Standard Operating Procedure for Quantitative Suspension Test Method for Determining Tuberculocidal Efficacy of Disinfectants Against Mycobacterium bovis (BCG) SOP Number: MB-16-02 Date Revised: 10-27-14


US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Quantitative Suspension Test Method for
Determining Tuberculocidal Efficacy of
Disinfectants Against Mycobacterium bovis (BCG)
SOP Number: MB-16-02
Date Revised: 10-27-14

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SOP No. MB-16-02
Date Revised 10-27-14
Page 1 of 12
SOP Number
MB-16-02
Title
Quantitative Suspension Test Method for Determining
Tuberculocidal Efficacy of Disinfectants Against Mycobacterium
bovis (BCG)
Scope
This SOP describes the methodology used to determine the efficacy
of disinfectants against Mycobacterium bovis (BCG) in suspension.
This SOP is based on references 15.1 and 15.2.
Application
Use of this SOP is limited to disinfectants with certain active
ingredients (e.g., glutaraldehyde).


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Date SOP withdrawn: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-16-02
Date Revised 10-27-14
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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
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
10

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1. Definitions
Additional abbreviations/definitions are provided in the text.
1.	QSTM = Quantitative Suspension Test Method
2.	CFU = Colony Forming Unit
3.	MPB/Tween = Modified Proskauer Beck Medium with 0.1% (v/v) Tween
80
2. Health and
Safety
1.	Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Safety Data Sheets
for specific hazards associated with products.
2.	All manipulations of the test organism are required to be performed in
accordance with biosafety practices stipulated in the SOP MB-01, Lab
Biosafety. All M. bo vis (BCG) manipulations are performed in a biosafety
level 3 isolation laboratory (i.e., room B202 or room B207).
3. Personnel
Qualifications
and Training
1. Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
1. Refer to SOP EQ-02 (thermometers) and EQ-04 (spectrophotometers) for
details on method and frequency of calibration.
5. Sample
Handling and
Storage
1. Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01,
Chain of Custody Procedures.
6. Quality Control
1. For quality control purposes, the required information is documented on
the appropriate form(s) (see section 14).
7. Interferences
1. Filters with colonies greater than -30 CFUs can be difficult to count.
Check filters regularly. Count filters with >30 CFUs frequently (e.g.,
every other day) once growth is observed by indicating colonies with a
marker on the lid of the Petri plate. At the end of the incubation period,
record total counts on the appropriate form (see section 14).
8. Non-
conforming
Data
1. Management of non-conforming data will be specified; procedures will be
consistent with SOP ADM-07, Non-Conformance Reports.
9. Data
Management
1. Data will be archived consistent with SOP ADM-03, Records and
Archives.
10. Cautions
1.	To ensure the stability of the disinfectant, perform testing within 3 hours
of preparation.
2.	Strict adherence to the procedure is necessary for valid test results.
3.	Use appropriate aseptic techniques for all test procedures involving the

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SOP No. MB-16-02
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manipulation of test organisms and associated test components.
11. Special
Apparatus and
Materials
1.	Filter Units: Nalgene Sterile Analytical Filter Units (0.45 |im pore size)
cat. no. 130-4045, 47mm diameter 0.45 |im filter membranes with
appropriate filtration apparatus, or equivalent.
2.	15 mL glass tissue grinders with glass pestles (Wheaton and/or Kontes)
3.	Spectrophotometer (Beckman DU Series 730 or equivalent)
4.	Colony Counter
12. Procedure and
Analysis
TABLE 1. Test Culture Preparation Summary
QSTM Test Culture Preparation
Step
Description*
Culture
Notation5
1. Monthly Stock M7H9
or M7H11 Slant used to
inoculate several tubes
of MPB (Sect. 12.1b)
Solid ^LlQUldstationary InCubate
inoculated tubes in a slanted, stationary
position until a pellicle forms
-QSTM-01
2. Use pellicle from Step
1 to inoculate several
tubes of MPB/Tween
(Sect. 12. Id)
LiqUldstationary ~LlQUldstationary ~
Incubate the inoculated tubes of
MPB/Tween upright in a stationary
position until turbid
-QSTM-02
3. Use stationary
MPB/Tween culture to
inoculate flask of
MPB/Tween (Sect.
12.If)
Liqilidstationary ^Li(Jllidaerated — Use 5
mL of the stationary MPB/Tween
culture to inoculate 50 mL flask of
MPB/Tween, incubate flasks on orbital
shaker (-150 rpm) for 5-7 days
-QSTM-03
4. Use aerated
MPB/Tween culture to
inoculate flask of
MPB/Tween (Sect.
12. lh)
Liqilidaerated > F1 C| 111daerated ~ Use 10 Or
15 mL of the aerated MPB/Tween
culture to inoculate 100 or 150 mL
flasks of MPB/Tween, incubate flasks
on orbital shaker (-150 rpm) until
OD500 is —0.6
-QSTM-04
5. Add Tween 80 to
culture -QSTM-04 (Sect.
12. lj)
One day prior to harvesting the aerated
flask culture from step 4 (-QSTM-04),
add Tween 80 (1 mL per liter of
culture)
N/A
6. Culture Harvest (Sect.
12.1k)
Harvest cells by homogenization in a
tissue grinder when OD500 is -0.6
N/A
7. Frozen Test Culture
(Sect. 12. In)
Dispense pooled homogenized culture
into cryovials and freeze at < -70°C
-QSTM-FTC

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*A11 incubations performed at 36±1°C
§ Culture notations should be added to the "Comments" section of the Organism Culture
Tracking Form for Mycobacterium bovis (BCG)
12.1 Frozen Test
Culture
Preparation
a.	Record all transfers and manipulations on the Organism Culture
Tracking Form for Mycobacterium bovis (BCG) (see section 14).
b.	Inoculate several 20 mL tubes of Modified Proskauer-Beck (MPB)
medium with Mycobacterium bovis (BCG) from a stock Middlebrook
7H9 (M7H9) or Mycobacteria 7H11 (M7H11) slant culture (see SOP
MB-07, Tuberculocidal Activity of Disinfectants Test).
c.	Incubate in a slanted position at 36±1°C until a pellicle forms
(approximately 19-23 days).
d.	Transfer a loopful of pellicle onto the surface of several 20 mL tubes
of MPB/Tween 80.
e.	Incubate stationary at 36±1°C until cultures are turbid. Cultures will
require agitation (by gentle shaking/vortexing) to assess turbidity.
f.	Transfer 5 mL of a stationary culture to 50 mL of MPB/Tween 80 in
a 250 mL flask.
g.	Incubate for 5-7 days at 36±1°C with aeration (on a shaker at slow
speed, approximately 150 rpm).
h.	Transfer 10 mL of the aerated culture to 100 mL of MPB/Tween 80
in a 500 mL flask. Alternately: Transfer 15 mL of the aerated
culture to 150 mL of MPB/Tween 80 in a 500 mL flask.
i.	Incubate for 10-15 days at 36±1°C with aeration (on a shaker 150
rpm) OR until the absorbance at 500 nm is about 0.6 (target stock
culture titer: -1-5 x 108 CFU/mL).
j. One day prior to harvesting, add Tween 80 to the culture (1 mL per L
of culture).
k. Harvest cells when absorbance at 500 nm is approximately 0.6.
1. Homogenize 10-20 mL aliquots in a tissue grinder.
m. Pool homogenized culture.
n. Dispense 1-2 mL aliquots of the homogenized suspension into
cryotubes.
o. Place in cryostorage at < -70°C. Check the concentration of viable
cells in the suspension by plating dilutions of the stock on M7H11

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SOP No. MB-16-02
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agar or M7H9 agar plates both before and after freezing. Check the
frozen test culture stock by acid-fast staining and record results.
12.2 Suspension
Test Culture
Preparation
a.	To prepare the suspension ofM bo vis (BCG), remove the necessary
number of vials of frozen stock culture and place on ice prior to
thawing.
b.	Quickly thaw the frozen vials in a 36±1°C water bath then place the
thawed vials back on ice. A vial of-1.8 mL of frozen test culture
requires -90-120 seconds to thaw completely. Details of the thawing
process should be recorded on the QSTM: Processing Sheet (see
section 14).
c.	Add an equal volume of buffered gelatin to the suspension and
homogenize with a sterile tissue grinder for 1 minute while keeping
the culture at 0-4°C in an ice bath.
d.	Dilute the homogenate with sterile saline plus 0.1% Tween 80 to
achieve the target density of approximately 1-5><107 CFU/mL.
e.	If organic soil is specified in the test parameters for the product test,
measure the culture and add the appropriate volume of soil to the
diluted homogenate. Swirl to mix.
12.3 Disinfectant
Sample
Preparation
a.	Turn on the recirculating chiller and allow the temperature of the
chiller unit and the test tube water bath to equilibrate to the
appropriate temperature (e.g., 20±1°C). Record the temperatures on
the QSTM: Information Sheet (see section 14).
b.	Follow chain of custody guidelines for disinfectant samples as
stipulated in SOP COC-01, Sample Login and Tracking and
disinfectant preparation guidelines as stipulated in MB-22.
c.	After preparation, dispense 9 mL aliquots of the disinfectant into
sterile 20x150 mm tubes.
12.4 Test Procedure
a. Suspension Test Procedure (see Attachment 1, Study Design for
QSTM Disinfectant Efficacy Evaluation):
i.	Allow four (one tube per replicate) 20x 150 mm tubes each
containing 9.0 mL of disinfectant to equilibrate for 10
minutes at 20±1°C in a water bath.
ii.	In a timed step, add 1.0 mL of the test culture to each tube of
disinfectant, and lightly vortex. Four replicates are necessary;
thus, this step will be repeated four separate times.
iii.	Following the specified exposure period, remove a 1.0 mL
aliquot of the disinfectant-organism mixture and transfer

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SOP No. MB-16-02
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Page 7 of 12
directly to a 9.0 mL tube of neutralizer (the 10° dilution
designated Tube A) and mix thoroughly.
iv.	Within 5 minutes of the transfer to the neutralizer tube, make
two additional ten-fold dilutions in saline blanks to achieve
10"1 and 10"2 dilutions (designated Tube B and Tube C
respectively); mix thoroughly between dilutions.
v.	Filter the three dilutions (tubes A, B, and C) separately. .
Pre-wet each filter with -20 mL saline and add 1 mL from
Tube A (10° dilution). Briefly swirl and filter. Rinse each
filter with -50 mL saline.
vi.	Repeat for Tube B (10"1) and Tube C (10"2).
vii.	Place each filter on the surface of an M7H11 agar plate.
Incubate at 36±1°C for 17-21 days (bag or parafilm plates to
prevent desiccation).
b. Enumeration of Inoculum (see Attachment 2, Study Design for
QSTM Culture Titer and Controls):
i.	Transfer 1.0 mL of the test culture (with soil if specified) to a
9.0 mL saline blank and vortex.
ii.	Serially dilute in saline: 10"1 through 10"7.
iii.	Pre-wet each filter with -20 mL saline. Filter 1.0 mL aliquots
of 10"5 through 10"7 dilutions in duplicate (6 total filters).
iv.	Briefly swirl and filter. Rinse each filter with -50 mL
saline.
v.	Place each filter on the surface of an M7H11 agar plate.
Incubate at 36±1°C for 17-21 days (bag or parafilm plates to
prevent desiccation).
Static Control: The Static Control is designed to confirm the
neutralization of the test substance (see Attachment 2, Experimental
Design for QSTM Culture Titer and Controls).
i.	Allow 0.9 mL of disinfectant to come to the specified test
temperature in a water bath.
ii.	Add 9.0 mL of neutralizer and mix by vortexing.
iii.	After 5 min, add 0.1 mL of the test culture and mix by
vortexing.
iv.	Serially dilute in saline: 10"1 through 10"5.
12.5 Quality
Control

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v. Filter dilutions 10"3 through 10"5 in duplicate as indicated in
Sections 12.4b, iii - 12.4b, v (6 filters total).
b.	Neutralizer Toxicity Control: The Neutralizer Toxicity Control must
demonstrate that the neutralizer does not impact the recovery of the
test organism (see Attachment 2, Experimental Design for QSTM
Culture Titer and Controls).
i.	Add 1.0 mL of the standardized test culture to a tube
containing 9.0 mL of saline at room temperature.
ii.	Remove 1.0 mL (of the saline/test culture mixture) and add to
a tube containing 9.0 mL neutralizer and mix.
iii.	After a 5 min contact period, serially dilute in saline 10"1
through 10"5.
c.	Filter dilutions 10"3 through 10"5 in duplicate as indicated in sections
12.4b, iii - 12.4b, v (6 filters total).
12.6 Reading Filters
and Recording
Results
a.	Examine filters after approximately 10 days and frequently thereafter
(see section 7). Record results after 17-21 days of incubation.
b.	Colonies appear initially as small buff colored accretions with
irregular borders. Record colony counts at the end of the incubation
period on appropriate test sheets.
12.7 Confirmation
Procedures and
Presumptive
Identification
of M bovis
(BCG)
a.	Presumptively confirm the identification of M bovis (BCG) using
acid fast staining and plating on selective media (e.g., M7H11).
b.	Take a smear for acid fast staining from a representative colony from
selected filters with growth on the day that final results are recorded.
For each set of filters from the Product Test, Enumeration of
Inoculum, Static Control, and Neutralizer Toxicity Control, choose
the filter with growth from the highest dilution (i.e., the smallest
number of colonies).
c.	Acid fast rods are typical forM bovis (BCG).
d.	In addition, streak the representative growth from the colony that was
used for Acid Fast staining over the surface of an M7H11 agar plate
and incubate for 17-21 days at 36±1°C.
e.	Following the incubation period, evaluate and record the colony
morphology of the organism on M7H11 agar. M. bovis (BCG)
typically appears as colorless to buff-colored, raised, rough growth
on M7H11 agar.
f.	Record results on the Test Microbe Confirmation Sheet (see section

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14)
13. Data Analysis/
Calculations
1.	See section 14, QSTM: Calculations Worksheet.
a.	The test substance must demonstrate > 1 .Ox 104 CFU kill of the test
organism at the stated contact time (i.e., a > 4 logio reduction of test
organism).
b.	The Static Control should demonstrate that the neutralizer adequately
neutralized the test substance (i.e., < 1 logio difference between the
Static Control and the Neutralizer Toxicity Control).
c.	The Neutralizer Toxicity Control must demonstrate that the
neutralizer does not impact the recovery of test organism (i.e., < 1
logio difference between the Neutralizer Toxicity Control and the
Organism Titer).
2.	The Organism Titer must be > 1 x 107 CFU/mL.
3.	When TNTC values are observed for each dilution filtered, substitute 200
for the TNTC at the highest (most dilute) dilution and scale up accordingly
for the calculations.
14. Forms and Data
Sheets
1. Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Attachment 1 Study Design for QSTM Efficacy JVIB16 02 A1 docx
Evaluation -
Attachment 2 Study Design for QSTM Culture , , , _ . „ .
, „ A , 5 v MB-16-02 A2.docx
Titer and Controls -
QSTM: Test Information Sheet MB-16-02_Fl.docx
QSTM: Time Recording MB-16-02 F2.docx
QSTM: Efficacy Evaluation Results Form MB-16-02 F3.docx
QSTM: Test Suspension Titer Form MB-16-02 F4.docx
QSTM: Static Control Form MB-16-02 F5.docx
QSTM: Neutralizer Toxicity Control Form MB-16-02 F6.docx
QSTM: Test Microbe Confirmation Sheet MB-16-02 F7.docx
QSTM: Processing Sheet MB-16-02 F8.docx
QSTM: Calculations Spreadsheet MB-16-02_F9.xlsx

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SOP No. MB-16-02
Date Revised 10-27-14
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1.	New Quantitative Tuberculocidal Procedure - Attachment C of USEPA
Data Call-in Notice for Tuberculocidal Effectiveness Data for all
Antimicrobial Pesticides with Tuberculocidal Claims, dated June 13,
1986.
2.	A More Accurate Method for Measurement of Tuberculocidal Activity of
Disinfectants (Ascenzi, J.M., et. al., Applied Environmental Microbiology,
Vol. 53, No. 9, 1987, pp.2189-2192).

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SOP No. MB-16-02
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Attachment 1
Study Design for QSTM Disinfectant Efficacy Evaluation
Buffered Gelatin
-80°C Ampules
M. bovis (BCG)
Culture
Grind on ice 1 minute
TEST CULTURE
1 mL
1 mL	1 mL	1 mL
1 mL	1 mL
1 mL
9 mL
9 mL
9 mL
9 mL
9 mL
9 mL
9 mL
10"1
lO"2
lO"3
104
10"5
10"6
lO"7
ill
Incubate plates @ 36±1°C
1 mL
9 mL
Germicide
T=0
1 mL
9 mL
1 mL
T=X
Neutralizer
1 mL
-Tube A TubeB-
10°
Incubate plates @ 36±1°C
¦TubeC
9 mL	9 mL
10"1	10"2
I 1
> ONE REPLICATE: The timer is
started when the test culture and
germicide are combined (in a water
bath at the specified temperature). At
Time X, the specified contact time, 1
mL of germicide/test culture is
removed and added to 9 mL of
neutralizer. The neutralized
suspension is diluted, filtered, and
plated as indicated. This scenario
represents one test replicate. The test
is repeated for a total of four
replicates.

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SOP No. MB-16-02
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Attachment 2 Study Design for QSTM Culture Titer and Controls
Buffered Gelatin
-80°C Ampules
M. bovis (BCG)
Culture
1 mL	1 mL	1 mL	1 mL	1 mL	1 mL
Grind on ice 1 minute
TEST CULTURE
1 mL
9 mL
9 mL
9 mL
9 mL
9 mL
9 mL
9 mL
10"1
lO"2
lO"3
104
10"5
10"6
10"7
ill
Incubate plates @ 36±1°C
1 mL
9 mL
Germicide
T=0
1 mL
9 mL
1 mL
T=X
Neutralizer
1 mL
¦Tube A TubeB-
10°
9 mL
9 mL
10"1
lO"2
| I
Incubate plates @ 36±1°C
-TubeC
> ONE REPLICATE: The timer is
started when the test culture and
germicide are combined (in a water
bath at the specified temperature). At
Time X, the specified contact time, 1
mL of germicide/test culture is
removed and added to 9 mL of
neutralizer. The neutralized
suspension is diluted, filtered, and
plated as indicated. This scenario
represents one test replicate. The test
is repeated for a total of four
replicates.

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