Food Protection Trends - January/February 2022 - 42

Figure 1. (a) The modified CDC reactor. (b) The holders placed with ideal abiotic surfaces.
The assembled reactor was treated with 1.2% sodium hypochlorite
and autoclaved after it was rinsed with distilled water
(22). The reactor was filled with 576 ml of sterile 10% reconstituted
skim milk. The sterilized abiotic surfaces were placed in
reactor holders, and the holders were then transferred to the
reactor. Finally, the entire volume of reconstituted skim milk
was inoculated with the active culture (4% inoculum ratio).
The reactor was then transferred to an incubator with a magnetic
stirrer. Incubation was performed at 60°C for 2 and 18
h (Geobacillus strains) and 5 and 18 h (Anoxybacillus strains).
Biofilm formation was accelerated at 2 h for Geobacillus strains
and at 5 h for Anoxybacillus strains (13). For a sufficient
amount of biofilm samples, 18 h was ideal. Longer incubation
times were unsuitable for CLSM analysis due to the accumulation
of thick biofilms on surfaces (unpublished data). Biofilm
collection, sanitizer treatment, and counting procedures were
performed as previously described.
CLSM imaging
The treated and untreated surfaces were stained with 5 μM
SYTO 9 (S34854, Molecular Probes, Eugene, OR) and 15
μM propidium iodide (PI) solutions (P1304MP, Molecular
Probes) after rinsing. The surfaces were incubated at 30°C
in a dark environment for 20 min and were washed twice
with sterile distilled water (5, 9). SYTO 9 fluorescent stain
(excitation, 488 nm; emission, 498 nm) interacts directly
with living cells and emits green fluorescent light, whereas PI
(excitation, 543 nm; emission, 617 nm) is absorbed by the
dead cells and gives them fluorescence properties in the red
spectrum (31). PI and SYTO 9 also allow extracellular DNA
in the biofilm matrix to appear with a yellowish color (21).
Stained samples were stored at 4°C in a dark environment until
they were observed using CLSM (15). In the CLSM study,
clean surfaces that served as negative controls were also stained
with SYTO 9 and PI.
The treated and untreated biofilm samples were examined
using CLSM (Zeiss LSM 510) and Plan-Neofluar 40X/1.3
differential interference contrast objectives (both from Carl
Zeiss, Jena, Germany). Because milk residues also react with
dyes, the green fluorescence of SYTO 9 was excited with an
argon laser source at 540 nm and the red fluorescence of PI
was excited with a helium or neon laser source at 660 nm.
Fluorescence was recorded in bandpass 505 to 530 nm and
longpass 650 nm with 30% laser power in two diachronic
mirrors to separate the two laser sources. Microscopic fields
were determined in each specimen with 2D (x-axis or y-axis
only, 230.34 × 230.34 μm) or 3D (x, 230.34 μm; y, 230.34
μm; z-axis, 4 μm) horizontal plane images. Live and dead cell
densities were determined, and 3D biofilm thicknesses (μm)
were also measured.
RESULTS AND DISCUSSION
Eradication of thermophilic biofilms sampled under
static and dynamic conditions
The eradication results for all thermophilic biofilms are
presented in Supplementary Tables S1 to S3; the results are also
summarized in Table 3.
In particular, the biofilms of A. kamchatkensis subsp. asaccharedens
F81 sampled on stainless steel and polypropylene surfaces could
not be eliminated by any of the tested chemical and enzymatic
sanitizers (Table 3). The biofilm cells and spores of A. flavithermus
DSM 2641T, which causes problems with its biofilms in the dairy
industry, were 100% removed by treatment with alkaline protease
and SDS. In addition, biofilm spores were removed from polyvinyl
chloride and polycarbonate surfaces with TCA and triclosan.
Both biofilm cells and spores on polyvinyl chloride surfaces were
removed with sodium metaperiodate alone, but removal rates on
polycarbonate surfaces remained lower for this strain (Table 3).
The biofilm cells of G. thermodenitrificans DSM 465T
stainless steel and polycarbonate surfaces could not be 100%
on
42
Food Protection Trends January/February

Food Protection Trends - January/February 2022

Table of Contents for the Digital Edition of Food Protection Trends - January/February 2022