mm s r4
US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Neutralization Confirmation Assay for Disinfectant
(Liquid or Spray) Products Tested against
Mycobacterium bovis (BCG)
SOP Number: MB-11-03
Date Revised: 09-02-10
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Date Revised 09-02-10
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EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Neutralization Confirmation Assay for Disinfectant (Liquid or Spray) Products Tested against
Mycobacterium bovis (BCG)
SOP Number: MB-11-03
Date Revised: 09-02-10
Initiated By: Date: / /
Print Name:
Technical Review: Date: / /
Print Name:
Technical Staff
QA Review: Date: / /
Print Name:
QA Officer
Approved By: Date: / /
Print Name:
Branch Chief
Effective Date: / /
Controlled Copy No.:
Withdrawn By:
Date: / /
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TABLE OF CONTENTS
Contents Page Number
1.0 SCOPE AM) APPLICATION 3
2.0 DEFINITIONS 3
3.0 HEALTH AND SAFETY 4
4.0 CAUTIONS 4
5.0 INTERFERENCES 4
6.0 PERSONNEL QUALIFICATIONS 4
7.0 SPECIAL APPARATUS AND MATERIALS 4
8.0 INSTRUMENT OR METHOD CALIBRATION 5
9.0 SAMPLE HANDLING AND STORAGE 5
10.0 PROCEDURE AM) ANALYSIS 5
11.0 DATA ANALYSIS/CALCULATIONS 12
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 12
13.0 QUALITY CONTROL 12
14.0 NONCONFORMANCE AND CORRECTIVE ACTION 12
15.0 REFERENCES 13
16.0 FORMS AM) DATA SHEETS 13
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1.0 SCOPE AND APPLICATION:
1.1 The neutralization of the active ingredients found in antimicrobial products is one
of the most important steps in efficacy testing. A neutralizing agent is used in
each test to inactivate the product's active ingredients, a process essential to
achieving the desired contact time. In addition, the neutralizer itself or in
combination with the recovery medium must not exhibit bacteriostatic activity
against the test microbe; bacteriostatic activity may bias the efficacy data.
1.2 This SOP describes methodology which will be used to determine the
effectiveness of neutralizers specified for efficacy tests of hard surface
disinfectants against Mycobacterium bovis (BCG).
1.3 This method can also be used to determine the effectiveness of an alternative
neutralizer, one not specified in the test parameters.
1.4 It is preferable to perform the neutralization assay prior to or concurrently with
product testing.
2.0 DEFINITIONS:
2.1 AO AC = AO AC INTERNATIONAL
2.2 CTB = Confirmative in vitro Test for Determining Tuberculocidal Activity
2.3 MPB = Modified Proskauer Beck
2.4 M7H9 = Middlebrook 7H9 agar and broth
2.5 M7H11 = Middlebrook 7H11 agar
2.6 K = Kirchners Medium
2.7 TB = TB Broth
2.8 CFU = colony forming unit
2.9 PBDW = phosphate buffered dilution water
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3.0 HEALTH AND SAFETY:
3.1 All manipulations of the test organism are required to be performed in accordance
with biosafety practices stipulated in the SOP MB-01 (see ref. 15.1). All
manipulations ofM bo vis (BCG) are performed in a biosafety level 3 laboratory
(e.g. room B202 or room B207).
3.2 Disinfectants may contain a number of different active ingredients, such as
quaternary ammonium compounds, halogens, phenolics, aldehydes, peroxides,
and heavy metals. Latex gloves and other personal protective clothing or devices
are worn during the handling of these items. A chemical fume hood or other
containment equipment is employed when performing tasks with products.
4.0 CAUTIONS:
4.1 To ensure the stability of the test disinfectant, prepare the disinfectant dilutions
within three hours of the disinfectant treatment step unless test parameters specify
otherwise.
4.2 Strict adherence to the protocol is necessary for validity of test results.
4.3 Use aseptic procedures for all test procedures involving manipulations of the test
organisms and associated test components.
5.0 INTERFERENCES:
5.1 For each assay, one batch (preparation) of each medium should be used for both
treatment and control groups. Differences in performance (quality) between
batches of media may lead to misleading neutralization results.
5.2 Presence of contamination (i.e. in media controls) will interfere with the
interpretation of results and may necessitate repeat analysis.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable of the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.
7.0
SPECIAL APPARATUS AND MATERIALS:
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7.1 Refer to section 7, SPECIAL APPARATUS AND MATERIALS, of each efficacy
test method SOP cited in this document.
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 Refer to section 8, INSTRUMENT OR METHOD CALIBRATION, of each
efficacy test method SOP cited in this document.
9.0 SAMPLE HANDLING AND STORAGE:
9.1 Disinfectants are stored according to manufacturers' recommendations or at room
temperature if the product label or testing parameters do not identify a storage
temperature. Those disinfectants requiring activation or dilution prior to use will
only be activated or diluted within three hours of testing unless test parameters
specify otherwise.
9.2 Follow chain-of custody guidelines during testing as stipulated in SOP COC-01,
Chain of Custody.
10.0 PROCEDURE AND ANALYSIS:
10.1 General Description of the Assay. The general procedure for conducting the
assay is the same for liquid and spray products. Most importantly, the test
parameters specified for product testing (e.g., H20 hardness, use dilution, organic
soil, neutralizer, contact time, temperature) must also be followed for the
neutralization confirmation assay.
In brief, this assay is designed to simulate the conditions of the efficacy test;
however, sterile carriers are used instead of inoculated carriers. Diluted M bo vis
(BCG) inoculum is added directly to the various sets of subculture media tubes.
The inoculum is quantified by plating on M7H9 or M7H11 agar. This provides
for a quantitative approach to assessing the effectiveness of the neutralizer and
any possible bacteriostatic action resulting from the neutralizer/subculture
media/disinfectant carryover combinations.
10.2 Preparation of Inoculum. In this assay, the inoculum is standardized by diluting
the pooled culture to yield 20.0% ± 1.0% transmittance at 650 nm; dilutions of the
standardized inoculum are applied directly to tubes of subculture media as
described in section 10.4.10. The term "inoculate" is used to describe this
process.
10.2.1
M bovis (BCG) cultures used in neutralization assays are
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generated in the same fashion as the cultures used for other
tuberculocidal assays. Culturing steps are described in section 10
of SOP MB-07 (see ref. 15.4).
10.2.2 For neutralizer assays conducted concurrently with product testing,
use the standardized inoculum prepared for inoculating carriers.
10.2.3 If the neutralizer assay is conducted independently of a product
test, harvest, homogenize and allow culture to settle 10-15
minutes; then, remove the culture remaining in suspension and
standardize (20.0% ± 1.0% transmittance at 650 nm) theM bovis
(BCG) culture as described in Test Culture Preparation section of
SOP MB-07 (see ref. 15.4). Record this information on the
Neutralization Confirmation Assay: Time Recording Sheet forM
bovis (BCG) Inoculum Preparation (see 16.0).
10.2.4 If the product test parameters specify the addition of an organic
soil load to the inoculum, then the neutralization assay will be
performed with the organic soil load added to the inoculum.
Otherwise, the inoculum should be prepared without the addition
of an organic soil load.
10.2.5 Initiate serial ten-fold dilutions of the inoculum by pipetting 1 mL
of the standardized broth culture into 9 mL of MPB or PBDW. Up
to four dilutions (e.g., final dilutions of 10"4 through 10"7) should
typically be used to inoculate the subculture media described
below. The performance of the neutralization assay will be
assessed based on the outcome of the inoculated tubes. Historical
media performance assays will be used as the baseline data for the
evaluation.
10.2.6 To estimate CFU/mL, plate (spread plate or pour plate method)
each of the dilutions in duplicate on M7H9 or M7H11 agar.
Briefly vortex each dilution tube prior to plating. Plate 0.1 mL
aliquots of each dilution for spread plating.
10.2.7 Record the dilution and plating information on the Neutralization
Confirmation Assay: Serial Dilution/Plating Tracking Form forM
bovis (BCG) (see 16.0).
10.2.8
When using the spread plate method for bacterial enumeration,
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M7H9 or M7H11 agar plates are prepared in advance and are
refrigerated prior to use to avoid possible contamination. Bring
refrigerated plates to room temperature before plating the
appropriate dilutions on the surface of the agar. The inoculum
from the respective dilutions is spread over the medium surface.
10.2.9 Incubate plates at 36±1°C for 21-25 days. Count colonies with aid
of a plate counter. Plates that have colony counts over 300 can be
estimated or labeled TNTC. Record the counts on the
Neutralization Confirmation Assay: Inoculum Enumeration Form
for M. bovis (BCG) (see 16.0).
10.3 Disinfectant Sample Preparation.
10.3.1 Follow guidelines for disinfectant sample preparation provided in
SOP MB-22.
10.4 Performing the Assay. The following instructions apply to the analysis of one
neutralizer with one product sample.
10.4.1 Each assay will require eight sterile carriers, four for the Primary
Subculture Treatment and four for the Subculture Media +
Fresh Neutralizer Control. One carrier is used per each inoculum
dilution described in 10.2.5. Use the carrier type required for the
product (i.e. porcelain penicylinders for liquid products or glass
slide carriers for spray products).
10.4.2 Four sterile carriers will be exposed to disinfectant; each will be
exposed to the disinfectant according to the contact time specified
in the test parameters for efficacy testing. The product must be
applied according to specific instructions (e.g., use-dilution,
contact time, spray distance, spray period) provided in the test
parameters. Procedures for treating carriers with liquid and spray
disinfectants are described in SOP MB-07 (see ref. 15.4) and SOP
MB-24 (see ref. 15.3), respectively.
10.4.3 As with product testing, expose the carriers to disinfectant at 30
second or one minute intervals (± 5 seconds of the exact time of
the treatment). If a specific contact time is stipulated by the
manufacturer other than 10 minutes, the interval is modified to
accommodate their claims. Record the carrier transfer information
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on the Neutralization Confirmation Assay: Time Recording Sheet
for M. bovis (BCG) Inoculum Preparation (see 16.0)
10.4.4 Allow product to remain on the carrier per the specified contact
time.
10.4.5 After the last carrier of the set (first 4 carriers) has been treated
with the disinfectant, and the contact time is complete, aseptically
transfer carriers in order in a timed fashion into tubes containing
the specified neutralizer within the ± 5 seconds time limit.
Transfer carriers according to methods specified in the appropriate
efficacy test method SOP. Drain the excess disinfectant from the
carrier prior to the transfer.
Note: For spray products, the amount of neutralizer is 20 mL per
tube (38 xl00 mm Bellco tubes) compared to 10 mL (25 x 100 mm
tubes) used in the CTB test methodfor liquid products.
10.4.6 Thoroughly shake or swirl the neutralizer tube with the carrier in it
and immediately transfer each carrier to a culture tube containing
the primary subculture medium (i.e. MPB). This set of MPB tubes
(4 total tubes) will represent the Primary Subculture Treatment.
Each tube will be inoculated with one of the four inoculum
dilutions.
10.4.7 From each neutralizer tube (4 total), transfer a 2 mL aliquot of
neutralizer to one tube of each subculture medium (M7H9, K or
TB) specified by the test parameters. This portion of the assay is
not timed, but the aliquots should be delivered to the subculture
media as quickly as possible. This set of eight tubes (4 tubes of
each of the two subculture media) represents the Secondary
Subculture + Exposed Neutralizer (exposed to the disinfectant)
Treatment. One tube of each medium will be inoculated with one
of the four inoculum dilutions (e.g., dilution tubes 10"3 through
10"6).
Note: For spray products, eight tubes of each of the additional
two subculture media specified (M7H9, K or TB) will be
inoculated with 2 mL of the exposed neutralizer. Duplicate tubes
for each additional subculture media will be inoculated with one of
the four inoculum dilutions (e.g., dilution tubes 10~3 through 10~6).
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10.4.8 Subculture Media + Fresh Neutralizer Control. The four
remaining sterile carriers will be exposed to neutralizer ONLY,
one carrier per tube of neutralizer) and transferred aseptically to
four tubes of MPB (this portion of the assay is not timed).
Thoroughly shake or swirl the neutralizer tube with the carrier in it
and immediately transfer each carrier to a culture tube containing
MPB. From each neutralizer tube (4 total), transfer a 2 mL aliquot
of neutralizer to one tube of each subculture medium (M7H9, K or
TB) specified by the test parameters. This portion of the assay is
not timed, but the aliquots should be delivered to the subculture
media as quickly as possible. One tube of each medium will be
inoculated with one of the four inoculum dilutions (e.g., dilution
tubes 10"3 through 10"6).
Note: For spray products, eight tubes of each of the additional
two subculture media specified (M7H9, K or TB) will be
inoculated with 2 mL of neutralizer. Duplicate tubes for each
additional subculture media will be inoculated with one of the four
inoculum dilutions (e.g., dilution tubes 10~3 through 10~6).
10.4.9 Subculture Media Only Control. This control contains four tubes
of each preparation of subculture media used in the neutralization
assay. Neutralizer is not added to the media. Each tube of each
medium will be inoculated with one of the four inoculum dilutions
(e.g., dilution tubes 10"3 through 10"6).
10.4.10 Inoculating Subculture Media. Inoculate the Primary Subculture
treatment, the Subculture + Exposed Neutralizer treatment, the
Subculture Media + Fresh Neutralizer control, and the
Subculture Media Only control with 0.1 mL of the diluted M.
bovis (BCG) inoculum (e.g., dilution tubes 10"3 through 10"6).
Inoculate the tubes following the transfer of all carriers and
neutralizer. In addition, refer to Table 1 and Table 2 for
organization of neutralization assays for liquid and spray products,
respectively.
10.4.11 Negative (uninoculated) media controls (one tube of each
medium) will also be included.
10.4.12
Incubate all tubes for 60 days at 36±1°C. If no growth or
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occasional (insufficient for confirmation) growth occurs, incubate
an additional 30 days before recording final results.
10.5 Record results as positive (+) or negative (0) as indicated by the presence or
absence of growth. Prior to entering (+) or (0), perform an acid fast stain. Record
results at 60 days and again at 90 days if necessary. Alternatively, tubes may be
evaluated for growth prior to 60 days (e.g., after 45 days of incubation).
10.6 Identification and Confirmation Testing:
10.6.1 The confirmatory tests used to verify the identity of M bovis
(BCG) are acid fast staining and plating on selective media.
10.6.2 For each medium in the Primary Subculture treatment, the
Subculture + Exposed Neutralizer treatment, the Subculture
Media + Fresh Neutralizer control, and the Subculture Media
Only control, select the tube with growth from the highest dilution
of inoculum (i.e., fewest CFU/mL delivered) and perform acid fast
staining on a sample of the growth. Acid fast rods are typical for
M bovis (BCG).
10.6.3 Additional confirmation can be conducted if necessary to include
isolation streaks on selective media such as M7H9 or M7H11 agar
plates.
10.6.4 Following the 21-25 day incubation period, evaluate the colony
morphology of the organism on M7H9 or M7H11 agar. On M7H9
or M7H11 agar, M. bovis (BCG) typically appears as colorless to
buff-colored, raised, rough growth.
10.6.5 Record confirmation results on the Neutralization Confirmation
Assay: Test Microbe Confirmation Sheet forM bovis (BCG) (see
16.0).
10.7 Interpretation of Results.
10.7.1 Review the plate count data. The plate counts are an essential
element of this assay. One of the four dilutions plated should
provide counts within the target range, 5-100 CFU/mL. Subculture
tubes inoculated from this dilution also received this low level of
challenge, an aspect critical to the determination of neutralization
effectiveness and bacteriostatic activity.
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10.7.1.1 The lack of complete neutralization of the
disinfectant or bacteriostatic activity of the
neutralizer itself may be masked when a high level
ofM bo vis (BCG) is added to the subculture tubes.
10.7.2 The Subculture Media Only Control tubes provide a basis for
comparison of growth in the subculture tubes to growth in the
Primary Subculture treatment, the Subculture + Exposed
Neutralizer treatment, and the Subculture Media + Fresh
Neutralizer control. Growth in Subculture Media Only control
tubes verifies the performance of the media and the presence of M
bovis (BCG) in the diluted inoculum.
No growth or only growth in tubes which received high levels of
inoculum (e.g., a dilution with plate counts which are too
numerous to count) indicates poor media performance.
10.7.3 The pattern of growth in the Subculture Media + Fresh
Neutralizer control tubes as compared to the Subculture Media
Only control tubes is used to assess any bacteriostatic effects
attributable to possible interactions between the neutralizer +
subculture media. Interactions between the media and neutralizer
may result in growth in some media and not others.
No growth or growth only in tubes which received a high level of
inoculum (e.g., the dilution with plate counts which are too
numerous to count) indicates the presence of bacteriostatic
properties as a result of the neutralizer-media interaction.
10.7.4 The pattern of growth in the Secondary Subculture + Exposed
Neutralizer treatment tubes as compared to the Subculture Media
Only control tubes is used to determine the effectiveness of the
neutralizer to inactivate the disinfectant when used under simulated
test conditions. Interactions between the media and neutralizer
may result in growth in some media and not others.
No growth or growth only in tubes which received a high level of
inoculum (e.g., the dilution with plate counts which are too
numerous to count) indicates ineffective neutralization and/or the
presence of bacteriostatic properties as a result of the neutralizer-
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media interaction.
10.7.5 The pattern of growth in the Primary Subculture treatment tubes
as compared to the Subculture Media Only control tubes is
similarly used to determine the effectiveness of the neutralizer
against the disinfectant when used under simulated test conditions.
No growth or growth only in tubes which received a high level of
inoculum (e.g., the dilution with plate counts which are too
numerous to count) indicates ineffective neutralization and/or the
presence of bacteriostatic properties as a result of the neutralizer-
media interaction.
10.7.6 Each tube of negative (uninoculated) media controls must show
no growth.
11.0 DATA ANALYSIS/CALCULATIONS: None
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1 Data will be recorded promptly, legibly, and in indelible ink on testing forms (see.
16.0). Completed forms are archived in notebooks kept in secured file cabinets in
D217. Only authorized personnel have access to the secured files. Archived data
is subject to OPP's official retention schedule contained in SOP ADM-03 (see ref.
15.2).
13.0 QUALITY CONTROL:
13.1 The OPP Microbiology Laboratory conforms to 40 CFRPart 160, Good
Laboratory Practice Standards. Appropriate quality control measures are
integrated into each SOP.
13.2 For quality control purposes, the required information is documented on the
appropriate form(s) (see 16.0).
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1 Any deviation from the standard protocol and the reason for the deviation will be
recorded on the appropriate record sheet (see 16.0); corrective action will be
expeditious.
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15.0 REFERENCES:
15.1 MB-01: Biosafety in the Laboratory
15.2 ADM-03: Records and Archives
15.3 MB -24: T esting of Mycobacterium bovis (B CG) Using the
Germicidal Spray Products as Disinfectants Test
15.4 MB-07: Tuberculocidal Activity of Disinfectants: II. Confirmative in vitro Test
for Determining Tuberculocidal Activity
16.0 FORMS AND DATA SHEETS:
16.1 Neutralization Confirmation Assay: Time Recording Sheet forM bovis (BCG)
Inoculum Preparation
16.2 Neutralization Confirmation Assay: Time Recording Sheet for Carrier Transfers
forM bovis (BCG)
16.3 Neutralization Confirmation Assay: Information Sheet forM bovis (BCG)
16.4 Neutralization Confirmation Assay: Results Sheet forM bovis (BCG)-Liquid
Products
16.5 Neutralization Confirmation Assay: Results Sheet forM bovis (BCG)-Spray
Products
16.6 Neutralization Confirmation Assay: Serial Dilution/Plating Tracking Form forM
bovis (BCG)
16.7
Neutralization Confirmation Assay: Test Microbe Confirmation Sheet forM
bovis (BCG)
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Table 1: Components of the Neutralization Confirmation Assay for Liquid
Products
Treatment/Control
M. bovis (BCG)
Inoculum Dilution
(0.1 mL added per tube)*
Media (O = Tube of Media)
MPB
(20 mL)
M7H9
(20 mL)
Kor TB
(20 mL)
h'imai'\ Siilviilluie
icr3
O/Carrier
\n Tulv
\n Tube
I'nmaiA Siilviilluie
10"4
O/Carrier
\n Tulv
\o liilv
I'riinaiA Subculture
10"5
O/Carrier
\n Tube
\o liilv
I'limaiA Subculture
10"6
O/Carrier
\o liilv
\o liilv
Secondary Subculture +
Exposed Neutralizer
10"3
No Tube
O
O
Secondary Subculture +
Exposed Neutralizer
10"4
No Tube
O
o
Secondary Subculture +
Exposed Neutralizer
10"5
No Tube
o
o
Secondary Subculture +
Exposed Neutralizer
10"6
No Tube
o
o
Subculture Media I'resli
\cuirali/cr ('i»utrol
10"3
O/Carrier
o
o
Subculture Media I'resli
\cuirali/cr ( 'i»iitrol
10"4
O/Carrier
o
o
Subculture Media I'resli
\cuirali/cr ( \»utrol
10"5
O/Carrier
o
o
Subculture Media I'resli
\cuirali/cr ( outm!
10"6
O/Carrier
o
o
Media Only Control
10"3
O
o
o
Media Only Control
10"4
O
o
o
Media Only Control
icr5
O
o
o
Media Only Control
10"6
O
o
o
Uninoculalcd Control
Not inoculated
O
o
o
*Final dilutions of 10"4 through 10"7; example only.
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Table 2: Components of the Neutralization Confirmation Assay for Spray Products
Treatment/Control
M. bovis (BCG)
Inoculum Dilution
(0.1 mL added per tube)*
Media (O = Tube of Media)
MPB
(20 mL)
M7H9
(20 mL)
Kor TB
(20 mL)
h'imai'\ Subculture
10"3
O/Slidc Carrier
No Tube
No Tube
himars Subculture
10"4
O/Slidc Carrier
No Tube
No Tube
hiuiars Subculture
10"5
O/Slidc Carrier
No Tube
No Tube
h'iiiiai'\ Subculture
10"6
O/Slidc Carrier
No Tube
No Tube
Secondary Subculture +
Exposed Neutralizer
10"3
No Tube
OO
OO
Secondary Subculture +
Exposed Neutralizer
lO"4
No Tube
OO
OO
Secondary Subculture +
Exposed Neutralizer
10"5
No Tube
OO
OO
Secondary Subculture +
Exposed Neutralizer
10"6
No Tube
OO
OO
Subculture Media I'rcsli
\euirali/cr ( \»utrol
10"3
O/Slide Carrier
OO
OO
Subculture Media I'rcsli
Ncutrali/cr ( \»utrol
10"4
O/Slide Carrier
OO
OO
Subculture Media I'rcsli
\cutrali/cr ( \>iiii\>I
10"5
O/Slide Carrier
OO
OO
Subculture Media I'rcsli
\cutrali/cr ( \»utrol
10"6
O/Slide Carrier
OO
OO
Media Only Control
10"3
O
O
O
Media Only Control
10"4
O
O
O
Media Only Control
10"5
O
O
O
Media Only Control
10"6
O
O
O
Uninoculated Control
Not inoculated
O
O
O
*Final dilutions of 10"4 through 10"7; example only.
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16.1
Neutralization Confirmation Assay: Time Recording Sheet for M. bovis
(BCG) Inoculum Preparation
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Dale
Type of Product (circle one)
Liquid Spray
EPA Reg. No.
Product Name
Sample No(s).
Nculralizcr
Date/Initials:
Inoculum Settle Time
(from clock/and a timer)
Test Culture %T
Start Time
End Time
/
/
/
/
Comments:
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16.2
Neutralization Confirmation Assay: Time Recording Sheet for Carrier Transfers for M. bovis (BCG)
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Dale
EPA Reg. No.
Product Name
Sample No(s).
Neutralizer
Initials/dale
Drop/Spray
Interval
Carrier Drop/Spray Start Time
(application of the disinfectant)
Carrier Drop/Spray End Time
(transfer to neutralizer then to MPB Tube)
Transfer of Neutralizer to
Additional Subculture Media
Clock
Timer
Clock
Timer
Start Time'
Analyst Dropping/Spraying Carriers:
Analyst Transferring Neutralizer into Subculture Media:
Comments:
1 Transfer of neutralizer into secondary subculture taken from the clock.
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16.3
Neutralization Confirmation Assay: Information Sheet for M. bovis (BCG)
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.
Test Date
Product Name
Lot No.
Sample No.
Expiration Date
Comments:
PARAMETERS FOR PRODUCT TESTING/Confirmed by:
H20 Hardness (CaCO.O ppm
Use Dilution
Specified
Specified
Organic Soil
Ncutralizcr
Specified
Specified
Titrated (Burcl)/Datc/Inil
/
/
HACH/Dalc/Inil
/
/
As Prcparcd/Dalc/Inil
/
As Prcparcd/Dalc/Inil
/
Temperature
Contact Time
Specified
Specified
Chiller Display
Before:
After:
Test tube Waterbalh
Before:
After:
As Tested
Other Parameters
Specified
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SOP No. MB-11-03
Date Revised 09-02-10
Page 21 of 26
TEST MICROBE INFORMATION/Confirmed by:
Org. Control No.
21-25 Day M. bovis (BCG) Culture
% Transmillancc
Dale Initialed:
Dale Harvested:
REAGENT/MEDIA INF ORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.
16.4
Neutralization Confirmation Assay: Results Sheet for M. bovis (BCG) -Liquid Products
OPP Microbiology Laboratory
TEST INF ORMATION/Confirmed by:
EPA Reg. No.
Test Date
Product Name
Ncutrali/cr
Sample No.
Product Type (circle one)
Spray Liquid
TEST RESULTS: Date Recorded/Initials: 60 Day
/90Day:
60 Day Results/90 Day Results
Treatment*
Dilutions of M. hovis
BCG (Final Dilutions)
Primary Subculture (MPB)
/
/
/
/
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SOP No. MB-11-03
Date Revised 09-02-10
Page 22 of 26
Secondary Subculture ( ) + Ex. Ncut.
/
/
/
/
Secondary Subculture ( ) + Ex. Ncul.
/
/
/
/
Subculture Media (MPB) + Fresh Ncul. Control
/
/
/
/
Subculture Media ( ) + Fresh Ncul. Control
/
/
/
/
Subculture Media ( ) + Fresh Ncul. Control
/
/
/
/
Subculture Media (MPB) Only Control
/
/
/
/
Subculture Media ( ) Only Control
/
/
/
/
Subculture Media ( ) Only Control
/
/
/
/
Uninoculalcd:
Pr. Subculture Media (MPB) Negative Control
/
Subculture Media ( ) Negative Control
/
Subculture Media ( ) Negative Control
/
*Fill in media type within the brackets "( )"
Pr. = Primary
SUMMARY OF RESULTS: Date/Initials:
Bacteriostatic Effect Observed?
Yes No
If ycs. which treatment/media?
Comments:
16.5
Neutralization Confirmation Assay: Results Sheet for M. bovis (BCG) - Spray Products
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
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SOP No. MB-11-03
Date Revised 09-02-10
Page 23 of 26
EPA Reg. No.
Test Dale
Product Name
Nculralizcr
Sample No.
Product Type (circle one)
Spray Liquid
TEST RESULTS: Date Recorded/Initials: 60 Day
/90Day:
60 Day Results/90 Day Results
Treatment*
Dilutions of M. hovis
BCG (Final Dilutions)
Primary Subculture (MPB)
/
/
/
/
Secondary Subculture ( ) + Ex. Neiit.**
1 / 1
1 / 1
1 / 1
1 / 1
Secondary Subculture ( ) + Ex. Ncul.**
1 / 1
1 / 1
1 / 1
1 / 1
Subculture Media (MPB) + Fresh Ncul. Control
/
/
/
/
Subculture Media ( ) + Fresh Ncul. Control**
1 / 1
1 / 1
1 / 1
1 / 1
Subculture Media ( ) + Fresh Ncut. Control**
1 / 1
1 / 1
1 / 1
1 / 1
Subculture Media (MPB) Only Control
/
/
/
/
Subculture Media ( ) Only Control
/
/
/
/
Subculture Media ( ) Only Control
/
/
/
/
Uninoculatcd:
Pr. Subculture Media (MPB) Negative Control
/
Subculture Media ( ) Negative Control
/
Subculture Media ( ) Negative Control
/
*Fill in media type within the brackets "( )".
** There are two tubes per subculture medium; #1 #2/#l #2 for 60 and 90 day results, respectively.
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SOP No. MB-11-03
Date Revised 09-02-10
Page 24 of 26
SUMMARY OF RESULTS: Date/Initials:
Bacteriostatic Effect Observed?
Yes No
If yes. which treatment/media?
Comments:
16.6
Neutralization Confirmation Assay: Serial Dilution/Plating Tracking Form for M. bovis (BCG)
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.
Test Dale
Name
Nculralizcr
Sample No.
Organism Control #
Confirmed bv:
Dilution Tube
10-1
10"2
10-3
icr4
10"5
10-6
Starting volume of diluent
Volume added to serial dilution tube (1 mL)
Volume plated (0.1 mL)
Final dilution factor (used for calculations)
Number of plates per dilution
Plating medium
Comments:
N/A = not applicable
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SOP No. MB-11-03
Date Revised 09-02-10
Page 25 of 26
TNTC = too numerous to count
RESULTS
Date Recorded/Initials:
CFU per Dilution Plate* (plate
1/plate 2)
/
/
/
/
Average CFU per inL
*Final dilution.
REAGENT/MEDIA INFORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.
16.7
Neutralization Confirmation Assay: Test Microbe Confirmation Sheet for M. bovis (BCG)
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.
Test Date
Name
Test Organism
Sample No.
Comments:
Source:
Date/
Slain
Media Information
Results
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SOP No. MB-11-03
Date Revised 09-02-10
Page 26 of 26
Tubc/Plalc ID
Initials
Results*
Type
Prep. No.
Inc. Time/
Temp.
Date/
Initials
Colony
Characteristics
*Record Acid Fast results as AFR=acid fast rods.
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