Food Protection Trends - November/December 2019 - 443

ease of decontaminating a surface (6, 8). Abraded surfaces
accumulate soil and prove more difficult to clean than
smooth surfaces (6).
In part because of the risks posed by improperly cleaned
and sanitized surfaces, the U.S. Department of Agriculture,
Food Safety and Inspection Service (USDA-FSIS) established the Pathogen Reduction; Hazard Analysis Critical
Control Point (PR/HACCP) Systems Final Rule (19).
Under this program, FSIS assesses industry performance
and controls for reducing contamination in raw meat and
poultry products, and processors must have and implement
a sanitation standard operating procedure (SSOP) that
addresses sanitation conditions and practices before,
during, and after processing. An SSOP may include a
pre-operational task during which selected areas of the
establishment and equipment that pose a high risk of
contamination are organoleptically, primarily visually,
inspected to assess sanitary conditions. Visual inspection
may be of little value because the residues, whether of food
or bacterial origin, may be present in quantities too small
to be detected by the human eye, although they still present
a contamination risk. A method to enhance the visual
assessment or inspection of potentially contaminated areas
in the processing line would therefore be of great value
to processors in identifying and measuring contaminated
areas in or on processing equipment as well as in identifying
design flaws that make sanitation of equipment difficult.
Various dyes have been employed to monitor total biofilm
biomass (13, 17). Some of those most commonly used,
such as crystal violet (11, 16) and safranin red (12), offer
simplicity and direct optical visualization. These dyes are
positively charged and bind to the negatively charged surfaces
of bacteria, both viable and non-viable, and to biofilm matrix
components including carbohydrates, DNA and proteins.
Although these dyes have been proven useful as screening
tools for measuring the effectiveness of biofilm removal by
various cleaning agents and sanitizers, they exhibit toxic
properties and are not approved by FDA for use in food
processing environments.
Erythrosin B, also known as FD&C Red #3, is an FDAapproved food coloring dye that has been utilized since
the early 1960s as a dental plaque disclosing agent (1).
The dye has also found use as a colorimetric reagent for
protein determination (15) because of its ability to form
intensely colored complexes. In addition to its characteristic
visible red color, the dye exhibits fluorescence in the visible
region centered at 550 nm (green-yellow). This property
has allowed erythrosin B to be utilized as a vital exclusion
stain for determination of cell viability after lethal treatment
(9). These traits suggest that erythrosin B has potential
use as a screening method for biofilm removal and in the
visual determination of cleaning efficacy in food processing
environments. The goal of the present study was to evaluate
the potential for adapting erythrosin B for use as a non-

specific dye for enhancing the visualization of biofilm
removal from stainless steel in addition to monitoring
removal of other organic soils potentially present in meat and
poultry processing environments. Two organisms commonly
associated with biofilm formation in food processing
environments are Listeria and Pseudomonas. Although they
do not generally occur as monospecies biofilms in nature,
both are regularly used as biofilm surrogates for research
purposes. Listeria innocua and Pseudomonas putida were
chosen as surrogates in the present work, partly because of
their non-pathogenicity and hence their safety.
MATERIALS AND METHODS
Cell attachment
Stock cultures of L. innocua and P. putida, obtained from
the U.S. National Poultry Research Center culture collection,
were inoculated into tryptic soy broth and incubated at 37°C
for 24 h and then plated onto Brain Heart Infusion (BHI) agar
(Oxoid GmbH, Wesel, Germany). Individual isolated colonies
were taken from fresh cultures of L. innocua and P. putida to
create lawns (plates entirely covered with growth) to make
10 lawn plates. Growth from these plates was transferred to
700 ml of PBS (phosphate buffered saline) with sterile swabs.
Final inoculum concentrations were 9 × 108 CFU/ml for
L. innocua and 2 × 108 CFU/ml P. putida. Stainless steel grade
316 L coupons having a 2B finish were cut to 2.5 × 7.5 cm
dimensions. A batch of coupons was prepared by cleaning the
surfaces with acetone, autoclaved, placed in a stainless-steel
microscope slide staining rack, and immersed approximately
halfway for 2 h into a slide tray containing the inoculated PBS
broth at 25°C. The coupon rack was then rinsed three times
with sterile deionized water and transferred to a second slide
tray containing 700 ml of sterile 1/10 TSB (tryptic soy broth)
medium (Becton Dickinson & Co., Franklin Lakes, NJ) at
25°C for 24 h, to promote cell attachment. After 24 h, the
slides were rinsed with sterile water and put into petri dishes
for temporary storage.
Cleaner and sanitizer treatments
Tween 80, α-amylase from Bacillus licheniformis, and
39% peroxyacetic acid were purchased from Sigma (SigmaAldrich, St. Louis, MO). Alcalase from Bacillus licheniformis
was purchased from EMD Millipore (MilliporeSigma,
Burlington, MA), and 30% non-stabilized hydrogen peroxide
was purchased from Acros Organics (Acros Organics, Morris,
NJ). Two commercial cleaning/disinfectant solutions were
provided by their manufacturers. Following cell attachment,
duplicate coupons were immersed in one of the eight
treatment solutions, shown in Table 1, for 15 m. Following
treatments, duplicate coupons were rinsed three times in
deionized water. One of the duplicates was swabbed with
a pre-moistened sterile swab, which was returned to the
remainder of 1 ml PBS, vortexed, and subjected to a series
of 1:10 dilutions for CFU/ml counts on BHI agar plates for

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