US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Disinfectant Towelette Test: Testing of Mycobacterium
bovis (BCG)
SOP Number: MB-23-04
Date Revised: 05-21-19

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SOP No. MB-23-04
Date Revised 05-21-19
Page i of 17
SOP Number
MB-23-04
Title
Disinfectant Towelette Test: Testing of Mycobacterium bovis (BCG)
Revisions Made
	Clarifications made to Section 11 (Special Apparatus and
Materials).
	Clarifications made to Section 12.4 (Enumeration of viable
bacteria from carriers (control carrier counts)).
	References updated.
	Minor editorial changes.

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SOP No. MB-23-04
Date Revised 05-21-19
Page 1 of 17
SOP Number
MB-23-04
Title
Disinfectant Towelette Test: Testing of Mycobacterium bovis (BCG)
Scope
Describes the methodology to determine tuberculocidal activity of
towelette-based disinfectants labeled to treat hard non-porous
surfaces against Mycobacterium bovis (BCG). The test is based on
AO AC Method 961.02 (Germicidal Spray Products as
Disinfectants), see 15.1.
Application
For official product testing, a study protocol is developed which
identifies the specific test conditions for a product sample such as
contact time, 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Date SOP withdrawn:


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SOP No. MB-23-04
Date Revised 05-21-19
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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
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
7
13.
DATA ANALYSIS/CALCULATIONS
13
14.
FORMS AND DATA SHEETS
13
15.
REFERENCES
14

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SOP No. MB-23-04
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1. Definitions
Additional abbreviations/definitions are provided in the text.
Carrier Set = One "carrier set" is defined as the primary MPB tube
containing the carrier and duplicate tubes of the two additional subculture
media (e.g., M7H9 broth, Kirchner's medium, or TB broth) inoculated from
the carrier's corresponding neutralizer tube for a total of 5 tubes per carrier.
There are 10 carrier sets per disinfectant tested.
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), EQ-05 (timers), and QC-19
(pipettes) 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
For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).
7. Interferences
1.	The carriers inside the Petri dishes should be dry prior to inoculation.
Moisture can interfere with the concentration and drying of the
inoculum on the glass slide carrier.
2.	Do not use any inoculated carrier that is wet at the conclusion of the
carrier drying period.
8. Non-
conforming
Data
1.	An assessment of media quality (performance) is necessary to ensure
the validity of the tuberculocidal efficacy results; tests will be
invalidated if any media exhibit unsatisfactory performance. The media
assessment may be conducted in advance of or concurrently with
efficacy testing; refer to SOP MB-10, Media and Reagents: Preparation
and Quality Evaluation.
2.	Sterility and/or viability controls do not yield expected results.
3.	The mean log density for control carriers falls outside the specified
range. Note: The prescribed minimum and maximum carrier counts
also account for the addition of 5% organic soil to the inoculum.
a. The mean TestLD must be at least 4.0 (corresponding to a
geometric mean density of l.OxlO4) and not above 6.0

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SOP No. MB-23-04
Date Revised 05-21-19
Page 4 of 17

(corresponding to a geometric mean density of l.OxlO6); a mean
TestLD below 4.0 or above 6.0 invalidates the test, except for two
retesting scenarios (outlined in the study protocol).
4. Management of non-conforming data will be consistent with SOP
ADM-07, Non-Conformance Reports.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
1.	There are time sensitive steps in this procedure including the use-period
of the inoculated carriers and the test chemical.
2.	Verify the volume of dilution blanks, neutralizer tubes, and subculture
tubes in advance and adjust accordingly.
11. Special
Apparatus and
Materials
1. Culture media.
a.	ModifiedProskauer-Beckmedium. Dissolve 2.5 g KH2PO4, 5.0 g
asparagine, 0.6 g MgS04x7H20, 2.5 g magnesium citrate, 20.0 mL
glycerol, 0.0046 g FeCb, and 0.001 g ZnS04 7H20 in 1 L H20.
Adjust to pH 7.2-7.4 with 1 N NaOH. Filter through Whatman
No. 4 (or equivalent) filter paper, place 20 mL portions in separate
25x150 mm tubes, and steam sterilize 20 min at 121C. Use this
broth for propagating test cultures grown statically (25 x 150 mm
tubes) and for recovery of test organism from treated carriers
(38x100 mm tubes).
b.	Middlebrook 7H9 broth (dehydratedM7H9 medium) with 0.1%
(v/v) polysorbate 80. Dissolve 4.7 g in 900 mL H2O containing 1
mL polysorbate 80, 2 mL glycerol, and 1.0 g Bacto agar. Heat to
boiling to dissolve completely. Steam sterilize 15 min at 121C.
Cool sterile medium to 45C, add 100 mL Middlebrook ADC
Enrichment under aseptic conditions and mix thoroughly. Store
prepared medium at 2-5C. Use this broth for propagating test
cultures grown with agitation.
c.	Middlebrook 7H11 agar (dehydratedM7H11 medium). Dissolve
21 g dehydrated M7H11 agar medium in 900 mL H2O containing
5 mL glycerol. Swirl to obtain a smooth suspension; boil if
necessary to completely dissolve the powder. Steam sterilize 15
min at 121C. Cool sterile medium to 50-55C, add 100 mL
OADC enrichment under aseptic conditions, and mix thoroughly.
Distribute in 20 mL portions in sterile 25x150 mm screw-capped
tubes and slant or dispense a minimum of 30 mL into sterile Petri
plates. Alternatively, pre-made M7H11 agar plates may be
purchased. Use slants to maintain stock culture and plates for

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SOP No. MB-23-04
Date Revised 05-21-19
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inoculum isolation and enumeration.
d.	Middlebrook 7H9 broth (dehydratedM7H9 medium). Dissolve
4.7 g in 900 mL H2O containing 2 mL glycerol and 1.0 g Bacto
agar. Heat to boiling to dissolve completely. Distribute 18 mL
portions in 25x150 mm tubes. Steam sterilize 10 min at 121C,
according to manufacturer's instructions. Cool sterile medium to
approximately 40-45C then add 2 mL Middlebrook ADC
Enrichment to each tube under aseptic conditions and mix
thoroughly. Store prepared medium at 2-5C. Use for recovery of
test organism from treated carriers.
e.	Kirchner's medium. Dissolve 5 g asparagine, 2.5 g sodium citrate,
0.6 g magnesium sulfate (heptahydrate), 2.5 g monopotassium
phosphate, and 1.5 g dipotassium phosphate, in 900 mL H2O
containing 20 mL glycerol and 1.0 g Bacto agar. Heat to boiling
to dissolve completely. Steam sterilize 15 min at 121C. Cool
sterile medium to 45C, add 100 mL Middlebrook ADC
Enrichment under aseptic conditions, and mix thoroughly.
Distribute in 20 mL portions in sterile 25 x 150 mm tubes. Use for
recovery of test organism from treated carriers.
f.	TB broth base. Dissolve 2.0 g yeast extract, 2.0 g proteose
peptone No. 3, 2.0 g casitone, 1.0 g potassium phosphate
monobasic, 2.5 g sodium phosphate dibasic, 1.5 g sodium citrate,
and 0.6 g magnesium sulfate (heptahydrate) in 900 mL H2O
containing 50 mL glycerol and 1.0 g Bacto agar. Heat to boiling
to dissolve completely. Steam sterilize 15 min at 121C. Cool
sterile medium to 45C, add 100 mL Dubos Medium Serum under
aseptic conditions, and mix thoroughly. Distribute in 20 mL
portions in sterile 25 x 150 mm tubes. Use for recovery of test
organism from treated carriers.
g.	Middlebrook 7H10 agar. Dissolve 19 g in 900 mL H2O
containing 5 mL glyerol. Heat to boiling to dissolve completely.
Steam sterilize 15 min at 121C. Cool sterile medium to 45C,
add 100 mL Middlebrook ADC Enrichment under aseptic
conditions and mix thoroughly. Use for initiating stock cultures.
2. Test organism.
a. Mycobacterium bovis (BCG) (ATCC #35743). For stock culture,
streak inoculate M7H11 agar slants. Incubate 15-20 days at
361C. Following incubation, maintain at 2-5C for up to 6
weeks.

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SOP No. MB-23-04
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3.	Reagents
a.	Sterile water. Use reagent-grade water free of substances that
interfere with analytical methods. Any method of preparation of
reagent-grade water is acceptable provided that the requisite
quality can be met. See Standard Methods for the Examination of
Water and Wastewater and SOP QC-01, Quality Assurance of
Purified Water for details on reagent-grade water.
b.	0.1%polysorbate 80 in saline. Add 0.1 mL polysorbate 80 to 100
mL sterile 0.85% aqueous saline (sodium chloride) solution, filter
sterilize. Used in test culture preparation and dilution of culture
grown with agitation.
4.	Apparatus.
a.	Specialized glassware. For neutralizer/primary subcultures, use
autoclavable 38x100 mm tubes (Bellco Glass Inc., Vineland, NJ).
Cap tubes with closures before sterilizing.
b.	Tissue grinder. Kimble glass tissue grinder (catalog number
885300-0015), for homogenization of the statically grown culture.
c.	Inoculating loop. For culture inoculation, 1 |iL sterile disposable
loops (Fisher Scientific). For culture harvest, 95% platinum, 3.5%
rhodium alloy, 18 or 19 gauge, 4 mm loop with 75 mm shank
(Baxter Scientific Products) or equivalent or disposable loops.
d.	Carriers. Glass Slide Carriers, 25 mm><75 mm (or comparable
size) borosilicate glass cover slips with number 4 thickness or
Fisherfinest Premium Frosted Microscope Slides (Fisher
Scientific, catalog number 12-544-2). Refer to SOP MB-03,
Screening of Stainless Steel Cylinders, Porcelain Cylinders and
Glass Slide Carriers Used in Disinfectant Efficacy Testing.
e.	Sterile surgical gloves. For handling the towelette.
f.	Forceps. For manipulating glass slides.
g.	Micropipettes. For performing serial dilutions.
h.	Positive displacement pipette. With corresponding sterile tips able
to deliver 10 |iL.
i.	Timer. Any certified timer that can display time in seconds.
j. Spectrophotometer. Calibrated; for preparing standardized test
culture.
k. Semi-microcuvette with cap. For measuring percent transmittance.

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Date Revised 05-21-19
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1. TB Stain Kit. For presumptive identification of test microbe.
m. Rotary Shaker. To provide rotation at 150 rpm for cultures grown
with agitation.
12. Procedure and
Analysis
One towelette is used to wipe ten carriers/slides. The area of the towelette
used for wiping is folded and rotated so as to expose a new surface of the
towelette for each carrier.
The method may be altered to accommodate various towelette/carrier
combinations (e.g., more than one towelette per set of ten slides).
The Disinfectant Towelette Test forM bo vis (BCG) Processing Sheet (see
section 14) must be used for tracking testing activities.
12.1 Test Culture
Preparation:
Agitated
Culture
Refer to SOP MB-02 for the test microbe culture transfer notation.
a.	Transfer a 10 |iL loopful of M. bovis (BCG) from an M7H11 stock
slant to a 25 x 150 mm tube containing 10 mL of Middlebrook 7H9
broth with 0.1% (v/v) polysorbate 80 (M7H9/P80), parafilm the
cap to the tube, and briefly vortex. Incubate the tube at 361C on
a rotary shaker at 150 rpm for 5-8 days. This represents a primary
(1) culture and is never used as a test culture.
b.	After incubation, vortex the 1 tube well and transfer 1 mL to a
250 mL flask containing 50 mL of M7H9/P80. Incubate at
361C on a rotary shaker at 150 rpm for 6-10 days. This
represents the secondary (2) culture and is the test culture.
c.	On the test day (following the 6-10 day incubation period), harvest
the culture:
i.	Transfer the 2 culture to sterile 25x 150 mm test tubes.
Allow the suspension to settle for 10-15 min.
ii.	Remove the upper portion of each culture (e.g., upper 3A),
leaving behind any debris or clumps, and transfer to a
sterile flask; pool cultures in the flask and swirl to mix.
iii.	Dilute the pooled culture with sterile saline with 0.1%
polysorbate 80 (saline/P80) to achieve 201%
transmittance at 650 nm. Use a semi-microcuvette with
cap while measuring transmittance. Blank the
spectrophotometer with M7H9/P80.
d.	If an organic soil load is specified in the test parameters for the
product test, add the appropriate amount of organic soil to the
pooled test culture prior to the inoculation of carriers. Swirl to
mix.

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e. Inoculate glass slide carriers with the standardized culture within
10 min of standardization. Briefly mix culture prior to use.
12.2 Test Culture
Preparation:
Static Culture
(alternative
culture
preparation
procedure)
Refer to SOP MB-02 for the test microbe culture transfer notation.
a.	Initiate test culture by inoculating a sufficient number of 25 x 150
mm tubes containing 20 mL MPB (approximately 10) from stock
culture slant(s) (M7H11 agar slants) by transferring 1-2 1 |iL
loopfuls from the stock culture onto the surface of the broth.
Record all transfers on the Organism Culture Tracking Form
(culture notation = -SL, indicating a transfer from slant to liquid).
b.	Note: Over-inoculation of MPB may lead to reduced viability due
to excessive growth after 212 days; the resulting carrier counts
may be negatively impacted.
c.	Incubate the tubes 212 days undisturbed at 361C in a slanted
position to increase surface area.
d.	On the test day: using a transfer loop, transfer culture to a sterile
glass tissue grinder, add 1.0 mL saline/P80, grind continuously for
approximately 1 min to break up large clumps or aggregates of the
test organism.
e.	Dilute the homogenized culture with 9 mL MPB broth and transfer
the suspension from the tissue grinder to a sterile test tube.
Harvest and homogenize culture from multiple MPB broth tubes.
f.	Repeat 12.2d-e as necessary to obtain enough concentrated culture.
g.	Note: Growth from multiple tubes may be harvested and combined
to prepare the concentrated culture prior to standardization.
i.	Allow the suspension to settle for 10-15 min.
ii.	Remove the upper portion of each culture (e.g., upper 3A),
leaving behind any debris or clumps, and transfer to a
sterile flask; pool cultures in the flask and swirl to mix.
iii.	Dilute the pooled culture with MPB broth to achieve
201% T at 650 nm. Use a semi-microcuvette with cap
while measuring transmittance. Blank the
spectrophotometer with MPB.
h.	If an organic soil load is specified in the test parameters for the
product test, add the appropriate amount of organic soil to the
pooled test culture prior to the inoculation of carriers. Swirl to
mix.

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i. Aliquot a sufficient volume of culture into a sterile test tube.
j. Inoculate glass slide carriers with the standardized culture within
10 min of standardization. Briefly mix culture prior to use.
12.3 Carrier
Inoculation
Inoculate approximately 20 carriers; 10 carriers are required for testing, 3
for control carrier counts, and 3 for the viability controls.
a.	Vortex-mix the inoculum periodically during the inoculation of
carriers. Use a calibrated positive displacement pipette to transfer
10 |iL of the test culture onto the sterile test carrier in the Petri
dish, at one end of the slide. Do not place inoculum in the middle
of the slide. Immediately spread the inoculum uniformly over one
third of the carrier surface using a sterile loop. Do not allow the
inoculum to contact the edge of the glass slide carriers. Cover dish
immediately.
b.	Dry carriers in incubator at 361C for 302 min. Record the
timed carrier inoculation activities on the Disinfectant Towelette
Test forM bovis (BCG) Processing Sheet (see section 14). Use
inoculated carriers for testing within 2 h of drying.
c.	After completion of all slide inoculations, thoroughly wipe the
micropipette with 70% ethanol prior to removal from the BSC.
12.4 Enumeration
of viable
bacteria from
carriers
(control
carrier counts)
a.	After inoculated carriers have dried, randomly select 3 inoculated
carriers for assay. Assay 1 carrier immediately prior to conducting
the efficacy test and 2 carriers immediately following the test.
b.	Place each of the inoculated, dried carriers in a 38 x 100 mm tube
or a sterile 50 mL polypropylene conical tube containing 20 mL of
MPB broth and vortex each tube for 15 s. Record the time of
vortexing on the Disinfectant Towelette Test for M bovis (BCG)
Processing Sheet (see section 14).
c.	After vortexing, make serial ten-fold dilutions in 9 mL phosphate
buffered dilution water. If the serial dilutions are not made and
plated immediately, keep the vortexed tubes at 2-5C until this
step can be done; however, perform dilution and plating within 2 h
of vortexing.
d.	Briefly mix each serial dilution tube prior to plating. Plate 0.1 mL
aliquots of appropriate dilutions in duplicate on M7H11 using
surface spread plating. Serial dilution tubes 10"1 through 10"3
should produce plates with CFU in the countable range. Spread
inoculum evenly over the surface of the agar. Plates must be dry
prior to incubation.

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SOP No. MB-23-04
Date Revised 05-21-19
Page 10 of 17

e.	Incubate plates (inverted) concurrently with the efficacy test
subculture tubes at 361C for 17-21 days. Place plates in sterile
bags to reduce dehydration during the incubation period.
f.	Count colonies. Record plates that have colony counts over 300 as
TNTC. Record counts on the Disinfectant Towelette Test forM
bovis (BCG) Carrier Counts Form (see section 14). See section 13
for data analysis.
12.5 Disinfectant
Sample
Preparation
a.	Prepare disinfectant sample per SOP MB-22.
b.	Wipe the outside of the towelette packet or dispenser with 70%
ethanol and allow to air dry prior to opening.
12.6 Test
Procedure
a.	Record timed events on the Disinfectant Towelette Test for M.
bovis (BCG) Time Recording Sheet for Carrier Transfers (see
section 14).
b.	Aseptically remove several towelettes before aseptically removing
a towelette to initiate testing. Fold towelette in half lengthwise
one to two times depending on the size. Beginning at the bottom,
fold up towards the top five times. The following steps in the
"procedure" section are more conveniently done with two analysts
- one to manage the Petri dishes and slides, and the other to
perform the wiping procedure.
c.	Remove the lid from the Petri dish and aseptically remove the
inoculated slide and hold it firmly against the rim of the Petri dish.
d.	Wipe the slide back and forth three times lengthwise with the
towelette for a total of six passes across the inoculum or as
specified by the study sponsor. Wiping should be done within 5
seconds of specified time. Place slide in Petri dish, close the lid,
and allow slide to sit undisturbed for the contact time. Maintain the
wiped carriers in a horizontal position.
e.	Repeat with four additional slides, folding the used section of the
towelette in such a way as to expose a new surface for wiping each
slide.
f.	After the fifth slide, unfold the vertical fold in the towelette and
reverse the towelette so that the used surface of the towelette faces
inward. Continue wiping an additional five slides, folding the
towelette between each slide to expose a new surface.
g.	After the last slide of a set (typically 10 slides) has been wiped and
the exposure time is complete, sequentially transfer each slide into
the neutralizer tube within the 5 second time limit. Drain the

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SOP No. MB-23-04
Date Revised 05-21-19
Page 11 of 17
excess disinfectant from each slide, without touching the Petri
dish, and transfer into the neutralizer tube. Perform transfers with
sterile forceps. Place the inoculated/wiped end of the slide into the
tube.
h.	After the slide is deposited, recap the neutralizer tube and shake
thoroughly; transfer the carrier to the tube containing 20 mL MPB
broth within 5-10 minutes. Sterilize forceps after each carrier
transfer.
i.	Once all carriers have been transferred to the MPB broth tubes,
sequentially transfer 2 mL aliquots from each neutralizer tube into
duplicate tubes of 2 additional subculture media, M7H9 broth,
Kirchner's medium, or TB broth, as specified. This portion of the
assay is not timed, but the aliquots should be sequentially
transferred to the subculture media within approximately 305
min. Repeat this with each tube of neutralizer. Shake each
subculture tube thoroughly. Slightly loosen caps of growth media
prior to incubation.
j. Incubate 60 days at 361C.
k. Report results as + (growth) or 0 (no growth).
1. Record results at 60 days. If the 60th day of incubation falls on a
weekend or holiday, record the results on the first workday
following the 60th day of incubation.
i.	Tubes may be monitored beginning at day 21 for evidence
of typical mycobacterial growth. If multiple tubes show
significant growth prior to the 60th day, confirmatory tests
(e.g., acid fast staining and streak isolation) may be
initiated prior to day 60. If the results of the confirmatory
test are indicative ofM bo vis (BCG), the results may be
recorded at that point to expedite the reporting process.
ii.	Provide justification when recording results on days other
than 60 in the comments section of the Disinfectant
Towelette Test forM bovis (BCG) Results Sheet (see
section 14).
m. If no growth or occasional growth (insufficient for confirmatory
tests) occurs within a set of tubes after 60 days, incubate the set an
additional 30 days and record the results. After 30 days, if growth
occurs check using standard confirmatory procedures (e.g., acid

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Date Revised 05-21-19
Page 12 of 17

fast staining and growth on M7H11 agar) to ensure that no
contamination is present.
n. Record results at 90 days. If the 90th day of incubation falls on a
weekend or holiday, record the results on the first workday
following the 90th day of incubation. Recording of results beyond
the 90th day should be notated in the Comments section on the
Disinfectant Towelette Test forM bo vis (BCG) Results Sheet (see
section 14).
12.7 Sterility and
viability
controls
a.	Sterility controls. Place one sterile, uninoculated carrier into a
tube of MPB broth. In addition, incubate 1 tube of each subculture
medium with 2 mL sterile neutralizer for quality control purposes.
Shake each tube thoroughly and incubate all tubes with the
efficacy test. Report results as + (growth) or 0 (no growth) as
determined by presence or absence of turbidity or presence of
culture growth. Growth should not occur in any tube. Record
results on the Disinfectant Towelette Test forM bo vis (BCG)
Results Sheet (see section 14).
b.	Viability controls. On the day of testing, place a dried inoculated
carrier into a tube of MPB broth and a tube of each subculture
medium. Incubate tubes as in the efficacy test. Report results as +
(growth) or 0 (no growth) as determined by presence or absence of
turbidity or presence of culture growth. Growth should occur in
all tubes. Record results on the Disinfectant Towelette Test forM
bovis (BCG) Results Sheet (see section 14).
12.8 Test microbe
identification
a.	Presumptively confirm at least one positive subculture tube for
each carrier set with growth. The maximum number of tubes
subjected to confirmatory tests per disinfectant tested is 10.
b.	If more than one subculture tube for a carrier set is positive,
confirm a minimum of one tube using acid fast staining and
isolation on selective media (M7H11 agar plates).
c.	If the MPB in the set is positive, it is the representative subculture
tube used for identification. If MPB is not positive, any of the
other subculture media may be used for identification.
d.	If growth is observed in only one carrier set, then all subculture
tubes showing growth for that carrier are subject to confirmatory
tests.
e.	Growth for acid fast staining is taken from the selected positive
tubes on the day that results are read. Acid fast rods are typical for
M bovis (BCG). The acid fast staining results should be read

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Date Revised 05-21-19
Page 13 of 17

promptly prior to assigning a + or 0 to the results. If acid fast rods
are observed from the selected tubes then a + is assigned to the
results. If no cells are observed for the acid fast stain, apply a 0 to
the results.
f.	In addition, streak isolate growth from positive tubes on M7H11
agar and incubate for 17-21 days at 361C.
g.	Following the 17-21 day incubation period, evaluate the colony
morphology on M7H11 agar. M bo vis (BCG) typically appears as
colorless to buff-colored, raised, rough growth on M7H11 agar
(see Attachment 1).
h.	If a satisfactory smear cannot be obtained directly from the tube,
take the smear for acid fast staining from the 17-21 day old
M7H11 agar plate that was inoculated with the growth from the
tube.
i.	In the event that no cells were observed with acid fast staining
initially but typical growth was observed on the M7H11, correct
the 0 to read + on the test sheet. An entry error will be noted in the
comments section of the Disinfectant Towelette Test for M bovis
(BCG) Results Sheet (see section 14).
j. Record results on the Disinfectant Towelette Test for M bovis
(BCG) Microbe Confirmation Sheet (see section 14).
13. Data Analysis/
Calculations
Calculations will be computed using a Microsoft Excel spreadsheet (see
section 14). Both electronic and hard copies of the spreadsheet will be
retained. Counts from 0 through 300 and their associated dilutions will be
included in the calculations.
14. Forms and
Data Sheets
1.	Attachment 1: Typical Growth Characteristics of Strains ofM bovis
(BCG)
2.	Attachment 2: Culture Initiation and Stock Culture Generation for
Mycobacterium bovis (BCG)
3.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:
Physical Screening of Carriers Record MB-03 Fl.docx
Organism Culture Tracking Form for MB-07 F5.docx
Mycobacterium bovis (BCG)
Test Microbe Confirmation Sheet (Quality MB-07 F6.docx
Control)

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Page 14 of 17

Disinfectant Towelette Test forM bovis (BCG) MB-23-04 Fl.docx
Carrier Counts Form
Disinfectant Towelette Test forM bovis (BCG) MB-23-04 F2.docx
Time Recording Sheet for Carrier Transfers
Disinfectant Towelette Test forM bovis (BCG) MB-23-04 F3.docx
Information Sheet
Disinfectant Towelette Test forM bovis (BCG) MB-23-04 F4.docx
Results Sheet
Test Microbe Confirmation Sheet MB-23-04 F5.docx
Carrier Count Spreadsheet MS Excel spreadsheet: MB-23-04_F6.xlsx
Carrier Count Template_CTBDTT_v4
Disinfectant Towelette Test forM bovis (BCG) MB-23-04 F7.docx
Processing Sheet
15. References
1.	Official Methods of Analysis. Revised 2013. AOAC
INTERNATIONAL, Gaithersburg, MD, (Method 961.02).
2.	Official Methods of Analysis. 2012. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, (Method 965.12 In vitro Test
for Determining Tuberculocidal Activity).
3.	Standard Methods for the Examination of Water and Wastewater. 23rd
Ed. American Public Health Association, 1015 15th Street, NW,
Washington, DC
4.	Holt, J., Krieg, N., Sneath, P., Staley, J., and Williams, S. eds. 1994.
Bergey's Manual of Determinative Bacteriology, 9th Edition. Williams
& Wilkins, Baltimore, MD.
5.	Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Bergey's
Manual of Systematic Bacteriology. Volume 2. Williams & Wilkins,
Baltimore, MD.

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SOP No. MB-23-04
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Attachment 1
typical Growth Characteristics of strains ofM bovis (BCG) (see ref. 15.4 and 15.5)

M. bovis (BCG)*
Gram slain reaction
weakly (+)
Acid Fast slain reaction
(+)
Typical Growth Characteristics on Solid Media
Middlcbrook 7H9
rough, raised, thick colonics with a nodular or wrinkled surface and an irregular thin
margin, off-white to faint buff, or even yellow
Typical Microscopic Characteristics
Cell dimensions
0.3-0.6 nm in diameter by 1-4 |im in length*
Cell appearance
rods, straight or slightly curved, occurring singly and in occasional threads
* After 15-20 days

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Attachment 2
Culture Initiation and Stock Culture Generation for Mycobacterium bovis (BCG)
Al. Culture initiation. Refer to SOP MB-02 for establishment of the organism control number.
a.	Initiate new stock cultures from lyophilized cultures of Mycobacterium bovis
(BCG) from ATCC after no more than 18 stock culture transfers.
b.	Open ampule of freeze-dried organism as indicated by ATCC. Using a tube
containing 5-6 mL of M7H9 broth, aseptically withdraw 0.5 to 1.0 mL and
rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet
back into the original tube of broth. Mix well.
c.	Use several drops of the suspension to inoculate two Middlebrook 7H10 agar plates
and streak for isolation.
d.	Incubate the tube of rehydrated culture and the plates at 361C for 282 days.
A2. Culture maintenance.
a.	Confirm the identity of a streak isolation plates and acid fast stain (see Attachment
1 for colony morphology and acid fast staining results).
b.	Use an M7H10 streak isolation plate to streak M7H11 agar slants (stock slants).
Based on anticipated use, streak approximately 10-20 stock slants.
c.	Incubate the new stock transfers for 15-20 days at 361C. Store at 2-5C.
d.	Every 6 weeks (42 days), generate an additional 10-20 M7H11 slants. Inoculate
new M7H11 slants by streaking a loopful ofM bovis (BCG) growth from an
established tube to each of the 10-20 tubes. Perform QC of stock cultures per
section A3.
e.	Incubate the stock culture slants at 361C for 15 to 20 days. Following incubation,
maintain stock cultures at 2-5C for up to 6 weeks.
A3. QC of stock cultures
a.	Up to every 6 weeks (42 days), streak a loopful of growth for isolation from the
existing M7H11 stock slant used to inoculate new agar slants on a plate of M7H11
agar. Incubate the plate for 17-21 days at 361C.
b.	Following the incubation period, record the colony morphology as observed on the
M7H11 plate. See Attachment 1 for details on cell and colony morphology and
stain reactions.
c.	Perform an acid fast stain from growth taken from the M7H11 streak isolation plate
	according to the manufacturer's instructions. Observe the acid fast reaction by

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using brightfield microscopy at 1000X magnification (oil immersion).
d. Record observations on the Test Microbe Confirmation Sheet (Quality Control) (see
section 14).

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