US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Tuberculocidal Activity of Disinfectants:
In vitro Test for Determining Tuberculocidal Activity
SOP Number: MB-07-09
Date Revised: 02-25-19

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SOP No. MB-07-09
Date Revised 02-25-19
Page 1 of 16
SOP Number
MB-07-09
Title
Tuberculocidal Activity of Disinfectants: In vitro Test for
Determining Tuberculocidal Activity
Scope
Describes the methodology to determine tuberculocidal activity of
disinfectants labeled to treat hard non-porous surfaces against
Mycobacterium bovis (BCG), 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, 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Date SOP withdrawn:


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SOP No. MB-07-09
Date Revised 02-25-19
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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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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 the two additional subculture media tubes (e.g., M7H9 broth,
Kirchner's medium, or TB broth) inoculated from the carrier's corresponding
neutralizer tube. 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
Transferring the inoculated carrier into the tube containing the disinfectant is a
critical, technique-sensitive step. False positives can result from transfer of
test microbe to sides of tubes due to inadvertent contact.
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 fail to 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 1.0 x 104) and not above 6.0 (corresponding to a
geometric mean density of 1.0 x 106); a mean TestLD below 4.0 or
above 6.0 invalidates the test, except for two retesting scenarios
(outlined in the study protocol).

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4. Management of non-conforming data will be consistent with SOP ADM-
07, Non-Conformance Reports.
9. Data
Management
Data will be archived per SOP ADM-03, Records and Archives.
10. Cautions
1.	There are time sensitive steps in this procedure including the use-periods
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-Beck medium. Dissolve 2.5 g KH2PO4, 5.0 g
asparagine, 0.6 g MgS04 7H2O, 2.5 g magnesium citrate, 20.0 mL
glycerol, 0.0046 g FeCb, and 0.001 g ZnS04 7H20 in 1 L H2O.
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 and for recovery of test
organism from treated carriers.
b.	Middlebrook 7H9 broth (dehydratedM7H9 medium) with 0.1% (v/v)
polysorbate 80.1 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 25 x 150 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 inoculum isolation and
enumeration.
d.	Middlebrook 7H9 broth (dehydratedM7H9 medium). Dissolve 4.7 g
1 Used for propagating test cultures grown with agitation. Step currently not in the official AO AC standard 965.12.

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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 glycerol. 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.
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

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Date Revised 02-25-19
Page 6 of 16
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.
c.	Octylphenoxypolyethoxyethanol nonionic surfactant (e.g. Triton X-
100).
4. Apparatus.
a.	Specialized glassware. For disinfectant, use autoclavable 25x100
mm tubes (Bellco Glass Inc., or equivalent). For glassware used to
prepare test chemical, refer to SOP MB-22.
b.	Tissue grinder. Kimble glass tissue grinder (885300-0015), for
homogenization of the statically grown culture.
c.	Recirculating chiller unit. For maintaining specified temperature of
the test chemical.
d.	Inoculating loop. For culture inoculation, 1 |iL sterile disposable
loops (Fisher Scientific).2 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.
e.	Wire hook. For carrier transfer. Make 3-5 mm bend (approximately
60) at end of suitable platinum or platinum alloy wire, No. 23 B&S
gauge, in appropriate holder (Johnson Matthey Inc., or equivalent).
f.	Carriers. "Penicylinders," porcelain, 81 mm outer diameter, 61
mm inner diameter, 101 mm long (CeramTec Ceramic; Cat. No.
LP15819 0645). Use only carriers that passed physical screening;
refer to SOP MB-03, Screening of Stainless Steel Cylinders,
Porcelain Cylinders and Glass Slide Carriers Used in Disinfectant
Efficacy Testing.
g.	Timer. Any certified timer that can display time in seconds.
h.	Spectrophotometer. Calibrated; for preparing standardized test
culture.
i.	Sonicator (ultrasonic cleaner). For conducting control carrier
counts.
2 Step currently not in the official AO AC standard 965.12.

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i. Verification of the sonicator is used to determine the impact
of sonication on the culture. If necessary, verify the sonicator
by placing the standardized broth culture into sonicator for 10
min, serially dilute, and plate. Compare sonicated counts to a
non-sonicated control. The sonicated and non-sonicated
counts should be comparable.
j. Semi-microcuvette with cap. For measuring percent transmittance.
k. TB Stain Kit. For presumptive identification of test microbe.
1. Rotary Shaker. To provide rotation at 150 rpm for cultures grown
with agitation.
12. Procedure and
Analysis
The AOAC Tuberculocidal Activity of Disinfectants Test Processing Sheet
(see section 14) must be used for tracking testing activities.
12.1 Test Culture
Preparation:
Agitated
Culture3
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.
3 Step currently not in the official AO AC standard 965.12.

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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.
e.	Inoculate porcelain cylinders 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) (M7H9 or M7H11 agar slants) by transferring 1-2 1 |iL
loopfuls4 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 min5 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.6
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%
transmittance at 650 nm. Use a semi-microcuvette with cap
while measuring transmittance. Blank the spectrophotometer
4	Step currently not in the official AO AC standard 965.12.
5	Step currently not in the official AO AC standard 965.12.
6	Step currently not in the official AO AC standard 965.12.

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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.
i.	Inoculate porcelain cylinders 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 viability controls.
a.	Inoculate sets of 10 sterile carriers with approximately 15-20 mL
standardized test culture in 25 x 150 mm test tubes.
b.	The test culture must completely cover the carriers. If a carrier is not
covered, gently shake the tube or reposition the carrier within the
tube with a sterile wire hook. Be sure to inoculate a sufficient
number of carriers for the test.
c.	After 151 min contact period, remove cylinders using flame-
sterilized wire hook and shake carriers vigorously against side of the
tube to remove excess culture. Place carriers on end in vertical
position in sterile Petri dishes matted with 2 layers of Whatman No. 2
(or equivalent) filter paper, making sure that carriers do not touch to
prevent improper drying. Place no more than 12 carriers in a Petri
dish.
d.	Carriers that touch or fall over cannot be used for testing and must be
removed, cleaned, and sterilized.
e.	Once all of the carriers have been transferred, cover and place in
incubator at 361C, and let dry 302 min. Record the time on the
AOAC Tuberculocidal Test Processing Sheet (see section 14).
f.	Use inoculated carriers for testing within 2 h of drying.
12.4 Enumeration
of bacterial
inocula (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 following the test.
b.	Place each inoculated carrier into a tube containing 10 mL of MPB
broth and sonicate in an ultrasonic cleaner for 10 min. Record the
time of sonication on the AOAC Tuberculocidal Test Processing
Sheet (see section 14).
c.	For sonication, place tubes into an appropriately sized glass beaker
with tap water to the level of the MPB broth in the tubes. Place the
beaker in an ultrasonic cleaner so that the water level in the beaker is

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SOP No. MB-07-09
Date Revised 02-25-19
Page 10 of 16

even with the water level fill-line on the tank. Fill the tank with tap
water to the water level fill-line. Hold the beaker so that it does not
touch the bottom of the tank and all 3 liquid levels (inside the test
tubes, inside the beaker, and inside the tank) are approximately the
same.
d.	After sonication, briefly mix each tube on a vortex mixer and make
serial ten-fold dilutions in 9 mL phosphate buffered dilution water.
If the serial dilutions are not made and plated immediately, keep the
sonicated tubes at 2-5C until this step can be done; however perform
dilution and plating within 2 h of sonication.
e.	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.
f.	Incubate plates (inverted) concurrently with the efficacy test
subculture tubes at 361C for 17-21 days.
g.	Count colonies. Record plates that have colony counts over 300 as
TNTC. Record counts on the AOAC Tuberculocidal Test 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.	Equilibrate the water bath and allow it to come to 201C or the
temperature specified (1C). Prepare the disinfectant dilution
within 3 hours of performing the assay unless test parameters specify
otherwise. Record the time of disinfectant preparation on the AOAC
Tuberculocidal Test Processing Sheet (see section 14).
c.	Dispense 10 mL aliquots of the disinfectant into 25 x 100 mm test
tubes, one tube per carrier. Place tubes in the equilibrated water bath
for approximately 10 min to allow disinfectant to come to the
specified temperature. Record the temperature of the water bath and
recirculating chiller before and after testing on the AOAC
Tuberculocidal Test Information Sheet (see section 14).
12.6 Test Procedure
a.	Sequentially transfer carriers from Petri dish to test tubes containing
disinfectant at appropriate intervals (e.g., 30 s intervals). Record
timed transfer activities on the AOAC Tuberculocidal Time
Recording Sheet for Carrier Transfers (see section 14).
b.	Add one carrier per tube. For a contact time of 10 min, the carrier
must be deposited in the tube within 5 s of the prescribed drop time.

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SOP No. MB-07-09
Date Revised 02-25-19
Page 11 of 16
c.	Using alternating hooks, sterilize the hook and allow it to cool after
each carrier transfer. When lowering the carrier into the disinfectant
tubes, neither the carrier nor the wire hook should touch the interior
sides of the tube. If the interior sides of the tube are touched, discard
the disinfectant tube and carrier and repeat.
d.	Following the exposure time, sequentially transfer the carriers into
neutralizer tubes using a sterile hook. Drain excess disinfectant from
the carrier prior to transfer.
e.	Shake tube containing carrier in neutralizer thoroughly; transfer the
carrier to the tube containing 20 mL MPB broth within 5-10
minutes.7 Sterilize hook after each carrier transfer. Avoid contact of
the carrier to the interior of the tube during transfer.
f.	Once all carriers have been transferred to the MPB broth tubes,
sequentially transfer 2 mL aliquots from each neutralizer tube into 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.8
g.	Incubate 60 days at 361C.
h.	Report results as + (growth) or 0 (no growth).
i.	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 AO AC Tuberculocidal
Test Results Sheet (see section 14).
j. If no growth or occasional growth (insufficient for confirmatory
7	Step currently not in the official AO AC standard 965.12.
8	Step currently not in the official AO AC standard 965.12.

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SOP No. MB-07-09
Date Revised 02-25-19
Page 12 of 16

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 fast
staining and growth on M7H11 agar) to ensure that no contamination
is present.
k. 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 AO AC
Tuberculocidal Test 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 AO AC
Tuberculocidal Test 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 media.
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 AO AC Tuberculocidal Test 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 forM
bovis (BCG). The acid fast staining results should be read promptly

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SOP No. MB-07-09
Date Revised 02-25-19
Page 13 of 16

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 then a 0 is applied 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. bovis (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 AO AC Tuberculocidal Test Results Sheet
(see section 14).
j. Record results on the AO AC Tuberculocidal Test 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
AO AC Tuberculocidal Activity of Disinfectants , _ n7 nQ p. j
Test: Time Recording Sheet for Carrier Transfers - ' CX
AO AC Tuberculocidal Activity of Disinfectants , _ 1
Test: Test Information Sheet MB-07-09_F2.docx
AO AC Tuberculocidal Activity of Disinfectants MB-07-09_F3.docx

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Date Revised 02-25-19
Page 14 of 16

Test: Results Sheet
AO AC Tuberculocidal Activity of Disinfectants , ^ ^  ^ 1
rpi , rpi j. a /T* U ci . MB-07-09 F4.docx
Test: Test Microbe Confirmation Sheet -
Organism Culture Tracking Form for MB-07-09 FS.docx
Mycobacterium bovis (BCCr) -
Test Microbe Confirmation Sheet (Quality , _ nn r, 1
 J MB-07-09 F6.docx
Control) -
AO AC Tuberculocidal Activity of Disinfectants , _ nn n 1
T .n  n , t-i y MB-07-09 F7.docx
Test Carrier Counts Form -
AO AC Tuberculocidal Activity of Disinfectants , _  1
 +T3 . . y MB-07-09 F8.docx
Test Processing Sheet -
Carrier Count Spreadsheet (MS Excel): Carrier - .
Count Template_CTB_v3 MB-07-09_F9.xlsx
15. References
1.	Official Methods of Analysis. 2012. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, (Method 965.12 In vitro Test for
Determining Tuberculocidal Activity).
2.	Standard Methods for the Examination of Water and Wastewater. 23rd Ed.
American Public Health Association, 1015 15th Street, NW, Washington,
DC
3.	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.
4.	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-07-09
Date Revised 02-25-19
Page 15 of 16
Attachment 1
typical Growth Characteristics of strains ofM bovis (BCG) (see ref. 15.3 and 15.4)

M. bovis (BCG)*
Gram slain reaction
weakly (+)
Acid Fast slain reaction
(+)
Typical Growth Characteristics on Solid Media
Middlcbrook 7H9
rough, raised, thick colonies 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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SOP No. MB-07-09
Date Revised 02-25-19
Page 16 of 16
Attachment 2
Culture Initiation and Stock Culture Generation tor 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 (M7H10)
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 plate and acid fast stain (see Attachment 1
for colony morphology and manufacturer's instructions for acid fast staining).
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
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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