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-23-02

Date Revised: 03-05-13


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SOP No. MB-23-02
Date Revised 03-05-13
Page 1 of 16

SOP Number

MB-23-02

Title

Disinfectant Towelette Test: Testing of Mycobacterium bovis (BCG)

Scope

Describes the methodology used to determine the efficacy of
towelette-based disinfectants against Mycobacterium bovis (BCG) on
hard surfaces. The test is based on AO AC Method 961.02 (Germicidal
Spray Products as Disinfectants). See 15.1.

Application

For product evaluations under the Antimicrobial Testing Program
(ATP), 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Date SOP withdrawn:




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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

6

13.

DATA ANALYSIS/CALCULATIONS

12

14.

FORMS AND DATA SHEETS

12

15.

REFERENCES

13


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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, Kirchners 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 Material 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 SOP EQ-01, EQ-02, EQ-03, EQ-04 and EQ-05 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.	Any inoculated carrier that is wet at the conclusion of the carrier drying
period should not be used.

8. Non-
conforming
Data

1.	Sterility and/or viability controls do not yield expected results.

2.	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 for carriers inoculated withM bovis (BCG)
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 x 106); a mean TestLD below 4.0 and above 6.0
invalidates the test, except for two retesting scenarios (outlined in
the study protocol).

3.	Management of non-conforming data will be specified in the study
protocol; procedures will be consistent with SOP ADM-07, Non-


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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
periods of the inoculated carriers and the test chemical.

2.	Poor media performance may invalidate the test.

3.	A spectrophotometer out of calibration may result in carrier counts
outside the range specified in this SOP.

4.	To ensure adequate volume of media and diluents, verify volumes 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 FeCl3, and 0.001 g ZnS04x7H20 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
25 x 150 mm tubes, and steam sterilize 20 min at 121C. Use this
broth for propagating test cultures (25 x 150 mm tubes) and for
recovery of test organism from treated carriers (38 x 100 mm
tubes).

b.	Middlebrook 7H9 agar (dehydrated M7H9 medium + agar).
Dissolve 4.7 g in 900 mL H20 containing 2 mL glycerol and 15.0
g agar. Heat to boiling to dissolve completely. Steam sterilize 15
min at 121C. Cool sterile medium to approximately 45C, add
100 mL Middlebrook ADC 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. Use slants to maintain stock culture
and plates for inoculum isolation and enumeration.

c.	Middlebrook 7H11 agar (dehydratedM7H11 medium). Dissolve
21 g dehydrated M7H11 agar medium in 900 mL H20 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


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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. 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.

e.	Kirchners 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.

2.	Test organism.

a. Mycobacterium bovis (BCG) (Organon Teknika Corp., Durham,
NC, USA, or equivalent). For stock culture, streak inoculate
M7H9 or M7H11 agar slants. Incubate 15-20 days at 36  1C.
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 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.


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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.

4. Apparatus.

a.	Specialized glassware. For neutralizer/primary subcultures, use
autoclavable 38 x 100 mm tubes (Bellco Glass Inc., Vineland, NJ).
Cap tubes with closures before sterilizing. For glassware used to
prepare test chemical, refer to SOP MB-22.

b.	Tissue grinder. Kimble glass tissue grinder (catalog number
885300-0015).

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 or
equivalent or disposable loops.

d.	Carriers. Glass Slide Carriers, 25 mm x 75 mm (or comparable
size) borosilicate glass cover slips with number 4 thickness. 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. Semimicrocuvette with cap. For measuring percent transmittance.

1. TB Stain Kit. For presumptive identification of test microbe.

12. Procedure and
Analysis

An assessment of media quality (performance) is necessary to ensure the
validity of the tuberculocidal efficacy results. The media assessment may
be conducted in advance of or concurrently with efficacy testing; refer to
SOP MB-10, Media and Reagents Used in Microbiological Assays
Including Performance Assessment and Sterility Verification.

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


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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

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 one
to two 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).

Note: Over-inoculation of MPB may lead to reduced viability due
to excessive growth after 21  2 days; the resulting carrier counts
may be negatively impacted.

b.	Incubate the tubes 21  2 days undisturbed at 36  1C in a slanted
position to increase surface area.

c.	On the test day: Using a transfer loop, transfer culture to a heat-
sterilized glass tissue grinder, add 1.0 mL 0.1% polysorbate 80 in
saline solution, grind to break up large clumps or aggregates of the
test organism.

d.	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.

Note: Growth from multiple tubes may be harvested and combined
to prepare the concentrated culture prior to standardization.

e.	Allow the suspension to settle for 10-15 min.

f.	Remove the upper portion of each culture, leaving behind any
debris or clumps, and transfer to a sterile flask; pool cultures in the
flask and swirl to mix.

g.	Dilute the pooled culture with MPB broth to achieve 20  1% T at
650 nm. Use a semimicrocuvette with cap while measuring
transmittance.

h.	If an organic soil load is specified in the test parameters for the
product test, the appropriate amount of organic soil is added to the
pooled test culture prior to the inoculation of carriers. Swirl to


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mix.

i. Aliquot a sufficient volume of culture into sterile test tubes,
j. Use standardized culture to inoculate glass slide carriers.

12.2 Carrier

Inoculation

Inoculate approximately 20 carriers; 10 carriers are required for testing, 3
for control carrier counts, and 3 for the viability controls.

a.	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.
Vortex-mix the inoculum periodically during the inoculation of
carriers. 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 during the
inoculation process. Cover dish immediately.

b.	Dry carriers in incubator at 36  1C for 30  2 min. Record the
timed carrier inoculation activities on the Disinfectant Towelette
Test forM bovis (BCG) Processing Sheet (see section 14).
Perform efficacy testing within two hours of drying.

c.	After completion of all slide inoculations, thoroughly wipe the
micropipette with 70% ethanol prior to removal from the BSC.

12.3 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 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 seconds. Record the time
of vortexing on the Disinfectant Towelette Test for M bovis
(BCG) Processing Sheet (see section 14).

c.	Make serial ten-fold dilutions in 9 mL phosphate buffered dilution
water. If the serial dilutions are not made and plated immediately,
the vortexed tubes are kept 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 100 |iL
aliquots of appropriate dilutions in duplicate on M7H9 or M7H11
using spread plating. Dilutions 10"1 through 10"3 should produce
plates with CFU in the appropriate range. Plates must be dry prior
to incubation.

e.	Incubate plates (inverted) concurrently with the efficacy test


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subculture tubes at 36  1C for 17-21 days.

f.	Count colonies. Plates that have colony counts over 300 will be
reported as TNTC. Record counts on the Disinfectant Towelette
Test forM bo vis (BCG) Carrier Counts Form (see section 14).
See section 13 for data analysis.

g.	The mean TestLD for carriers inoculated withM bovis (BCG)
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 x 106).

12.4 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.

12.5 Test

Procedure

a.	Record timed events on the Disinfectant Towelette Test forM
bovis (BCG) Time Recording Sheet for Carrier Transfers (see
section 14).

b.	Wipe the outside of the towelette dispenser or packet with 70%
ethanol and allow to air dry.

c.	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.

d.	Remove the lid from the Petri dish and aseptically remove the
inoculated slide and hold it firmly against the rim of the Petri dish.

e.	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.

f.	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.

g.	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


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towelette between each slide to expose a new surface.

h.	Following the contact time, drain the excess disinfectant from each
slide without touching the Petri dish and sequentially transfer into
the neutralizer tube within the 5 second time limit. Perform
transfers with sterile forceps. Place the inoculated/wiped end of the
slide into the tube.

i.	After the slide is deposited, shake tube containing carrier in
neutralizer thoroughly; transfer the carrier to the tube containing
20 mL MPB broth within 5-10 minutes. Sterilize forceps after
each carrier transfer.

j. 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,
Kirchners 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 30  5
min. Repeat this with each tube of neutralizer. Shake each
subculture tube thoroughly. Slightly loosen caps of growth media
prior to incubation.

k. Incubate 60 days at 36  1C.

1. Report results as + (growth) or 0 (no growth).

m. 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).

n. If no growth or occasional growth (insufficient for confirmatory
tests) occurs within a set of tubes, incubate the set an additional 30
days and record the results. Growth should be checked by using


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standard confirmatory procedures (e.g., acid fast staining and
growth on M7H9 or M7H11 agar) to ensure that no contamination
is present.

o. 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).

12.6 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).

12.7 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 (M7H9 or 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


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M. bovis (BCG). The acid fast staining results should be read
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 M7H9 or
M7H11 agar and incubate for 17-21 days at 36  1C.

g.	Following the 17-21 day incubation period, evaluate the colony
morphology on M7H9 or M7H11 agar. M. bovis (BCG) typically
appears as colorless to buff-colored, raised, rough growth on
M7H9 and 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 M7H9
or 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 M7H9 or 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
forM bovis (BCG) Results Sheet (see section 14).

j. Record results on the Disinfectant Towelette Test forM 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 F6.docx
Mycobacterium bovis (BCG)

Test Microbe Confirmation Sheet (Quality MB-07 F7.docx
Control)


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Disinfectant Towelette Test forM bovis (BCG) MB-23-02 Fl.docx
Carrier Counts Form

Disinfectant Towelette Test forM bovis (BCG) MB-23-02 F2.docx
Time Recording Sheet for Carrier Transfers

Disinfectant Towelette Test forM bovis (BCG) MB-23-02 F3.docx
Information Sheet

Disinfectant Towelette Test forM bovis (BCG) MB-23-02 F4.docx
Results Sheet

Test Microbe Confirmation Sheet MB-23-02 F5.docx

Carrier Count Spreadsheet MS Excel spreadsheet: MB-23-02_F6.xlsx
Carrier Count Template_CTBDTT_v3

Disinfectant Towelette Test forM bovis (BCG) MB-23-02 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. 2005.
21st Ed., American Public Health Association, Washington, D.C.

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.

6.	Package Insert - TB Stain Kits and Reagents. Becton, Dickinson and
Company. Part no. 8820201JAA. Revision 03/2011.


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Attachment 1

Typical Growth Characteristics of strains of M. bo vis (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.	Obtain lyophilized culture ofM bovis (BCG).

b.	Reconstitute the lyophilized culture with ~1 mL of sterile DI water. Inoculate two
M7H9 or M7H11 agar plates by streaking for isolation.

c.	Add -0.2 mL of the rehydrated culture to each of 4 tubes of MPB.

d.	Incubate the M7H9 or M7H11 agar plates and MPB broth tubes for 15 to 20 days at
36  1C or until there is sufficient growth. Incubate MPB broth tubes in a slanted
position.

A2. Culture maintenance.

a.	Confirm the identity of the streak isolation plates and acid fast stain (see
Attachment 1 for colony morphology and section 15.5 for acid fast staining).
Afterwards, use the 15-20 day old MPB broth cultures (from section Al) to initiate
stock cultures.

b.	Streak M7H9 or M7H11 agar slants (stock slants) using 1-4 tubes of MPB broth
cultures ofM bovis (BCG). Based on anticipated use, streak approximately 10-20
stock slants.

c.	Incubate the new stock transfers for 15-20 days at 36  1C. Store at 2-5C.

d.	Every 6 weeks (42 days), generate an additional 10-20 M7H9 or M7H11 slants.
Inoculate new M7H9 or M7H11 slants by streaking a loopful of M 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 36  1C 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 M7H9 or M7H11 stock slant used to inoculate new agar slants on a plate of
M7H9 or M7H11 agar. Incubate the plate for 17-21 days at 36  1C.

b.	Following the incubation period, record the colony morphology as observed on the
M7H9 or M7H11 plate. See Attachment 1 for details on cell and colony
morphology and stain reactions.

c.	Perform an acid fast stain (refer to 15.5) from growth taken from the M7H9 or

	M7H11 streak isolation plate according to the manufacturer's instructions. Observe


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SOP No. MB-23-02
Date Revised 03-05-13
Page 16 of 16

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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