Standard Operating Procedure for Neutralization Confirmation Assay for Disinfectant Products Tested against Mycobacterium bovis (BCG)



US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Neutralization Confirmation Assay for Disinfectant
Products Tested against Mycobacterium bovis (BCG)
SOP Number: MB-11-05
Date Revised: 06-20-16

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 1 of 11
SOP Number
MB-11-05
Title
Neutralization Confirmation Assay for Disinfectant Products Tested
against Mycobacterium bovis (BCG)
Scope
Describes the methodology for determining the effectiveness of a
neutralizer used when testing the tuberculocidal activity of
disinfectants against Mycobacterium bovis (BCG) on hard surfaces
using liquid, sprays, or towelettes.
Application
For official product testing, a study protocol is developed which
identifies the specific test conditions for a product sample such as
contact time, dilutions, neutralizers, etc.


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Date SOP withdrawn:

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 2 of 11
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
3
9.
DATA MANAGEMENT
3
10.
CAUTIONS
3
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
4
13.
DATA ANALYSIS/CALCULATIONS
9
14.
FORMS AND DATA SHEETS
9
15.
REFERENCES
9

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 3 of 11
1. Definitions
Additional abbreviations/definitions are provided in the text.
1.	"Exposed" Neutralizer: Neutralizer solution which has been used to
inactivate the product.
2.	"Unexposed" Neutralizer: Neutralizer solution which has not been
used to inactivate the product.
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
Refer to SOPs EQ-01 (pH meters), EQ-02 (thermometers), EQ-03 (weigh
balances), EQ-04 (spectrophotometers) and EQ-05 (timers) for details on
method and frequency of calibration.
5. Sample
Handling and
Storage
Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
6. Quality Control
Appropriate quality control measures are integrated into each SOP. For
quality control purposes, the required information is documented on the
appropriate forms (see section 14).
7. Interferences
1.	An interaction between a product's active ingredient(s) and the
neutralizer can interfere with the reading of results.
2.	Presence of contamination will interfere with the interpretation of
results and may necessitate repeat analysis.
8. Non-
conforming
Data
1.	Management of non-conforming data will be consistent with SOP
ADM-07, Non-Conformance Reports.
2.	Media performance (Subculture Media Control) must be acceptable in
order to interpret the neutralization results.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1.	The lack of complete neutralization of the disinfectant or bacteriostatic
activity of the neutralizer itself may be masked when a high level of M.
bovis (BCG) is added to the subculture tubes.
2.	There are time sensitive steps in this procedure including the use-
period of the test chemical.
3.	Verify the volume of dilution blanks, neutralizer tubes, and subculture

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 4 of 11

tubes in advance and adjust accordingly.
4. Interactions between the media and neutralizer may result in growth in
some media and not others; if this occurs, a more suitable neutralizer
should be selected.
11. Special
Apparatus and
Materials
1. Refer to section 11 (Special Apparatus and Materials) of MB-07, MB-
23, and MB-24 depending on the type of carrier used in the
neutralization assay.
12. Procedure and
Analysis
1.	For an overview of the setup of this procedure, see Tables 1 and 2.
2.	Sterile carriers are used for this assay. Sterile growth medium with or
without soil load is applied to the test carriers (via carrier submersion
or application using a pipette) in advance of testing to simulate
inoculation.
3.	Perform the neutralization assay in advance of or concurrently with
product testing to verify that the prescribed neutralizer is suitable for
the efficacy evaluation. It is preferable to use the same preparation of
media for conducting the neutralization and efficacy assays.
4.	The general procedure for conducting the assay is the same for liquid,
spray, and towelette products. Follow the test parameters specified for
product testing (e.g., H2O hardness, use dilution, soil load, neutralizer,
contact time, temperature) for the neutralization confirmation assay.
5.	The Neutralization Confirmation Assay for M bovis (BCG):
Processing Sheet (see section 14) must be used for tracking testing
activities.
12.1 Preparation of
Inoculum
a. Prepare standardized inoculum per section 12.1 or 12.2 of MB-
07, MB-23, or MB-24.
12.2 Preparation of
inoculum
dilutions and
enumeration
a.	Prepare serial ten-fold dilutions of the standardized inoculum in 9
mL of Modified Proskauer Beck medium (MPB) or phosphate
buffered dilution water (PBDW). Use four dilutions (e.g., 10"2,
10"3, 10"4, and 10"5) to inoculate the subculture media described in
sections 12.5-12.7. The target number of cells is 5-100 CFU/mL;
this level should be seen in one of the two highest dilutions.
b.	To estimate CFU/mL, plate 0.1 mL aliquots of each of the four
dilutions in duplicate on M7H11 agar using spread plating.
Briefly mix each serial dilution tube prior to plating. Spread
inoculum evenly over the surface of the agar. Plates must be dry
prior to incubation.

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 5 of 11

c.	Record the dilution and plating information on the Neutralization
Confirmation Assay for M bovis (BCG): Inoculum Enumeration
Form (see section 14).
d.	Incubate plates (inverted) concurrently with the neutralization test
subculture tubes at 36±1°C for 17-21 days. An evaluation prior
to 17 days may be made for plates with high colony counts.
e.	Count colonies. Plates that have colony counts over 300 CFU
will be reported as TNTC. Record the counts on the
Neutralization Confirmation Assay for M bovis (BCG):
Inoculum Enumeration Form (see section 14).
12.3 Product
Sample
Preparation
a. Prepare the product according to the test parameters; follow
guidelines for disinfectant sample preparation provided in SOP
MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
12.4 Carrier
Preparation
a.	Use carrier type required for the specific test.
b.	Application of sterile medium with or without soil load.
i.	For CTB: Add 10 sterile carriers to a tube containing 15-
20 mL of either MPB or Middlebrook 7H9 broth with
0.1% (v/v) polysorbate 80 (M7H9/P80); use the same
sterile medium used to grow the test culture. Add soil
load to the sterile medium as necessary per the test
parameters.
ii.	For GSPT: Using a calibrated positive displacement
pipette, transfer 10 |iL of either MPB or M7H9/P80 onto
approximately 1 square inch of the sterile test carrier in
the Petri dish; use the same sterile medium used to grow
the test culture. Add soil load to the sterile medium as
necessary per the test parameters. Immediately spread the
medium uniformly over the majority of the carrier surface
using a sterile loop. Cover dish immediately. Prepare at
least 10 carriers.
iii.	For DTT: Using a calibrated positive displacement
pipette, transfer 10 |iL of either MPB or M7H9/P80 onto
the sterile test carrier in the Petri dish at one end of the
slide; use the same sterile medium used to grow the test
culture. Add soil load to the sterile medium as necessary
per the test parameters. Immediately spread the medium
uniformly over one third of the carrier surface using a

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 6 of 11

sterile loop. Cover dish immediately. Prepare at least 10
carriers.
c. Dry carriers in incubator at 36±1°C for 30±2 min.
12.5 Subculture
Media +
Exposed
Neutralizer
Treatment
a.	Requires four dried carriers (with medium added). Each carrier
will be associated with one dilution of inoculum.
b.	Apply the product to the carriers according to the efficacy method
SOP and the specific instructions provided in the test parameters.
Record the carrier transfer information on the Neutralization
Confirmation Assay for M. bovis (BCG): Time Recording Sheet
for Carrier Transfers (see section 14).
c.	After the last carrier of a set (4 total carriers) has been treated
with the disinfectant and the contact time is complete, aseptically
transfer carriers in order in a timed fashion into tubes containing
the specified neutralizer, in the same manner as product efficacy
testing. Drain excess liquid from the carrier prior to the transfer.
Note: For spray and towelette products, use 20 mL neutralizer
per 38 x 100 mm tube. For liquid products use 10 mL neutralizer
per 25 x 100 mm tube.
d.	Shake the tube containing the carrier in neutralizer thoroughly
and transfer the carrier to the primary subculture medium tube
containing 20 mL MPB broth within 5-10 minutes.
e.	For liquid products, sequentially transfer 2 mL aliquots from each
of the four neutralizer tubes into one tube of each of the 2
additional subculture media (M7H9, K or TB) specified by the
test parameters. Do not add neutralizer to the MPB tube. This
portion of the assay is not timed, but the aliquots should be
transferred to the subculture media within approximately 30
minutes. Inoculate one tube of each medium with one of the four
inoculum dilutions prepared in section 12.2.
f.	For spray and towelette products, sequentially transfer 2 mL
aliquots from each of the four neutralizer tubes into two tubes of
each of the 2 additional subculture media (M7H9, K or TB)
specified by the test parameters. Inoculate two tubes of each
medium with one of the four inoculum dilutions prepared in
section 12.2.
12.6 Subculture
Media +
Unexposed
a. Requires four dried carriers (with medium added). Each carrier
will be associated with one dilution of inoculum.

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 7 of 11
Neutralizer
Treatment
b.	Expose four of the carriers to neutralizer, one carrier per tube of
neutralizer (this portion of the assay is not timed).
c.	Shake the tube containing the carrier in neutralizer thoroughly
and transfer the carrier to the primary subculture medium tube
containing 20 mL MPB broth within 5-10 minutes.
d.	For liquid products, sequentially transfer 2 mL aliquots from each
of the four neutralizer tubes into one tube of each of the 2
additional subculture media (M7H9, K or TB) specified by the
test parameters. Do not add neutralizer to the MPB tube. This
portion of the assay is not timed, but the aliquots should be
transferred to the subculture media within approximately 30
minutes. Inoculate one tube of each subculture media with one of
the four inoculum dilutions prepared in section 12.2.
e.	For spray and towelette products, transfer 2 mL aliquots from
each of the four neutralizer tubes into two tubes of each of the 2
additional subculture media (M7H9, K or TB). Inoculate two
tubes of each subculture media with one of the four inoculum
dilutions prepared in section 12.2.
12.7 Subculture
Media
Controls
a.	Subculture Media Control. This control contains four tubes of
each preparation of subculture media used in the neutralization
assay. Do not add neutralizer to the media. Inoculate each tube
of each medium with one of the four inoculum dilutions prepared
in section 12.2.
b.	Negative (uninoculated) Media Control. Incubate one
uninoculated tube of each medium along with the assay.
12.8 Inoculating
Subculture
Media
a. Within 30 minutes after all carriers and neutralizer aliquots have
been transferred, inoculate the Subculture Media + Exposed
Neutralizer treatment, the Subculture Media + Unexposed
Neutralizer treatment, and the Subculture Media Control with
0.1 mL of the diluted M bo vis (BCG) inoculum (dilution tubes
10"2 through 10"5), beginning with the least concentrated dilution.
12.9 Incubation and
Presumptive
Confirmation
Testing
a.	Incubate tubes at 36±1°C for up to 60 days; tubes may be
monitored for growth prior to 60 days.
b.	Record results between 45-60 days as positive (+) or negative (0)
as indicated by the presence or absence of growth.
c.	For each medium in the Subculture Media + Exposed
Neutralizer treatment, the Subculture Media + Unexposed
Neutralizer treatment, and the Subculture Media Control,

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 8 of 11

select the tube with growth from the highest dilution of inoculum
(i.e., fewest CFU/mL delivered) and perform acid fast staining on
a sample of the growth. Acid fast rods are typical forM bo vis
(BCG).
d.	If necessary, conduct additional confirmation to include isolation
streaks on selective media such as M7H11 agar plates. Following
the 17-21 day incubation period, evaluate the colony morphology
of the organism on M7H11 agar. On M7H11 agar, M. bovis
(BCG) typically appears as colorless to buff-colored, raised,
rough growth.
e.	Record confirmation results on the Neutralization Confirmation
Assay forM bovis (BCG): Test Microbe Confirmation Sheet (see
section 14).
12.1 ©Interpretation
of Results
a.	Plate count data. One of the four dilutions elated should provide
counts within the target range, 5-100 CFU/mL.
b.	Growth in the Subculture Media Control (media performance)
verifies the presence of the test microbe, performance of the
media, and provides a basis for comparing growth in the
subculture tubes to growth in the Subculture Media + Exposed
Neutralizer treatment and the Subculture Media + Unexposed
Neutralizer treatment.
c.	The occurrence of growth in the Subculture Media +
Unexposed Neutralizer treatment as compared to the
Subculture Media Control tubes is used to assess any
bacteriostatic effects attributable to possible interactions between
the neutralizer and subculture media.
d.	The occurrence of growth in the Subculture Media + Exposed
Neutralizer treatment tubes as compared to the Subculture
Media Control tubes is used to determine the effectiveness of the
neutralizer to inactivate the disinfectant when used under
simulated test conditions.
e.	Verification of neutralizer effectiveness.
i.	Growth in tubes that were inoculated with dilutions
yielding plate counts ranging from TNTC to 5-100 CFU;
growth in at least one tube receiving a desired target of 5-
100 CFU is required.
ii.	Growth in the Subculture Media + Unexposed
Neutralizer treatment and Subculture Media + Exposed

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 9 of 11
Neutralizer treatment should be comparable to the
Subculture Media Control.
f.	Lack of verification of neutralizer effectiveness.
i.	Growth in tubes that were inoculated with dilutions
yielding plate counts of TNTC and no growth in tubes that
were inoculated with dilutions yielding plate counts with a
desired target of 5-100 CFU.
ii.	No growth in any tubes.
g.	For the data to be deemed valid, each Negative (uninoculated)
Media Control tube must show no microbial growth.
13. Data Analysis/
Calculations
None.
14. Forms and Data
Sheets
Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Neutralization Confirmation Assay for M
bovis (BCG): Time Recording Sheet for
Carrier Transfers
MB-11-05 Fl.docx
Neutralization Confirmation Assay for M
bovis (BCG): Information Sheet
Neutralization Confirmation Assay for M
bovis (BCG): Results Sheet - Liquid Products
Neutralization Confirmation Assay for M
bovis (BCG): Results Sheet - Spray/Towelette
Products
Neutralization Confirmation Assay for M
bovis (BCG): Inoculum Enumeration Form
Neutralization Confirmation Assay for M
bovis (BCG): Test Microbe Confirmation
Sheet
Neutralization Confirmation Assay for M
bovis (BCG): Processing Sheet
MB-11-05 F2.docx
MB-11-05 F3.docx
MB-11-05 F4.docx
MB-11-05 F5.docx
MB-11-05 F6.docx
MB-11-05 F7.docx
15. References
Official Methods of Analysis. Revised 2013. 18th Ed., AO AC
INTERNATIONAL, Gaithersburg, MD, (Method 965.12 In vitro Test for
Determining Tuberculocidal Activity).

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 10 of 11
Table 1: Components of the Neutralization Confirmation Assay for Liquid
Products
1 iv;ilmciil/( 'oni nil
M. Iwvis (lii ii)
1 nociiIn in Dilution
(0.1 ml. ;iri(k'(l per in ho)
Modi;i (O = TiiIr1 of Mi'di.i)
M 1*15
(20 ml.)
\ni«>
(20 ml.)
km- Tli
(20 ml.)
Subculture Media +
Exposed Neutralizer
lO"2
O/Carrier
O
O
lO"3
O/Carrier
O
O
10"4
O/Carrier
o
o
10"5
O/Carrier
o
o
Subculture Media +
Unexposed Neutralizer
10-2
O/Carrier
o
o
10"3
O/Carrier
o
o
10"4
O/Carrier
o
o
10"5
O/Carrier
o
o
Subculture Media Control
10-2
O
o
o
10-3
O
o
o
10"4
O
o
o
10"5
O
o
o
Negative Uninoculaled
Control
Not inoculated
O
o
o

-------
SOP No. MB-11-05
Date Revised 06-20-16
Page 11 of 11
Table 2: Components of the Neutralization Confirmation Assay for
Spray/To welette Products
1 iv;ilmciil/( 'oni nil
M. Iwvis (lii ii)
1 nociiIn in Dilution
(0.1 ml. ;iri(k'(l per in ho)
Modi;i (O = TiiIr1 ol' Mi'di.i)
M 1*15
(20 ml.)
\ni«)
(20 ml.)
k or Tli
(20 ml.)
Subculture Media +
Exposed Neutralizer
lO"2
O/Carrier
OO
OO
lO"3
O/Carrier
oo
OO
10"4
O/Carrier
OO
oo
10"5
O/Carrier
oo
oo
Subculture Media +
Unexposed Neutralizer
to-2
O/Carrier
oo
oo
10"3
O/Carrier
oo
oo
10"4
O/Carrier
oo
oo
10"5
O/Carrier
oo
oo
Subculture Media Control
10-2
O
o
o
10-3
O
o
o
10"4
O
o
o
10"5
O
o
o
Negative Uninoculalcd
Control
Not inoculated
O
o
o

-------