US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for
Screening of Polished Stainless Steel Penicylinders,
Porcelain Penicylinders, and Glass Slide Carriers Used in
Disinfectant Efficacy Testing

SOP Number: MB-03-07

Date Revised: 02-14-17

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SOP No. MB-03-07
Date Revised 02-14-17
Page 1 of 8

SOP Number

MB-03-07

Title

Screening of Polished Stainless Steel Penicylinders, Porcelain
Penicylinders, and Glass Slide Carriers Used in Disinfectant Efficacy
Testing

Scope

This protocol describes the procedures for the preparation of carriers
for the AO AC Use-Dilution Methods, the AO AC Tuberculocidal
Activity of Disinfectants Test, the AO AC Germicidal Spray Products
as Disinfectants Test, the Disinfectant Towelette Test, and the AOAC
Sporicidal Activity of Disinfectants Test. See section 15.

Application

The carriers discussed in this SOP are used in methods designed to
evaluate the performance of disinfectants and sterilants against the
prescribed microbes.





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Date SOP withdrawn: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-03-07
Date Revised 02-14-17
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TABLE OF CONTENTS
Contents	Page Number

1.

DEFINITIONS

3

2.

HEALTH AND SAFETY

3

3.

PERSONNEL QUALIFICATIONS AND TRAINING

3

4.

INSTRUMENT CALIBRATION

3

5.

SAMPLE HANDLING AND STORAGE

3

6.

QUALITY CONTROL

3

7.

INTERFERENCES

3

8. NON-CONFORMING DATA

3

9.

DATA MANAGEMENT

3

10.

CAUTIONS

3

11.

SPECIAL APPARATUS AND MATERIALS

4

12.

PROCEDURE AND ANALYSIS

4

13.

DATA ANALYSIS/CALCULATIONS

8

14.

FORMS AND DATA SHEETS

8

15.

REFERENCES

8

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1. Definitions

Abbreviations/definitions are provided in the text.

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
balance), EQ-04 (spectrophotometer) and EQ-05 (timers).

5. Sample

Handling and
Storage

1.	Refer to SOP MB-22, Preparation and Sampling Procedures for
Antimicrobial Test Substances, and SOP COC-01, Chain of Custody
Procedures.

2.	Store BTC 835 at room temperature in a cabinet designed to contain
flammable materials.

3.	Dilute BTC 835 prior to use. Conduct testing within three hours of
dilution. Store diluted product at room temperature.

6. Quality Control

For quality control purposes, the required information is documented on the
appropriate form(s) (see section 14).

7. Interferences

Failing a physical screen may involve varying levels of physical damage or
imperfection. Thus, examples of carriers passing and failing physical screens
are studied during the training process.

8. Non-
conforming
Data

1.	Manage non-conforming data as specified in the study protocol;
procedures will be consistent with SOP ADM-07, Non-Conformance
Reports.

2.	Do not use polished stainless steel, porcelain, and glass slide carriers that
fail physical screening.

3.	Do not use polished stainless steel carriers that fail the biological
screening, unless carriers are physically re-screened, re-cleaned and pass
biological screening.

4.	All polished stainless steel carriers used for product efficacy testing that
subsequently give a positive (with growth) result must be cleaned and
biologically rescreened in the same manner before reusing.

9. Data

Management

Data will be archived consistent with SOP ADM-03, Records and Archives.

10. Cautions

All carriers used for disinfectant testing must be screened in advance
according to procedures outlined in this protocol.

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11. Special

Apparatus and
Materials

1.	Disinfectant for biological screening of steel penicylinders: BTC 835:
50% n-Alkyl (50% C14, 40% C12, 10% Ci6) Dimethyl Benzyl Ammonium
Chloride (Stepan Co.).

2.	Carriers, polished stainless steel penicylinders for the AO AC Use-dilution
Method. Polished stainless steel cylinders, 8±1 mm outer diameter, 6±1
mm inner diameter, 10±1 mm length; type 304 stainless steel, SS 18-8 (S
& L Aerospace Metals, or Fisher Scientific Cat. No. 07-907-5Q).

3.	Carriers, porcelain penicylinders for the AO AC Tuberculocidal Activity of
Disinfectants Test and the AOAC Sporicidal Activity Test. Porcelain, 8±1
mm outer diameter, 6±1 mm inner diameter, 10±1 mm long (CeramTec
Ceramic; Cat. No. LP15819 0645).

4.	Carriers, glass slide carriers for the AOAC Germicidal Spray Products as
Disinfectants Test. 25x25 mm (or comparable size) borosilicate glass
cover slips with number 4 thickness (Chemglass Life Sciences, Cat. No.
EPA-1112-151CLSR).

5.	Carriers, glass slide carriers for the Disinfectant Towelette Test. 25x75
mm (or comparable size) borosilicate glass cover slips with number 4
thickness (Bellco Glass, Inc., item number: 1916-S0134) or Fisherfinest®
Premium Frosted Microscope Slides (Fisher Scientific, Cat. No. 12-544-

2).

6.	Phenolphthalein. For detecting the presence of NaOH in the rinse water
during cleaning of polished stainless steel penicylinders. Phenolphthalein
1% (w/v) solution in alcohol.

7.	Sterile deionized water. For preparing 500 ppm solution of BTC 835.

8.	Sodium hydroxide (NaOH) IN solution. For cleaning polished stainless
steel penicylinders.

9.	Triton X-100. For washing used porcelain penicylinders for the AOAC
Sporicidal Activity Test.

12. Procedure and
Analysis



12.1 Polished

Stainless Steel
Penicylinders
(AOAC Use
Dilution
Methods)

a.	Physical Screening: Visuallv screen polished stainless steel carriers.
Discard carriers that fail physical screening due to visible damage
(dull, chipped, dented, or gouged). Place carriers that pass physical
screening in a container and label with date and "Physically
Screened."

b.	Cleaning: Soak the physically screened carriers overnight (approx. 12
hr) in IN NaOH and rinse several (3-4) times with tap water. Collect
a portion of the last rinse water and add 2-3 drops of 1%

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phenolphthalein. If any NaOH remains, the phenolphthalein turns
pink, indicating the need for additional rinsing. Continue to rinse the
carriers until the addition of phenolphthalein to the collected portion
of the rinse water does not produce a color change (to pink). Rinse
twice more with DI water. Allow carriers to air dry and store in a
closed container marked with date and "Cleaned Carriers/Not
Biologically Screened."

c.	Biological Screening:

i.	Place the cleaned carriers into 25x 150 mm test tubes, 20 per
tube.

ii.	Cover the carriers with reagent grade water (i.e., deionized
water) and cap.

iii.	Autoclave at 121°C for 20 min; cool and store at room
temperature.

iv.	Perform AO AC Use-Dilution testing (see SOP MB-05, Use
Dilution Method) on each carrier using the following
parameters: Staphylococcus aureus, 500 ppm solution of
BTC 835 prepared using sterile deionized water, 20±1°C, no
organic soil, ten-minute exposure period, and letheen broth as
the neutralizer. Use primary subculture tubes only. Use test
forms from SOP MB-05, Use Dilution Method, as necessary
(e.g., Processing Sheet, Carrier Counts Form, etc.).

v.	Conduct control counts (e.g., six total carriers per test day) as
per section 12.3 of SOP MB-05, Use Dilution Method.

vi.	Record screening results in the Use-Dilution Test Results
Sheet for Screening Carriers (see section 14).

vii.	If growth is observed in the subculture tube, the carrier fails.
Autoclave, physically screen, wash, and repeat biological
screening of failed carriers.

viii.	Collect all carriers that pass biological screening. Autoclave
and re-wash carriers as per section 12.1.b. After air drying,
assign a master media/reagent preparation number (see SOP
QC-15, Media Prep and Sterilization Run Numbers) to the
set. These passing carriers represent a master pool of official
carriers to be used in efficacy evaluations.

d.	Preparation of Penicylinders for Efficacy Testing.

i. Prior to conducting an AO AC Use-Dilution test, remove the
	required number of cleaned carriers from the master pool of

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biologically screened carriers (i.e., current master prep; see
section 12.1 .c.viii).

ii.	Place the cleaned carriers into 25x 150 mm test tubes, 20 per
tube.

iii.	Cover the carriers with reagent grade water (i.e., deionized
water) and cap.

iv.	Autoclave at 121°C for 20 min; cool and store at room
temperature.

v.	Fill out a media/reagent preparation sheet and assign a
preparation number (see SOP QC-15, Media Prep and
Sterilization Run Numbers) to the autoclaved carriers.

vi.	Following efficacy testing, identify/separate (e.g., place all
positive carriers in one container and mark with tape) carriers
giving a positive result (i.e., growth). Autoclave all carriers.
Re-clean (see section 12.1 .b) the following: carriers for which
test results were negative, carriers used for enumeration, and
unused carriers still in 25 x 150 mm test tubes. Allow carriers
to air dry and return to the master pool of biologically
screened carriers.

vii.	Carriers giving a positive result (i.e., growth) in efficacy
testing must be cleaned and biologically rescreened prior to
subsequent reuse in testing. Do not return these carriers to
the master pool.

12.2 Porcelain
Penicylinders
(AOAC
Tuberculocidal
Activity of
Disinfectants
Test)

a.	Physical Screening: Examine porcelain carriers individually for
scratches, nicks, spurs, and discolorations. Do not use carriers that
do not pass the screening process. Record screening results in the
Physical Screening of Carriers Record Form (see section 14).

b.	Cleaning: Rinse unused carriers gently in water three times to
remove loose material and drain.

i.	Place clean porcelain carriers in multiples of 10 or 20 in
capped Erlenmeyer flasks or 20x 150 mm tubes.

ii.	Fill out a media/reagent preparation sheet to assign a
preparation number to the carriers. Sterilize 20 minutes at
121°C or for 2 hours at 180°C in air oven; cool and store at
room temperature. Handle porcelain carriers with care.
Minimize carrier movement and avoid excessive contact
between carriers that might result in damage.

iii.	Discard all porcelain carriers used in TB product testing;

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carriers are single use.

12.3 Porcelain
Penicylinders
(AOAC
Sporicidal
Activity Test
Method)

a.	Physical Screening: Prior to use, examine porcelain carriers
individually and discard those with scratches, nicks, spurs, or
discolorations. Record screening results in the Physical Screening of
Carriers Record Form (see section 14).

b.	Cleaning: Rinse unused carriers gentlv in water three times to
remove loose material and drain. Place rinsed carriers into Petri
dishes matted with 2 layers of filter paper in groups of 15 carriers per
Petri dish or place carriers into 25x150 mm tubes (10 carriers per
tube).

i.	Sterilize 20 minutes at 121 °C or for 2 hours at 180°C in air
oven; cool and store at room temperature. Note: Handle
porcelain carriers with care when placing in Petri dishes.
Minimize carrier movement and avoid excessive contact
between carriers that might result in chips and cracks.

ii.	Wash carriers with Triton X-100 and rinse with water four
times for reuse.

iii.	Carriers for this test may be re-used after cleaning and
autoclaving.

12.4 Glass Slide
Carriers
(AOAC
Germicidal
Spray Products
as

Disinfectants
Test and
Disinfectant
Towelette
Test)

a.	Physical Screening: Visuallv screen glass slide carriers (25x25 mm
and 25x75 mm) for scratches, chips or cracks and discard those
which are damaged or defective. Record screening results in the
Physical Screening of Carriers Record Form (see section 14).

b.	Cleaning: Prior to carrier preparation for testing, rinse the carriers
once with DI water, rinse three times with 95% ethyl alcohol, and
finally rinse three times with DI water.

i.	Drain and allow carriers to dry before use.

ii.	For slides prepared for use in the AOAC Germicidal Spray
Products as Disinfectants Test (25x25 mm), place one glass
slide carrier into a glass Petri dish with 2 pieces of Whatman
No. 2 filter paper. Fill out a media/reagent preparation sheet
to assign a preparation number to a set of carriers.

iii.	For slides prepared for use in the Disinfectant Towelette Test
(25x75 mm), place one glass slide carrier into a glass Petri
dish, directly onto the glass surface of the dish (no filter paper
will be added to the Petri dishes). Fill out a media/reagent
preparation sheet to assign a preparation number to a set of
carriers.

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iv.	Autoclave for 45 minutes at 121°C with a 30-minute dry
cycle; cool; store at room temperature.

v.	Discard all glass slide carriers used in testing. They may not
be reused.

13. Data Analysis/
Calculations

1.	For control counts for biological screening, calculations will be computed
using a Microsoft Excel spreadsheet (see section 14 of SOP MB-05, Use
Dilution Method). Both electronic and hard copies of the spreadsheet will
be retained. For direct plating, counts up to 300 and their associated
dilutions will be included in the calculations.

2.	Alternatively, Petrifilm may be used for enumeration of bacterial
organisms. Follow manufacturer's instructions for preparation and
incubation of Petrifilm cards. Note: At a minimum, conduct a culture
purity check (isolation streak) using suspension from one dilution tube of
one carrier or pooled set.

14. Forms and Data
Sheets

Test Sheets. Test sheets are stored separately from the SOP under the
following file names:

Physical Screening of Carriers Record Form MB-03-07 F1 .docx

AO AC Use-Dilution Test Information Sheet for
Biological Screening of Carriers

AO AC Use-Dilution Test Results Sheet for
Biological Screening of Carriers

MB-03-07 F2.docx

MB-03-07 F3.docx

15. References

1.	Official Methods of Analysis. Method 955.14 - Salmonella enterica.
Posted March 2013. AO AC INTERNATIONAL, Gaithersburg, MD.

2.	Official Methods of Analysis. Methods 955.15 - Staphylococcus aureus.
Posted September 2013. AO AC INTERNATIONAL, Gaithersburg, MD.

3.	Official Methods of Analysis. Method 964.02 - Pseudomonas aeruginosa.
Posted September 2013. AO AC INTERNATIONAL, Gaithersburg, MD.

4.	Official Methods of Analysis. 2012. 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, (Method 965.12 In vitro Test for
Determining Tuberculocidal Activity).

5.	Official Methods of Analysis. Method 961.02. Posted March 2013.

AOAC INTERNATIONAL, Gaithersburg, MD.

6.	Official Methods of Analysis. Method 966.04. Posted March 2013.

AOAC INTERNATIONAL, Gaithersburg, MD.

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