Standard Operating Procedure for Disinfectant Product Preparation and Sampling Procedures SOP Number: MB-22-03 Date Revised: 10-30-13


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 Product Preparation and
Sampling Procedures
SOP Number: MB-22-03
Date Revised: 10-30-13

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 1 of 7
SOP Number
MB-22-03
Title
Disinfectant Product Preparation and Sampling Procedures
Scope
This SOP describes procedures for the preparation and sampling of
liquid, spray, and towelette disinfectants for efficacy testing.
Application
Procedures are applicable to all disinfectant efficacy test methods
performed in the laboratory.


Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:
Data SOP issued:

Controlled copy number:

Date SOP withdrawn:

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 2 of 7
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
4
7.
INTERFERENCES
4
8. NON-CONFORMING DATA
4
9.
DATA MANAGEMENT
4
10.
CAUTIONS
4
11.
SPECIAL APPARATUS AND MATERIALS
4
12.
PROCEDURE AND ANALYSIS
4
13.
DATA ANALYSIS/CALCULATIONS
7
14.
FORMS AND DATA SHEETS
7
15.
REFERENCES
7

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 3 of 7
1. Definitions
1.	Product sample = Representative container of a disinfectant sample.
Multiple containers from the same production lot are all considered to be
the same sample.
2.	Sampling = Procedure in which part of a product sample is removed from
a container for testing, (see sectionl5.1).
3.	Ready-to-use product = Product that requires no activation or dilution.
4.	Concentrated liquid product = Liquid product that requires a dilution
prior to use.
5.	Spray products = trigger, aerosol or pump based products.
6.	Towelette products = a pre-moistened wipe-based disinfectant.
7.	COC = chain of custody.
2. Health and
Safety
1.	Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Material Safety Data
Sheet for hazards associated with products.
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 disinfectants.
3.	A chemical fume hood or other containment equipment, such as a BSC is
employed when performing tasks with products.
3. Personnel
Qualifications
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Calibration
Refer to SOP EQ-03 (weigh balances) and QC-19 (pipettes) for details on
method and frequency of calibration.
5. Sample Storage
1.	Store disinfectants according to the manufacturer's recommendations if
stipulated, or at room temperature, in Room D204. Store flammable
products in the flammable cabinet located in Room B204.
2.	Activate or dilute disinfectant products within three hours of testing to
ensure stability of the product unless test parameters specify otherwise.
3.	Follow chain-of-custody (COC) guidelines in SOP COC-01, Disinfectant
Sample Log-in, Tracking and Disposal.
4.	Store products that require activation and have an extended shelf life prior
to testing in a fume hood using secondary containment. Identify the
product by name, sample number and preparation number. Establish a
new for activated product. Once product is used archive COC seal in the
COC laboratory notebook.
5.	Use permanent marker to record the test date on the container used for

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 4 of 7

testing.
6. Prepare a product-specific Media/Reagent Preparation Sheet.
6. Quality Control
Required information is documented on the appropriate record form(s). See
section 14.
7. Interferences
None
8. Non-conforming
Data
Errors in the preparation of the disinfectant, if not corrected prior to the
efficacy test, will result in a repeat of the study.
9. Data
Management
Data will be archived consistent with SOP ADM-03, Records and Archives.
10. Cautions
a.	Strict adherence to the protocol is necessary for the validity of the
test results.
b.	Prepare products inside a BSC and follow aseptic techniques. A
fume hood maybe necessary for strong samples or caustic
materials such as strong acids.
c.	Do not place a pipette or any other instrument inside the product
container.
11. Special
Apparatus and
Materials
a.	Sterile 25 x 100 mm tubes - used for testing liquid disinfectants.
b.	Volumetric glassware (pipettes, flasks, etc.) - to measure liquids
for product dilution and for dispensing disinfectants.
c.	Ethanol - for cleaning the outside of the sample container
(70%) and preparation of spray bottles (100%).
d.	Weigh Balance (Weighs 0 to 5100 g) - to weigh sample containers
prior to and after sample removal.
e.	Sterile spray bottles - used for products requiring application by
sprays.
f Forceps - to open sample container or feed towelettes through
container aperture.
g. Product diluent - Sterile liquid used to make product dilutions, (e.g.
tap water, de-ionized water or hard water).
12. Procedure and
Analysis
a.	Prepare a product-specific Media/Reagent Preparation Sheet.
Calculate the volume of product needed based on the method in use
or as defined in the study protocol and adjust as necessary.
b.	Retrieve product from sample storage.
c.	If applicable, remove COC seal and record on COC sample log-in
and tracking book.
d.	For products requiring the use of hard water as the diluent refer to
MB-30 (AOAC hard water) orMB-25 (OECD hard water) for
instructions.

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 5 of 7
12.1 Sampling and
Preparation of
Liquid
Disinfectants
a.	Weigh the product container and record the weight on the
Media/Reagent Preparation Sheet. Gently shake the container of a
liquid product prior to opening. Remove the cap. Do not touch the
inside surface of the cap. If present, carefully remove the seal
attached to the lip of the spout with sterile instruments (i.e., razor
blade, forceps).
b.	Aseptically pour the necessary amount of product to make the use
dilution into a sterile beaker based on the calculations on the
Media/Reagent Preparation Sheet. If the sample is not used
immediately, cover the beaker with foil. After dispensing, place
cap on the product container and secure tightly.
c.	Weigh the sample container and record weight on the
Media/Reagent Preparation Sheet.
d.	For ready-to-use products dispense 10 ml aliquots directly from the
beaker into sterile 25 x 100 mm tubes.
e.	For concentrated products, aseptically prepare the disinfectant
dilution required for the test using the product dispensed in the
beaker (see step 12.1 .b). Prepare all dilutions using sterile
standardized volumetric glassware.
f Examples of disinfectant product dilutions:
i.	If a product requires a 1:10 dilution, 1 part product is added to
9 parts diluent.
ii.	'/2 ounce into gallon of diluent = 1:256 dilution (1 part product
+ 255 parts diluent)
iii.	1 ounce into gallon of diluent = 1:128 dilution (1 part product
+ 127 parts diluent)
iv.	3/4 cup (6 oz.) into gallon of diluent = 6:128 dilution (6 parts
product +122 parts diluent)
g.	For diluted products, use >1.0 mL or 1.0 g of the product sample to
prepare the final solution to be tested. Use v/v dilutions for liquid
products and w/v dilutions for solids. Round to 2 decimal places
toward a more concentrated product.
h.	Dispense 10 mL aliquots of the diluted disinfectant into sterile 25 x
100 mm test tubes. Place tubes in a water bath for approximately
10 minutes to allow product to equilibrate to the required
temperature. Record the test date on the container using permanent
marker. Complete the Media/Reagent Preparation Sheet and COC
forms and return the container to the appropriate storage location.
12.2 Sampling and
Preparation of
a. For spray products which require dilution proceed as described in

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 6 of 7
Spray
Disinfectants
sections 12. la through 12.1g.
b.	For spray products not supplied with their own spray bottles,
prepare a sterile spray bottle used to dispense the test chemical, as
follows:
c.	Working in a BSC, add approximately 10 oz. (~300mL) of ethanol
to the spray bottle. Pump the trigger several times to fill the
nozzle/sprayer with ethanol. Let stand approximately 10 minutes.
d.	Spray out approximately 50 mL of ethanol into a sterile beaker.
e.	Add 10 oz. of sterile DI water to each bottle. Spray out
approximately 50 mL of the water into a sterile beaker. Repeat this
step one more time.
f Add a small volume (approximately 25 mL) of sterile DI water to
the spray bottle and spray approximately 10 mL of the water out
into a sterile tube.
g.	To check sterility, filter 10 mL of water through a 0.45 |im or 0.2
|im filter unit and apply filter to surface of TS A plate. Incubate at
36±1°C for 3-10 days.
h.	Close nozzle/lid of bottle and leave in the BSC until the efficacy
test is completed to ensure sterility.
i.	Dispense an appropriate amount of product into the spray bottle (s)
to conduct the test. Using a permanent marker add the product
name and the test date on the spray bottle. Discard remaining
product at the end of the test day. A new preparation of the
product is required for each test day.
j. For aerosol cans, shake the can 25 times prior to use, unless
otherwise specified by the manufacturer.
k. Spray the product for 10-15 seconds prior to testing to ensure
sprayer is operating correctly and product is dispensed properly.
1. Record the test date on the spray bottle or container using
permanent marker. Complete the Media/Reagent Preparation Sheet
and COC forms and return the container to the appropriate storage
location.
12.3 Sampling and
Preparation of
Towelette
Disinfectants
a.	Weigh container and record weight on Media/Reagent Preparation
Sheet.
b.	For towelette canisters, carefully remove the seal attached to the lip
of the cover with sterile instruments (i.e., razor blade, forceps).
c.	Perform all manipulations of the towelettes aseptically.
d.	Dispense towelette samples as specified on the product label.

-------
SOP No. MB-22-03
Date Revised 10-30-13
Page 7 of 7

e. For dispenser-fed towelettes in canisters, using sterile gloves or
flame sterilized forceps start towelette feed. Thread a corner of the
first towelette, from the center of the roll, through the container
dispenser, if applicable, and pull out first towelette. The remaining
towelettes should automatically feed through the dispenser.
Remove and discard 2-3 towelettes.
f For canisters, invert 3-4 times or roll container to distribute the
liquid before removing towelettes for the wiping procedure.
g.	Close the lid of the towelette dispenser when not actively removing
towelettes.
h.	Products formulated as single towelettes in packets are sampled
from the product box, opened, and the towelette removed
aseptically (using sterile gloves or flame-sterilized forceps).
i.	After completion of the test or study, weigh the towelette canister
and record the weight.
j. Record the test date on the container using permanent marker.
Complete the Media/Reagent Preparation Sheet and COC forms
and return the container to the appropriate storage location.
12.4 Documentation
on
Media/Reagent
Preparation
Sheet
a. Record all required information on the Media/Reagent Preparation
Sheet (refer to SOP QC-15, Media Prep and Sterilization Run
Numbers).
13 Data Analysis/
Calculations
None
14 Forms and Data
Sheets
1. Media/Reagent Preparation Sheets. Sheets are stored separately from the
SOP under the following file names:
Media/Reagent Preparation Sheet for Liquid MB-22-03 F1 .xlsx
Products
Media/Reagent Preparation Sheet for Spray MB-22-03_F2.xlsx
Products
Media/Reagent Preparation Sheet for Towelette MB-22-03 F3 .xlsx
Products
Media/Reagent Preparation Sheet for Sterile MB-22-03 F4.xlsx
Spray Bottle
15 References
1. ISO/EEC: 17025 (2005): Section 5.7 Sampling.

-------