the existence of air-liquid interface for enhanced
development of the bacterial biofilms (14, 37). Coupons
stayed half-submerged in the food vehicle in an upright
tilted position and were incubated statically under aerobic
conditions at 7 or 25°C for up to 14 days. The time, biofilm
formation vehicle, and conditions of attachment for this
experiment were selected based on preliminary studies (11).
Tubes were incubated at 7 or 25°C for both wild-types and
pressure-stressed phenotypes, for 0, 1, 4, 7, 8, 11, and 14
days.
Formation and decontamination of biofilms
For investigating the formation of biofilms at 7 and 25°C,
2 h after inoculation (day 0) and on days 1, 4, 7, 8, 11, and
14, inoculated coupons were analyzed for microbial populations. Decontamination of the coupons was examined
on day 0 (counts of inoculated pathogen on stainless steel
coupons prior to biofilm formation, i.e., planktonic cells),
day 7 (1-week-mature biofilm), and day 14 (2-week-mature
biofilm). Pressure-stressed phenotypes of the pathogens
were prepared in the Public Health Microbiology Laboratory
of Tennessee State University by exposing the four-strain
mixtures of each pathogen to 15,000 lb/in2 (103.4 MPa) of
elevated hydrostatic pressure (Barocycler Hub880 Explorer,
Pressure BioScience Inc., South Easton, MA) at 25°C for

20 min. This was achieved by controlling the pressure and
temperature as explained in our open-access study (2), using
HUB Explorer PBI 1.0.8 software (Pressure BioScience Inc.)
inside a no-disk PULSE tube (Pressure BioScience Inc.).
The disinfectant used in the current study (Bleach-Rite,
catalog no. BRSPRAY128, Fisher Scientific) is registered as a
hospital-grade cleaner and contained at least 0.525% sodium
hypochlorite (bleach). The sanitizer was a premix and was
used without dilution, i.e., at the manufacturer's maximum
registered concentration for disinfection. In addition to
receiving a certificate of analysis from the supplier, the free
chlorine concentration was verified by measurements using
a colorimeter (model HI701, Hanna Instruments, Woonsocket, RI). Ten independent measurements were compared
statistically (one-sample t-test) with a target concentration of
5,250 ppm (i.e., 0.525%) and were not statistically (P ≥ 0.05)
different from this target value. Concentrations of each batch
were, in addition, semiquantitatively confirmed to be at least
>5,000 ppm using a chlorine sanitizer test strip.
For the enumeration of wild-type and pressure-stressed
pathogens on surfaces of coupons after inoculation, each
side of the coupons was rinsed with 10 mL of sterilized
distilled (total soluble solids <30 ppm) water for removal of
loosely attached cells (11). For treated samples, after rinsing
with sterilized distilled water, 10 mL of the chlorine-based
sanitizer (0.525% sodium hypochlorite) was applied to each
side of the coupons. Exposure time of the treatment was 60
s at 25°C. Subsequently, each coupon was placed in 30 mL
of Dey-Engley neutralizing broth (Difco, BD) containing 10
sterile glass beads (4-mm diameter). The tubes were then
vortexed for 2 min (3,200 rpm) to detach biofilm cells (11,
32). After this, a 1-mL aliquot was 10-fold serially diluted
in maximum recovery diluent (Difco, BD) and was spread
plated onto TSA + YE. Colonies were manually counted after
incubation of plates at 37°C for 24 h.
Design and statistical analyses
The experiments were carried out in two biologically
independent repetitions, each considered as a blocking factor
in a randomized complete block design. Each biologically
independent repetition (each block) consisted of two replications, and each replication further consisted of two microbiological repetitions per sample. Thus, each presented value
is the mean of eight independent observations (two blocks,
two replicates, two microbiological repetitions). Microbiological data were converted to log CFU/cm2, and, after log
transformation of the bacterial counts, the data were analyzed
using analysis of variance followed by Tukey-adjusted mean
separation for comparison of microbial counts. The statistical
analyses were carried out using the general linear models
procedure of SAS version 9.4 software (SAS Institute, Inc.,
Cary, NC) at type 1 error level of 5% (α = 0.05). Experiments
involving pathogens at 7 and 25°C were conducted independently and, thus, were analyzed and reported separately.

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