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 Measuring Disinfectant Efficacy
Against Biofilm Grown in the CDC Biofilm Reactor
SOP Number: MB-20-01
Date Pulished: 08-06-13

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SOP No. MB-20-01
Date Revised 07-31-13
Page 1 of 12
SOP Number
MB-20-01
Title
Single Tube Method for Measuring Disinfectant Efficacy Against
Biofilm Grown in the CDC Biofilm Reactor
Scope
Describes the Single Tube Method (see 15.1) used to determine the
efficacy of disinfectants against Pseudomonas aeruginosa biofilm
grown in the CDC biofilm reactor.
Application
This SOP may be used for additional organisms like S. aureus;
however, the growth and recovery parameters may need to be
adjusted.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:


Date SOP issued:

Controlled copy number:

Date SOP withdrawn:


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SOP No. MB-20-01
Date Revised 07-31-13
Page 2 of 12
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
5
13.
DATA ANALYSIS/CALCULATIONS
8
14.
FORMS AND DATA SHEETS
8
15.
REFERENCES
8

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SOP No. MB-20-01
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1. Definitions
Additional abbreviations/definitions are provided in the text.
1.	CDC = Centers for Disease Control and Prevention
2.	Biofilm = e.g., 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 = materials used to support the growth of biofilm (e.g.,
polycarbonate, borosilicate, stainless steel, etc.)
4.	Residence Time = the time that it takes for the entire volume of the
reactor to exchange once (during continuous flow operation) and is
equal to the inverse of the dilution rate. For example: an operating
volume of 325 mL with a flow rate of 10.8 mL/min has a residence
time of 30 min. Residence time is proportional to the volume and
inversely proportional to the flow rate. In addition, refer to section 12.
5.	Continuous Flow Operation = continuously stirred tank reactor (CSTR)
mode, where growth is broadly controlled by the dilution rate.
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 SOPsEQ-01, EQ-02, EQ-03, EQ-04, EQ-05, andEQ-10 for
details on method and frequency of calibration.
2.	Refer to MB-19 section 4 to confirm the operating volume of the
reactor and for pump calibration using Linkable Instrument Network
software.
5. Sample
Handling and
Storage
Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
6. Quality Control
For quality control purposes, the required information is documented on
the appropriate form(s) (see section 14).
7. Interferences
1. The speed at which the baffled stir bar rotates directly determines the
strength of the shear stress that the biofilm experiences. Biofilm
accumulation on the coupons is sensitive to changes in the baffle's
rotational speed. The baffle rotational speed is a critical factor that

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SOP No. MB-20-01
Date Revised 07-31-13
Page 4 of 12
must be controlled. If baffle speed is not maintained correctly, it may
impact the quality of the biofilm.
2.	Due to the deterioration of the materials, it may be necessary to change
the tubing and filters on the reactor and carboys after 5-6 autoclaving
processes.
3.	Inspect all parts of the reactor system frequently because reuse of worn
parts may cause variability in the data.
4.	Do not place any plastic or rubber pieces of the reactor system under
UV light due to potential degradation of the material.
5.	Overuse of carriers or carriers not prescreened adequately may cause
variability in the 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.	The mean TestLD for carriers inoculated with P. aeruginosa must be at
least 8.0 (corresponding to a geometric mean density of 1.0 x 108); a
mean TestLD below 8.0 invalidates the test.
9. Data
Management
1. Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1. Use appropriately sized secondary containment for contaminated waste
to prevent a biohazard spill.
11. Special
Apparatus and
Materials
1.	Dilution blanks. Standard Method Dilution Water (SMDW). Method
9050 C.lor (0.0425 g/L KH2P04 and 0.405 g/L MgCl2-6H20) steam-
sterilized for 15 min at 120°C (see ref. 15.2).
2.	Vortex. Any vortex that will ensure proper mixing of tubes.
3.	Micropipettes. For making dilutions.
4.	Ultrasonic water bath. Any bath capable of maintaining a
homogeneous sound distribution of 45 kHz with a variable power
setting and a volume large enough to accommodate 50 mL conical
tubes in a wet environment. For removing biofilm from coupons.
5.	Reactor components, carboys, and other associated materials. Refer
to SOP MB-19, section 11.
6.	Recirculating chiller unit and water bath. For maintaining specified
temperature of the test chemical.
7.	Detergent. Micro-90 Concentrated Cleaning Solution for Critical
Cleaning; International Products Corporation. For cleaning coupons

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SOP No. MB-20-01
Date Revised 07-31-13
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and reactor parts.
12. Procedure and
Analysis
This method is used for evaluating the efficacy of liquid disinfectants
against Pseudomonas aeruginosa biofilms. Three randomly selected
coupons are evaluated for efficacy and three are evaluated as controls.
In advance of testing, verify the performance of the neutralizer using the
procedure in Attachment 1.
12.1 Test culture
preparation
a.
Prepare mature P. aeruginosa biofilm per SOP MB-19, sections
12.1 through 12.6.
12.2 Disinfectant
a.
Prepare disinfectant sample per SOP MB-22.
sample
preparation
b.
Equilibrate the water bath and allow it to come to 20 ± 1 °C or the
temperature specified (±1 °C). Use the disinfectant within three
hours of preparation unless test parameters specify otherwise.
Record the time of disinfectant preparation on the Biofilm Single
Tube Method Processing Sheet (see section 14).

c.
Place prepared disinfectant in the equilibrated water bath for
approximately 10 min to allow disinfectant to come to specified
temperature. Record the temperature of the water bath and
recirculating chiller before and after testing on the Biofilm Single
Tube Method Test Information Sheet (see section 14).
12.3 Test procedure
a.
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.
i. Rods are numbered clockwise from 1 -8, beginning with
the rod to the right of the bacteria air vent (located on the
reactor top).

b.
Rinse the coupons to remove planktonic cells.
i.	Orient the rod in a vertical position directly over a 50 mL
conical tube containing 30 mL SMDW.
ii.	Immerse the rod with a continuous motion into the
SMDW with minimal to no splashing, then immediately
remove.
iii.	Use a new 50 mL conical tube with 30 mL SMDW for
each rod.

c.
Hold the rod with one of the randomly selected coupons centered
over an empty, sterile 50 mL conical tube.

d.
Loosen the set screw using a flame-sterilized Allen wrench and

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SOP No. MB-20-01
Date Revised 07-31-13
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allow the coupon to drop directly to the bottom of the tube.
i.	If the coupon does not freely drop, press directly in the
center of the coupon with the Allen wrench used to loosen
the set screw.
ii.	For each treatment or control, repeat coupon removal
twice more for a total of three tubes each containing one
coupon.
iii.	Record the rod number and coupon position on the
Biofilm Single Tube Method Results Sheet for each
coupon used in the study.
Note: Upon transfer, avoid contact of the coupon with the
lip or inner sides of the tube. Discard any tubes where the
coupon touched the inner side of the tube and replace them
with new tubes and coupons.
e.	After removing the appropriate number of coupons, slowly
pipette 4 mL prepared and equilibrated disinfectant (treatment) or
SMDW (untreated control) into the tubes containing the coupons,
being careful to completely cover the coupons. Record the time
on the Biofilm Single Tube Method Time Recording Sheet (see
section 14).
i. For a 10 minute contact time, a 1 minute interval between
coupons is recommended.
f.	Gently tap each tube to release any air bubbles trapped below the
coupon. Do not shake the tubes.
i. To ensure that the maximum biofilm surface area is in
contact with the disinfectant, the coupon should be at an
angle in the bottom of the tube. The gentle tap used to
release any air bubbles can correct the orientation of the
coupon to allow full exposure to the disinfectant.
g.	Place the tubes at 20 ± 1 °C or other specified temperature for the
specified contact time.
h.	At the end of the contact time, add 36 mL of the appropriate
neutralizer (e.g., Dey/Engley (D/E) broth) to each tube. Replace
the cap and mix thoroughly by vigorously shaking the tube
several times. Allow the coupons to remain in the neutralized
disinfectant at room temperature.
i.	After all tubes have been neutralized, vortex each tube on the

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SOP No. MB-20-01
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highest setting for 30 ± 5 s.
j. Place all tubes into a 50 mL conical tube rack and suspend the
rack in the ultrasonic water bath so that the liquid level in the
tubes is even with the liquid level in the tank of the bath.
Sonicate the tubes at 45 kHz for 30 ± 5 s.
k. Vortex the tubes as described in 12.3i.
1. Sonicate the tubes as described in 12.3j.
m. Vortex the tubes as described in 12.3i. These tubes are the 10°
dilution.
n. Serially dilute each 10° dilution in 9 mL blanks of SMDW.
o. Briefly vortex each serial dilution tube prior to plating. Plate 0.1
mL aliquots of appropriate dilutions in duplicate on R2 A using
spread plating. Plate appropriate dilutions to achieve colony
counts in the range of 30-300 colony forming units (CFU) per
plate. Spread inoculum evenly over the surface of the agar.
Plates must be dry prior to incubation.
p. Incubate plates (inverted) at 36 ± 1 °C for 24-48 h.
12.4 Recording
Results
a.	Count colonies. Plates that have colony counts over 300 will be
reported as too numerous to count (TNTC). Record counts on the
Biofilm Single Tube Method Results Sheet (see section 14).
b.	Inspect the growth on the plates for purity and typical
characteristics of the test microbe. Gram stain one representative
colony per carrier set with growth for treated and controls.
Record results on the Biofilm Test Microbe Confirmation Sheet.
Isolation streaks may be performed for additional verification of
the test organism.
i. P. aeruginosa is a Gram negative rod. It may display
three colony types: a) circular, undulate edge, convex,
rough and opaque; b) circular, entire edge, convex,
smooth and translucent; c) irregular, undulate edge,
convex, rough, spreading, and translucent.
12.5 Coupon and
reactor reuse
a.	After use, remove the coupons from each rod and place in an
autoclavable container. Steam-sterilize the reactor, coupons, and
necessary tubing for 30 min.
b.	After sterilization, clean the reactor components with a 1:100
dilution of laboratory soap (e.g., Micro-90 Concentrated Cleaning
Solution) and tap water. After washing, rinse all components

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SOP No. MB-20-01
Date Revised 07-31-13
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with deionized water,
c. Clean and rescreen the coupons per SOP MB-19, section 12.2.
13. Data Analysis/
Calculations
1.	All colony counts are recorded and used in calculations to determine
log reductions.
2.	To calculate the CFU/carrier use the following equation:
(
CFU for 10 r + CFU for 10 ' + CFU for 10"
10 " + 10 ^ + 10 z
c 10x40, where 10" , 10"y and
10"z are the dilution tubes plated, "10" accounts for the volume plated
(0.1 mL) , and "40" is the volume of medium originally in the tube
with the carrier (40 mL).
3.	Calculate the log density of each carrier by taking the logio of the
density (per carrier).
4.	Calculate the mean logio density across treated carriers.
5.	Calculate the mean logio density across control carriers.
6.	Calculate the logio reduction (LR) for treated carriers:
logio reduction = mean logio control - mean logio treated
14. Forms and Data
Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Biofilm Single Tube Method Test Information
&	MB-20-01 Fl.docx
Sheet	-
Biofilm Single Tube Method
Timing/Dilution/Plating Form
MB-20-01 F2.docx
Biofilm Single Tube Method Results Sheet MB-20-01_F3.docx
Biofilm Single Tube Method Processing Sheet MB-20-01_F4.docx
Biofilm Test Microbe Confirmation Sheet MB-20-01 F5.docx
Biofilm Neutralization Test Information Sheet MB-20-01_F6.docx
MB-20-01 F7.docx
Biofilm Neutralization Dilution/Plating
Tracking Form
Biofilm Neutralization Timing Sheet
Biofilm Neutralization Results Sheet
MB-20-01_F8.docx
MB-20-01 F9.docx
15. References
1. ASTM International, 2012. E2871-12: Standard Test Method for
Evaluating Disinfectant Efficacy against Pseudomonas aeruginosa
Biofilm Grown in CDC Reactor using Single Tube Method.

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SOP No. MB-20-01
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2. Standard Methods for the Examination of Water and Wastewater. 21st
Edition. Eaton, A.D., Clesceri L.S., Rice E.W., Greenberg A.E. (Eds.)
2005. American Public Health Association, 1015 15th Street, NW,
Washington, DC.

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SOP No. MB-20-01
Date Revised 07-31-13
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Attachment 1
Biofilm Neutralization Assay
A1. 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 ± 1 °C for 24 ± 2 h.
b.
Prepare serial dilutions in 9 mL blanks of SMDW 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. These dilutions
should result in counts of 30-300 CFU/plate (refer to the Biofilm Neutralization
Assay Flowchart).
A2. Neutralization confirmation assay
a.
Test Culture Titer (TCT). At timed intervals, add 0.1 mL of test organism diluted to
106 CFU/mL to 40 mL SMDW and vortex to mix thoroughly. Repeat with the test
organism diluted to 105 CFU/mL. Proceed with section A2.e.
b.
Neutralizer Toxicity Treatment (NTT). At timed intervals, add 0.1 mL of the test
organism diluted to 106 CFU/mL to 40 mL neutralizer and vortex to mix
thoroughly. Repeat with the test organism diluted to 105 CFU/mL. Proceed with
section A2.e.
c.
Neutralization Confirmation Treatment (NCT). At timed intervals, add 4 mL
disinfectant to 36 mL neutralizer, briefly mix, add 0.1 mL of the test organism
diluted to 106 CFU/mL, and vortex to mix thoroughly. Repeat with the test
organism diluted to 105 CFU/mL. Proceed with section A2.e.
d.
Test Chemical Control (TCC). At timed intervals, add 0.1 mL of the test organism
diluted to 106 CFU/mL to 4 mL disinfectant and vortex to mix thoroughly. Proceed
with section A2.e.
e.
Hold all treatments at room temperature for 10 minutes.
i. For the Test Chemical Control only: after the contact time, add 36 mL
neutralizer to the Test Chemical Control tube and vortex to mix thoroughly.
f.
After the contact time, vortex each tube thoroughly and prepare one 10-fold dilution
in 9 mL SMDW.
g-
Briefly vortex the dilution tube prior to plating. Plate 0.1 mL aliquots from each
tube on R2A using spread plating. Spread inoculum evenly over the surface of the
agar. Plates must be dry prior to incubation.

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SOP No. MB-20-01
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h. Incubate plates (inverted) at 36 ± 1 °C for 24-48 h.
A3. Results
a.	For calculation purposes, use the dilution that resulted in 30-300 CFU/plate.
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 at least 0.5 logs of the Test Culture Titer (see section
A2.a). A count lower than 0.5 logs indicates that the neutralizer is harmful
to the test organism. Note: counts higher than the Test Culture Titer (e.g.,
0.5 logs above the Test Culture Titer) are also deemed valid.
ii.	The recovered number of CFU in the Neutralizer Confirmation Treatment
(see section 12.4c) is within 0.5 logs of the Test Culture Titer; this verifies
effective neutralization. Note: counts higher than the Test Culture Titer
(e.g., 0.5 logs above the Test Culture Titer) are also deemed valid.

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SOP No. MB-20-01
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Biofilm Neutralization Assay Flowchart
1 mL
1 mL
1 mL
(10 mL TSB inoculated
with 10 |iL P.a. FSC)
24 ± 2 h P. a. culture, 9 mL
9 mL
9 mL
~108 CFU/mL 101
10-2
10-3
1 I
106 CFU/mL 105 CFU/mL
V.
J
V
Use each tube to inoculate one
of the two tubes for each
treatment shown below.
--TREATMENTS-
[==] [==]
V V
Test
Culture
Titer
V V
Neutralizer
Toxicity
Treatment
V V
Neutralizer
Confirmation
Treatment

V V
Test
Chemical
Control

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