Poster ID
#642
07/ 14
Bias Assessment of the Single Tube Method (ASTM E2871-12)
Center for
Biofilm
Engineering
D. Goveia, B. Fritz, L. Lorenz, A. Parker, D. Walker, D. Goeres
Sponsored by: CBE Industrial Associates
Treatments provided by: CBE Member Company, Sealed Air
a National Science Foundation Engineering Research Center in the MSU College of Engineering
SUMMARY
RESULTS
Bias is defined as the systematic deviation from a true value, i.e.,
the measured value not hitting the bulls eye (Figure 1). Bias leads
to inaccurate conclusions about experimental results. In disinfectant
testing, the log reduction value will be biased if and only if there are
differential removal or disaggregation efficiencies for treated
samples as compared to controls. If a disinfectant has a fixative
effect, conventional removal techniques may inadequately remove
the biofilm from a surface, resulting in artificially low viable cell
counts for treated coupons (Hamilton et al. JOAC 2009).
A
B
C
A
B
C
B
C
1.25X Control
1.25X Control
1.25X Control
1.25X 1:10
Biased
A
1.25X 1:10
1.25X 1:10
Unbiased
Figure 1. Target representation of biased vs. unbiased.
The Single Tube Method (STM) is the latest ASTM Standard
(E2871-12) for use in biofilm disinfectant efficacy testing. Our
research goal was to demonstrate the feasibility of microscopy and
the crystal violet assay (CRV) for the assessment of bias in
disinfectant efficacy testing using the STM.
1.25X 1:100
1.25X 1:25
1.25X 1:100
1.25X 1:1000
Statistically significant bias in the log reductions was detected for
two of the disinfectants tested (Statistical Analysis section). The
results demonstrate that visualizing biofilm after treatment exposure
can identify bias due to differential biofilm removal between the
control coupons and those coupons treated with a disinfectant.
1.25X 1:50
Figure 3. Divosan MH crystal violet assay (A) before sonication/vortexing;
(B) after sonication/vortexing (3 replicates); and (C) confocal images
stained with BacLight live/dead after sonication/vortexing.
METHODS
(1)
Rinse rod
 Add x mL disinfectant
 Incubate for specified
contact time, temperature
 Add y mL neutralizer
 Incubate for specified
contact time, temperature
Figure 4. Divosan OSA-N crystal violet assay (A) before sonication/vortexing;
(B) after sonication/vortexing (3 replicates); and (C) confocal images stained
with BacLight live/dead after sonication/vortexing.
(2)





Vortex 30s
Sonicate 30s
Vortex 30s
Sonicate 30s
Vortex 30s
(3)
Dilute & plate
Figure 2. Diagram of Single Tube Method and sampling procedure.
Table 1. The bias of the log reduction (LR) for each of the
3 products at their test concentrations.
0.30
Product
Bias = calculated LR - true LR
2.5
2.0
OD
Figure 5. TSA Acid Sanitizer crystal violet assay (A) before
sonication/vortexing; (B) after sonication/vortexing (3 replicates); and (C)
confocal images stained with BacLight live/dead after sonication/vortexing.
Bias of the LR
3.0
• A Pseudomonas aeruginosa ATCC 15442 biofilm was grown on
borosilicate glass coupons according to the CDC method
• The STM was conducted as shown in Figure 2
• Three different disinfectants (Divosan MH, Divosan OSA-N and
TSA Acid Sanitizer) were tested at high, medium, and low
concentrations
• A control coupon was stained with crystal violet before biofilm
removal (1)
• Replicate coupons were treated at each concentration for 10 min
and neutralized with 2X Letheen broth
• A combination of vortexing and sonication was used to remove the
biofilm from the coupon and disaggregate clumps
• Replicate coupons were evaluated for biofilm removal using the
CRV and quantified using a spectrophotometric absorbance
measurement (2)
• A coupon was stained with BacLight live/dead and imaged using
confocal microscopy (2)
• The cell suspension was serially diluted and plated on R2A agar
for viable cell counts (3)
Place coupon w/
biofilm in vial
1.25X 1:800
1.5
1.0
0.5
Before/After
Conc
Product
B A
0
Control
B A
1:10
B A
B A
1:100 1:1000
Divosan MH
B A
B A
B A
1:10
1:25
1:50
Divosan OSA-N
B A
B A
B A
B A
1:10
1:100
1:800
0
TSA Acid Sanitizer
Water Only
Figure 6. Either before (B) or after (A) the STM removal step was applied, an optical density (OD)
measurement was taken of each coupon. In every instance, the OD after removal was less than the OD
before removal (i.e., the percent reductions (PR) are always positive).
LR
bias
LR
corrected
for bias
1:10
2.71
0.19
2.53
1:25
1.64
0.24
1.40
1:50
3.29
0.19
3.11
1:100
3.51
0.25
3.26
1:1000
2.29
-0.01
2.30
1:10
4.64
0.07
4.57
1:100
2.58
0.09
2.49
1:800
1.56
0.01
1.55
0.25
0.20
Divosan OSA-N
0.15
Divosan MH
0.10
0.05
0.00
0.0
Conc
LR
observed
Conc
Product
TSA Acid
Sanitizer
1:10 1:100 1:1000
Divosan MH
1:10 1:25
1:50
Divosan OSA-N
1:10 1:100 1:800
TSA Acid Sanitizer
Figure 7. The bias of the log reduction (LR) for each of the 3 products tested.
For all 3 products, perhaps not surprisingly, the LR bias was smallest at the
least efficacious disinfectant concentration.
STATISTICAL ANALYSIS
INDUSTRIAL RELEVANCE
Percent reductions (PR) of the biofilm due to removal were
measured for each set of control and treated coupons in each
experimental run. A 100% reduction after removal would indicate
total removal of the biofilm from the coupons. While a 100%
reduction was never observed, the log reduction for a disinfectant
remains an unbiased estimator of disinfectant efficacy as long as
the PR for the controls is equal to the PR fro the treated coupons.
For Divosan MH and Divosan OSA-N, due to differential percent
reduction of bacteria from the control coupons (~90% reductions)
and from coupons treated with high concentrations of products (4560% reductions), statistically significant bias in the log reductions
(LR) was detected. The bias in the LR for Divosan MH was between
0.25 and 0.3 for the higher concentrations tested (p-value < 0.001);
the bias in the LR for Divosan OSA-N was between 0.18 and 0.25
for all concentrations tested (p-value = 0.029. Visually, this can be
seen in Figures 3 and 4.
The timing of this research is of particular importance, as the US
EPA has recently referenced both the Single Tube Method and
ASTM Method 2562-12 The Quantification of Pseudomonas
aeruginosa Biofilm Grown with High Shear and Continuous Flow
using CDC Biofilm Reactor Method in their proposed guidelines for
registering products with a biofilm claim. This research will provide
the laboratories who plan to conduct product testing with a
reference that justifies the validity of their log reduction results (i.e.,
that the results are unbiased (and repeatable or reproducible)) from
disinfectant efficacy testing specific to biofilm. In addition, this study
demonstrates the power of using statistics to clearly establish
whether a treatment is significantly biased with respect to different
use concentrations.
REFERENCE
Hamilton, M., Buckingham-Meyer, K. and Goeres, D.M. 2009.
Checking the validity of the harvesting and disaggregating steps in
laboratory tests of surface disinfectants. Journal of AOAC
International. 92: 1755-1762.
ACKNOWLEDGMENTS
We are grateful to the CBE Industrial Associates whose member
fees contributed to making this study possible.
Special thanks to B. Glembocki, Sealed Air, for initial bias
investigations and supplying the chemistries for this study.