US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for

Single Tube Method for Determining the Efficacy of
Disinfectants against Bacterial Biofilms

SOP Number: MB-20-04

Date Revised: 09-19-22


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SOP No. MB-20-04
Date Revised 09-19-22
Page i of 17

SOP Number

MB-20-04

Title

Single Tube Method for Determining the Efficacy of Disinfectants
against Bacterial Biofilm

Revisions Made

•	Minor editorial changes for clarification purposes.

•	Removed Dey Engley (D/E) Neutralizing broth as an example of
neutralizer.

•	Took out footnotes that steps were not currently in ASTM E2871-
19.

•	Updated ASTM International Standard reference from 2019 to
2021 and updated version number from E2871-19 to E2871-21.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 1 of 17

SOP Number

MB-20-04

Title

Single Tube Method for Determining the Efficacy of Disinfectants
against Bacterial Biofilm

Scope

Describes the operational parameters required to perform a
quantitative liquid disinfectant efficacy test against bacterial biofilm
grown in the CDC biofilm reactor.

Application

For use in determining the efficacy of aqueous disinfectants against
biofilm grown on borosilicate glass coupons.





Approval

Date

SOP Developer:



09/19/22



Print Name: Rebecca Pines

SOP Reviewer



09/19/22



Print Name: Lisa Smith

Quality Assurance Unit



09/19/22



Print Name: Michele Cottrill

Branch Chief



09/19/22



Print Name: Rebecca Pines



Date SOP issued:

09/19/22

Controlled copy
number:

0

Date SOP withdrawn:




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SOP No. MB-20-04
Date Revised 09-19-22
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TABLE OF CONTENTS
Contents	Page Number

1.

DEFINITIONS

3

2.

HEALTH AND SAFETY

3

3.

PERSONNEL QUALIFICATIONS AND TRAINING

3

4.

INSTRUMENT CALIBRATION

3

5.

SAMPLE HANDLING AND STORAGE

3

6.

QUALITY CONTROL

3

7.

INTERFERENCES

3

8. NON-CONFORMING DATA

4

9.

DATA MANAGEMENT

4

10.

CAUTIONS

4

11.

SPECIAL APPARATUS AND MATERIALS

4

12.

PROCEDURE AND ANALYSIS

6

13.

DATA ANALYSIS/CALCULATIONS

11

14.

FORMS AND DATA SHEETS

12

15.

REFERENCES

12


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SOP No. MB-20-04
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1. Definitions

Additional abbreviations/definitions are provided in the text.

1.	CDC = Centers for Disease Control and Prevention

2.	Biofilm = microorganisms living in a self-organized community
attached to surfaces, interfaces, or each other, embedded in a matrix of
extracellular polymeric substances of microbial origin, while
exhibiting altered phenotypes with respect to growth rate and gene
transcription.

3.	Coupon = biofilm growth surface

4.	Disinfectant = antimicrobial test substance applied to coupons

5.	Control substance = innocuous liquid applied to coupons

2. Health and
Safety

Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Safety Data Sheet for
specific hazards associated with products.

3. Personnel
Qualifications
and Training

Refer to SOP ADM-04, OPP Microbiology Laboratory Training.

4. Instrument
Calibration

1.	Refer to SOPs EQ-01 (pH meters), EQ-02 (thermometers), EQ-03
(weigh balances), EQ-05 (timers), QC-19 (pipettes), and QC-22
(Vitek) for details on method and frequency of calibration.

2.	Refer to MB-19 section 4 to confirm the operating volume of the
reactor and residence time verification.

5. Sample

Handling and
Storage

Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures as necessary.

6. Quality Control

1.	For quality control purposes, the required information is documented
on the appropriate form(s) (see section 14).

2.	See MB-19 for purity check of test organism.

7. Interferences

1.	Coupons must be screened prior to use; replace coupons that are
compromised (presence of nicks or scratches).

2.	Touching the rod to the top of the conical tube will cause
contamination of the test system.

3.	Improper use of the splashguard may cause erroneous data (e.g.,
unexposed inoculum).


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SOP No. MB-20-04
Date Revised 09-19-22
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4.	Improper placement of the conical tubes with the coupons in the
sonicator during the recovery steps may interfere with removal of the
biofilm from the coupon.

5.	Conduct neutralizer confirmation in advance of the disinfectant assay
to ensure validity of the efficacy results.

8. Non-
conforming
Data

1.	Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-
Conformance Reports.

2.	For the purpose of conducting the Single Tube Method, the mean
TestLD for coupons inoculated with P. aeruginosa must be 8.0-9.5
CFU/coupon (corresponding to a geometric mean density of l.Ox 108 to
3.2x 109), with each coupon exhibiting a LD of 8.0 to 9.5. A mean
TestLD below 8.0 or above 9.5 invalidates the test.

3.	For the purpose of conducting the Single Tube Method, the mean
TestLD for coupons inoculated with S. aureus must be 7.5-9.0
CFU/coupon (corresponding to a geometric mean density of 3.2x 107 to
l.OxlO9), with each coupon exhibiting a LD of 7.5 to 9.0. A mean
TestLD below 7.5 or above 9.0 invalidates the test.

9. Data

Management

Archive the data consistent with SOP ADM-03, Records and Archives.

10. Cautions

1. Evaluate treated coupons prior to evaluating control coupons to
mitigate cross-contamination.

11. Special

Apparatus and
Materials

1.	Test organisms.

a. Coupons with mature Pseudomonas aeruginosa ATCC 15442 or
Staphylococcus aureus ATCC 6538 biofilm grown per MB-19. A
mature biofilm of P. aeruginosa or S. aureus meets the criteria
identified in 8.2 and 8.3, respectively.

2.	Bacterial plating medium. R2A agar for P. aeruginosa, tryptic soy agar
(TSA) for S. aureus.

3.	Dilution buffer. Prepare stock phosphate buffer solution by dissolving
34.0 g KH2PO4 in 500 mL reagent-grade water, adjust to pH 7.2±0.5
with 1 N NaOH, and dilute to 1 L with reagent-grade water. Prepare
stock magnesium chloride solution: 81.1 g MgCh 6FI2O/L reagent-
grade water. Filter sterilize both stock solutions. Prepare buffered
dilution water by combining 1.25 mL KH2PO4 stock solution and 5.0
mL MgCh 6FI2O, and dilute to 1 L with reagent-grade water (for final
concentrations of 0.0425 g/L KH2PO4 and 0.405 g/L MgCh 6FI2O)


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SOP No. MB-20-04
Date Revised 09-19-22
Page 5 of 17

and sterilize appropriately (see ref. 15.2).

a. Alternatively, phosphate buffered dilution water (PBDW), or
phosphate buffered saline (PBS) may be used for rinse tubes
(with 30 mL), control coupon exposure fluid, dilution blanks, and
filtration fluid, provided that the same buffer is used for each step.

4.	Neutralizer. One specific to the disinfectant being evaluated as
determined for effectiveness and toxicity according to Attachment 1.

a. Some treatments may require additional neutralizer volume (for
example, up to 196 mL). In these instances, use 250 mL conical
tubes.

5.	Small Allen wrench (1.27 mm, hex). For loosening set screws and
pushing coupons out of reactor rods.

6.	Vortex mixer. Any vortex that will ensure proper mixing of tubes.

7.	Calibratedmicropipettes. Continuously adjustable pipettes with
volume capacity of 100 |iL and 1000 |iL.

8.	Ultrasonic water bath. Any capable of maintaining a homogeneous
sound distribution of 45±5 kHz and a volume large enough to
accommodate 50 mL or 250 mL conical tubes in a wet environment.

a. Prior to using the sonicating bath for the first time, verify that the
sonicating bath does not kill viable cells by placing the
standardized broth culture into the sonicator for 60 s, serially
dilute, and plate. Compare sonicated counts to a non-sonicated
control. The sonicated and non-sonicated counts should be
comparable.

9.	Detergent. Laboratory detergent for cleaning coupons and reactor parts
(e.g., Micro-90 Concentrated Cleaning Solution for Critical Cleaning;
International Products Corporation).

10.	Conical centrifuge tubes. Sterile, any with 50 mL volume capacity and
secure leak-proof lids.

a. For foaming disinfectants or for disinfectants requiring a larger
volume of neutralizer, 250 mL conical tubes are used to preserve
the required geometry and allow for greater neutralization
capacity.

11.	Polyethersulfone (PES) filter membranes. Sterile 47 mm diameter
membranes with 0.45 |im pore size for microbe recovery from treated
coupons. Filtration units (reusable or disposable) may be used.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 6 of 17



12.	Forceps. Any appropriate for handling membrane filters.

13.	Splashguard inserts (from BioSurface Technologies). Used during
coupon deposition. Two sizes to fit the 50 mL and 250 mL conical
tubes used during coupon deposition. Equivalent splashguards (2.54
cm tapered to 2.39 cm (outer diameter) x 10.48 cm long for the 50 mL
conical tubes and 2.50 cm (outer diameter) x 14.80 cm long for the 250
mL conical tubes) from other suppliers may also be used.

12. Procedure and
Analysis

Each test includes three untreated control coupons (exposed to buffered
dilution water) and five treated coupons (per
disinfectant/concentration/contact time combination).

In advance of testing, verify the performance of the neutralizer using the
procedure in Attachment 1.

12.1 Test culture
preparation

a. Prepare mature biofilm per SOP MB-19, section 12.

12.2 Reaction tube
preparation

a.	Prior to steam sterilization, verify that the splashguards will sit
properly in the conical tubes so that the end of the splashguard sits
at the straight/conical interface of the tube.

b.	Steam sterilize the splashguards appropriately (e.g., place
splashguards into an autoclave pouch or wrap with aluminum foil
and steam sterilize for at least 25 min).

c.	Splashguards are only required for reaction tubes with coupons
treated with disinfectants.

d.	For disinfectants requiring larger neutralizer volumes, use 250
mL conical tubes with corresponding splashguards.

12.3 Disinfectant
sample
preparation

a.	Prepare disinfectant per SOP MB-22. When preparing
disinfectant, ensure that the disinfectant is adequately mixed. Use
within 3 h of preparation or as specified in the manufacturer's
instructions. Record the time of disinfectant preparation on the
Biofilm Single Tube Method Processing Sheet (see section 14).

b.	Evaluate the disinfectant at room temperature (21±2°C). If
necessary, place disinfectant in water bath prior to use to
equilibrate to the appropriate temperature for 10-15 min. Record
temperature on the Single Tube Method Processing Sheet (see
section 14).

c.	Bring the neutralizer to room temperature prior to use.

12.4 Removal of
coupons from

a. Prepare sampling materials: reaction tubes with splashguards,
rinse tubes, and flame-sterilized Allen wrench.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 7 of 17

the CDC	b. Turn off growth medium flow and baffled stir bar. After growth

biofilm reactor	medium flow and baffled stir bar have been turned off, use

coupons for testing (that is, exposed to disinfectant/control)
within 30 min.

i. If necessary for experiments in which more than 8

coupons are evaluated or removed from the reactor and
evaluated in batches over a period of time, the growth
medium flow and baffled stir bar may remain on during
coupon removal such that the total amount of time during
the CSTR phase does not exceed 24±2 h. Insert sterile
coupon holder blank in place of those rods removed for
coupon evaluation to maintain the appropriate flow
dynamics within the reactor.

c.	Aseptically remove a randomly selected rod containing coupons
with biofilm from the CDC Biofilm Reactor by firmly pulling it
straight up out of the reactor.

d.	Rinse the coupons to remove planktonic cells. Orient the rod in a
vertical position directly over a 50 mL conical tube containing 30
mL sterile buffered water. With one continuous motion, immerse
the rod into the buffered water with minimal to no splashing, then
immediately remove. Use a new 50 mL conical tube with 30 mL
sterile buffered water for each rod.

e.	Hold the rod with one of the randomly selected coupons centered
over an empty, sterile 50 mL or 250 mL conical tube containing a
splashguard (for coupons exposed to a disinfectant).

i. During coupon deposition, do not allow the rod to contact
the tube or splashguard for treated or control samples.
Refer to Attachment 2 for proper rod orientation. If
contact occurs, replace the coupon and associated tube
and/or splashguard.

f.	Loosen the set screw using a flame-sterilized Allen wrench and
allow the coupon to drop directly to the bottom of the tube.

i. If the coupon does not freely drop, press in the center of
the coupon with the Allen wrench used to loosen the set
screw.

g.	Remove an appropriate number of coupons for testing. Obtain a
set of five coupons for each treatment and a set of three coupons

	for the controls (one set of control coupons per reactor run) as


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SOP No. MB-20-04
Date Revised 09-19-22
Page 8 of 17



described in section 12.4c-f.

h. After removing the coupons for testing, gently remove the

splashguard from each tube using sterile forceps. Cap the reaction
tube to mitigate dehydration. Splashguards are not required for
control coupons.

12.5 Conduct
efficacy
evaluation

a.	Slowly pipette 4 mL previously prepared disinfectant (treatment)
or buffered dilution water (untreated control) down the side of
each of the conical tubes containing the coupons, avoiding direct
contact with the coupon during application and being careful to
completely cover the coupon. Record the time of coupon
exposure and the room temperature (21±2°C), see section 14.
Refer to Attachment 2 for proper treatment application
positioning. Process coupons treated with disinfectant first
followed by controls.

i.	If necessary for experiments in which more than 8
coupons are evaluated, evaluate one control coupon prior
to the start of the disinfectant treatment(s) and the
remaining two control coupons after the disinfectant
treatment(s).

ii.	For a 10 min contact time, a 1 min interval between
coupons is recommended.

b.	Immediately after deposition of disinfectant or control substance,
gently swirl the tube 1-2 times to fully expose the biofilm on the
coupon to the liquid, ensuring the coupon is fully covered by the
test substance and that there are no air bubbles trapped beneath
the coupon. The coupon is invalid if it is not fully exposed to the
test substance due to trapped air bubbles; replace with new
coupon and tube if this occurs. For those test substances that
cause effervescence, the presence of the effervescence does not
invalidate the coupon.

c.	Allow tubes to remain at room temperature (21±2°C) for the
duration of the contact time.

d.	At the end of the contact time, add the appropriate volume of
neutralizer (for example, 36 mL or 196 mL) to each tube. Replace
the cap and briefly vortex the tube.

12.6 Remove and
disaggregate
biofilm

a.	Vortex each tube on the highest setting, ensuring a complete
vortex for 30±5 s.

b.	Place all tubes into a wire or plastic conical tube rack and suspend


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SOP No. MB-20-04
Date Revised 09-19-22
Page 9 of 17



the rack in the ultrasonic water bath so that the liquid level in the
tubes is even with the water level in the tank of the bath. Do not
allow the tubes or the rack to touch the bottom or sides of the
ultrasonic water bath. Within the conical tube rack, allow space
between the tubes.

c.	Sonicate the tubes at 45±5 kHz for 30±5 s at room temperature
(21±2°C) (use normal mode if sonicator has variable settings).

d.	Vortex the tubes a second time as described in 12.6a.

e.	Sonicate the tubes a second time as described in 12.6b and 12.6c.

f.	Vortex the tubes a third time as described in 12.6a. These tubes
are the 10° dilution.

12.7 Dilute and
recover
disaggregated
biofilm
samples

a.	Dilute and recover treated samples followed by controls samples.
Initiate dilutions within 30 min of neutralization.

b.	Serially dilute the disaggregated biofilm from control samples in
buffered water. Test coupons may be serially diluted, if
necessary, to achieve countable filters in the target range of 20-
200 CFU. Make serial ten-fold dilutions in 9 mL buffered water.

c.	For treated coupons, filter at least 25% of the total volume of
neutralizer + disinfectant from the 10° reaction tube through 0.45
|im PES membrane filters. Initiate filtration within 30 min of
making dilutions.

i.	Vortex the reaction or dilution tube prior to filtration.

ii.	Pre-wet the membrane filter with approximately 10 mL
dilution buffer.

iii.	Filter the appropriate volume. Liquid should pass through
the filter quickly (for example, within approximately 1
min of addition) with limited pooling of liquid in the filter
apparatus. If necessary to lessen this occurrence, use
multiple membrane filters per sample.

iv.	Separate filters but the same filtration unit can be used for
a given coupon provided the dilutions are filtered in order
starting with the most dilute.

d.	If filtering the entire contents of a tube, rinse the tube with
approximately 10 mL dilution buffer, vortex, and filter the
rinsate.

e.	Rinse the sides of the filter funnel with additional dilution buffer
(for example, approximately 40 mL) and transfer the membrane


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SOP No. MB-20-04
Date Revised 09-19-22
Page 10 of 17



filter to the recovery medium. Gently roll the filter onto the
surface of the agar to prevent trapping air bubbles between the
agar and the membrane; use sterile forceps to reposition the filter
if necessary.

f.	For control coupons, briefly vortex each tube and spread-plate
aliquots of the appropriate dilutions in duplicate on the recovery
medium.

i.	Plate 0.1 mL aliquots of appropriate dilutions in duplicate
on agar plates (R2A for P. aeruginosa or TSA for S.
aureus) for spread plating. Spread inoculum evenly over
the surface of the agar. Ensure plates are dry prior to
incubation.

ii.	Alternatively, 1 mL aliquots may be plated on Petrifilm.1

g.	Incubate plates from control coupons at 36±2°C for 48±4 h.
Incubate plates with filters from treated coupons for 72±4 h.

h.	If necessary, monitor recovery media for growth and assess the
number of visible colonies beginning at 24 h.

i.	Count the appropriate number of CFU according to the recovery
method used (for example, up to 200 CFU for filters and up to
300 CFU for plating). Record counts on the Biofilm Single Tube
Method Results Sheet (see section 14). Use CFU to calculate log
reduction. Log reduction is used to determine disinfectant
effectiveness.

j. Inspect the growth on the plates and filters for purity and typical
characteristics of the test microbe.

k. Gram stain one representative colony per coupon set with growth
for treated and controls. Record results on the Biofilm Test
Microbe Confirmation Sheet.

i.	P. aeruginosa is a Gram-negative rod.

ii.	S. aureus is a Gram-positive coccus.

1. Isolation streaks, biochemical and antigenic analyses, and/or
Vitek may be performed for additional verification of the test
organism.

12.8 Coupon,
reactor, and

a. After use in the reactor, place contaminated coupons in an

appropriate vessel, cover with liquid (for example, water), and

1 Option not provided in ASTM E2871-21.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 11 of 17

splashguard
reuse

autoclave with the other parts of the contaminated reactor system
(including splashguards) for 30 min.

After sterilization, clean the reactor components and splashguards
with a 1:100 dilution of detergent and tap water. After washing,
rinse all components with deionized water.

Clean and rescreen the coupons per SOP MB-19, section 12.1.

13. Data Analysis/
Calculations

1.	All colony counts are recorded and used in calculations to determine
log reductions.

2.	Calculate X., the mean CFU from the replicate samples plated.
The logio density (LD) for each control coupon is calculated as

a.

follows: Log10
where:

n= i(*i)

LSf=1(BxDi)J

x V

= number of dilutions

= average CFU of the replicate sample plates,

= volume plated,

= total volume of buffered dilution water plus neutralize^
= 10_k, and
= dilution.

3.	Calculate the mean LD for control coupons as follows:

Mean LD = [Logio (Coupon A) + Logio (Coupon B) + Logio (Coupon C)]/3

4.	Calculate biofilm density for treated coupons.

The LD for each treated coupon is calculated as follows:

n
X
B
V
D
k

a.

Logio
where:



U^iCQXD;)

x V

b.

= number of dilutions
= CFU per filter,

= volume filtered,

= total volume of disinfectant plus neutralize^

= 10_k, and
= dilution.

For the purpose of calculation, if no organism is recovered from a
treated carrier, the log density for that coupon is 0 provided that
the entire contents of the 10° reaction tube was filtered.

n

Y
C

V
D
k

c. If no organism is recovered from a treated carrier and only a


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SOP No. MB-20-04
Date Revised 09-19-22
Page 12 of 17



fraction of the 10° reaction tube was filtered, substitute 0.5 CFU
at the 10° dilution and scale up accordingly.

5.	Calculate the mean LD for each set of treated coupons as follows:

Mean LD = [Logio (Coupon A) + Logio (Coupon B) + Logio (Coupon C) + Logio
(Coupon D) + Logio (Coupon E)]/5

6.	Calculate the logio reduction (LR) for each disinfectant as follows:
LR = Mean LD (Control Coupons) - Mean LD (Treated Coupons)

a. If no organism is recovered from each of the five treated coupons
and the entire contents of the 10° reaction tube was filtered, the
log reduction is greater than or equal to the mean control carrier
log density.

7.	Use values with at least three significant figures when performing
calculations. Report log reduction values with at least two significant
figures.

14. Forms and Data
Sheets

1.	Attachment 1: Neutralization Assay

2.	Attachment 2: Method Photographs

3.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:

Biofilm Single Tube Method Test Information , .

& MB-20-04 Fl.docx
Sheet -

Biofilm Single Tube Method Dilution/Plating ,

m i • -i-! iviD"Zv"vT' rz.QOvX

Tracking Form -

Biofilm Single Tube Method Results Sheet MB-20-04 F3.docx

Biofilm Single Tube Method Processing Sheet MB-20-04 F4.docx

Biofilm Test Microbe Confirmation Sheet MB-20-04 F5.docx

Biofilm Calculations Spreadsheet MB-20-04_F6.xlsx

Biofilm Neutralization Test Information Sheet MB-20-04 F7.docx

Biofilm Neutralization Dilution/Plating - ,
_ , . _ 5 MB-20-04 F8.docx
Tracking Form -

Biofilm Neutralization Timing Sheet MB-20-04 F9.docx

Biofilm Neutralization Results Sheet MB-20-04 FlO.docx

Biofilm Neutralization Processing Sheet MB-20-04 Fll.docx

15. References

1. ASTM International, 2021. E2871-21: Standard Test Method for
Determining Disinfectant Efficacy Against Biofilm Grown in CDC


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SOP No. MB-20-04
Date Revised 09-19-22
Page 13 of 17

Reactor Using the Single Tube Method.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 14 of 17

Attachment 1

Biofilm Neutralization Assay

Al. Culture preparation

a.	Defrost a single cryovial at room temperature and briefly vortex to mix. Add 10 |iL
of the thawed frozen stock (single use) to a tube containing 10 mL of TSB (30 g/L),
vortex, and incubate at 36±2°C for 24±2 h.

b.	Prepare serial dilutions in 9 mL blanks of dilution buffer to achieve concentrations
of approximately 106 and 105 CFU/mL per dilution tube; these concentrations are
typically observed in the 10"2 and 10"3 dilution tubes, respectively. At least one of
these dilutions when diluted and plated should result in counts of 30-300 CFU/plate
(refer to the Biofilm Neutralization Assay Flowchart).

A2. Neutralization confirmation assay

a.	Neutralization Confirmation Treatment (NCT). At timed intervals, add 4 mL
disinfectant to the appropriate volume of neutralizer (for example, 36 mL or 196
mL) in triplicate and briefly mix, within 10 s add 0.1 mL of the test organism
diluted to 105 CFU/mL, and vortex to mix thoroughly. Repeat with the test
organism diluted to 106 CFU/mL if desired. Proceed with section A2.d.

b.	Neutralizer Toxicity Treatment (NTT). At timed intervals, add 0.1 mL of the test
organism diluted to 105 CFU/mL to the appropriate volume of neutralizer (for
example, 40 mL or 200 mL) in triplicate and vortex to mix thoroughly. Repeat with
the test organism diluted to 106 CFU/mL if desired. Proceed with section A2.d.

c.	Test Culture Titer (TCT). At timed intervals, add 0.1 mL of test organism diluted to
105 CFU/mL to the appropriate volume of dilution buffer (for example, 40 mL or
200 mL) in triplicate and vortex to mix thoroughly. Repeat with the test organism
diluted to 106 CFU/mL if desired. Proceed with section A2.d.

d.	Hold all treatments at room temperature (e.g., 21±2°C) for 10 min±30 s.

e.	After the contact time, vortex each tube thoroughly and prepare one 10-fold dilution
in 9 mL dilution buffer.

f.	Briefly vortex the dilution tube prior to plating; initiate plating within 30 min of
making dilutions. Plate 0.1 mL aliquots from each tube in duplicate on R2A (fori5.
aeruginosa) or TSA (for S. aureus) using spread plating. Spread inoculum evenly
over the surface of the agar. Plates must be dry prior to incubation.

g.	Alternatively, filter 10 mL from each of the NCT, NTT, and TCT treatment tubes
through individual 0.45 |im polyethersulfone membranes; no additional dilution is
necessary.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 15 of 17

i.	In advance of testing, make adjustments to the initial dilution series to
achieve a target of 20-200 CFU per filter.

ii.	For disinfectants that require additional neutralizer volume, filter a
minimum of 25% of the total volume of neutralizer + disinfectant. If
necessary, use multiple filters to assay these larger volumes.

iii.	To filter, pre-wet the membrane with approximately 20 mL dilution buffer
then add the appropriate volume from the treatment tube. Rinse the sides of
the filter funnel with additional dilution buffer and place the filter membrane
on R2A (fori5, aeruginosa) or TSA (for S. aureus). Gently roll the filter
onto the surface of the agar to prevent trapping air bubbles between the agar
and the membrane; use sterile forceps to reposition the filter if necessary.

h. Incubate plates (inverted) at 36±2°C for 48±4 h.

A3. Results

a.	For calculation purposes, use the dilution that resulted in 30-300 CFU/plate (or 20-
200 CFU/filter). Average between spread plates for a given dilution (if using), then
average results from the three tubes per treatment.

b.	For determining and verifying the effectiveness of the neutralizer, ensure that:

i.	The recovered number of CFU in the Neutralizer Toxicity Treatment (see
section A2.b) is within 50% of the Test Culture Titer (see section A2.c). A
count less than 50% indicates that the neutralizer is harmful to the test
organism. Note: counts higher than the Test Culture Titer (e.g., 120% of the
Test Culture Titer) are also deemed valid.

ii.	The recovered number of CFU in the Neutralizer Confirmation Treatment
(see section A2.a) is within 50% of the Test Culture Titer; this verifies
effective neutralization. Note: counts higher than the Test Culture Titer
(e.g., 120% of the Test Culture Titer) are also deemed valid.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 16 of 17

Biofilm Neutralization Assay Flowchart - Direct Plating2 (for one dilution of the test
organism)

1 mL

1 mL

1 mL

24±2 h culture, ~108
CFU/mL
(10 mL TSB
inoculated with 10 |iL
FSC)

9 mL
10"1

9 mL

9 mL

lO"2

lO"3

I I

105 CFU/mL

J

106 CFU/mL

V~

Use 0.1 mL of one or both of
these tubes to inoculate each
treatment shown below.

-TREATMENTS-

V V V

Neutralizer
Confirmation
Treatment

V V V

Neutralizer

Toxicity
Treatment

V V V

Test
Culture
Titer

Prepare one 10-fold dilution from each tube

J V

J	I	I	I	I	L

Plate 0.1 mL from each tube; incubate plates for 48±4 h at 36±2°C.

I V

2 For recovery via filtration (see section A2.g), make adjustments to the initial dilution series of the 24±2 h culture
(~108/mL) to achieve a target of 20-200 CFU per filter.


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SOP No. MB-20-04
Date Revised 09-19-22
Page 17 of 17

Attachment 2
Method Photographs

Appropriate location of
splashguard insert (arrow
indicates appropriate
position of bottom of insert
in conical tube)

Gentle swirl of tube with
carrier after addition of 4 mL
disinfectant/control
substance.

Splashguard insert in 50 mL
conical tube

Addition of disinfectant
down the side of the reaction
tube (10° tube).

Desirable rod positioning.

Application of filter to agar
surface (rolling filter onto agar
plate).


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