US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Sterility Assessment of Disinfectant
Product Samples
SOP Number: QC-18-05
Date Revised: 11-01-11
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SOP No. QC-18-05
Date Revised 11-01-11
Page 1 of 11
EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Sterility Assessment of Disinfectant Product Samples
SOP Number: QC-18-05
Date Revised: 11-01-11
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Withdrawn By: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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SOP No. QC-18-05
Date Revised 11-01-11
Page 2 of 11
TABLE OF CONTENTS
Contents Page Number
1.0 SCOPE AM) APPLICATION 3
2.0 DEFINITIONS 3
3.0 HEALTH AND SAFETY 3
4.0 CAUTIONS 3
5.0 INTERFERENCES 3
6.0 P ERSONNEL QUALIFICATIONS 3
7.0 SPECIAL APPARATUS AND MATERIALS 3
8.0 INSTRUMENT OR METHOD CALIBRATION 4
9.0 SAMPLE HANDLING AND STORAGE 4
10.0 PROCEDURE AM) ANALYSIS 4
11.0 DATA ANALYSIS/CALCULATIONS 8
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 8
13.0 QUALITY CONTROL 8
14.0 NONCONFORMANCE AND CORRECTIVE ACTION 8
15.0 REFERENCES 8
16.0 FORMS AM) DATA SHEETS 8
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1.0 SCOPE AND APPLICATION:
1.1 This protocol describes quality control practices that may be performed on
disinfectant product samples to assess their sterility.
2.0 DEFINITIONS: None
3.0 HEALTH AND SAFETY:
3.1 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: None
5.0 INTERFERENCES:
5.1 Aseptic procedures must be followed during this assay to avoid accidental
contamination of the product. Exposing the product to external contaminants
during opening and dispensing, and the use of non-sterile laboratory supplies may
interfere with the outcome of this analysis. Quality control measures for media,
reagents and pre-sterilized supplies used in this evaluation must be followed as
outlined in SOP QC-11, Performance and Sterility of Media and Reagents.
5.2 Cloudiness of media tubes caused by interaction of the disinfectant and medium
may interfere with the evaluation of the media tubes. A trypticase soy agar (TSA)
plate is streaked for isolation and a Gram stain is performed to verify the presence
or absence of microbial growth.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable about 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:
7.1 Incubator with temperature reading at the appropriate temperatures, 36±1°C and
55±1°C.
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7.2 VITEK® 2 Compact Automated Identification System for microorganisms.
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 Refer to the laboratory equipment calibration and maintenance SOPs (SOP EQ
series) for details on method and frequency of calibration.
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.
10.0 PROCEDURE AND ANALYSIS:
10.1 General Guidelines:
10.1.1 Procedures such as opening the product container, preparing serial
dilutions, and inoculating media must be performed under aseptic
conditions in a biological safety cabinet.
10.1.2 The sterility assessment should be performed when the product
container is initially opened.
10.1.3 Sterility assessments may be performed prior to or concurrently with
an efficacy test.
10.1.4 Always follow appropriate chain of custody procedures as stipulated in
SOP COC-01, Sample Login and Tracking.
10.1.5 A neutralizer recommended for the product=s active ingredient(s)
should be used as the diluent (see 10.4). Information on the
appropriate neutralizer is included in the product=s test parameter.
10.2 Preparation and Opening the Sample Container:
10.2.1 The container must be opened under aseptic conditions in a biological
safety cabinet.
10.2.2 For liquids, prior to opening the container, gently shake the container
and thoroughly clean the area around the cap and spout with 70%
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ethanol. Allow the surface to dry. Remove the cap. Do not touch the
inside surface of the cap. If a seal is present, carefully remove the seal
attached to the lip of the spout with sterile instruments (i.e., razor
blade, forceps).
10.2.3 For spray products, shake the container at least 25 times immediately
prior to assay. Remove cap and clean the nozzle and wipe top of can
with 70% alcohol. Allow the surface to dry. Don sterile gloves.
Spray the product for 10-15 seconds prior to collection of sample.
10.2.4 For towelette products, clean the dispenser or packaging with 70%
alcohol. Allow the surface to dry. Wearing sterile latex gloves,
aseptically remove a towelette with sterile forceps.
10.3 Collection of the Sample:
10.3.1 For liquids, pour approximately 10 mL of the sample into a sterile
beaker. Do not place a pipette or any other instrument inside the
product container. Place cap on the product container and secure
tightly. Initiate serial dilutions from this sample (see 10.4).
10.3.2 For a spray product, spray the product into a sterile beaker for 20-30
seconds. Allow the product to settle (i.e., run down the sides of the
beaker). Approximately 10 mL of liquid should be collected by this
method. Initiate serial dilutions from this sample (see 10.4). When
collecting the sample (i.e., spraying), hold the spray can in one hand,
perpendicular to the biological safety cabinet surface. Hold the beaker
in the other hand, positioning it so that it is parallel to the biological
safety cabinet surface with the open end facing the nozzle of the spray
can. The potential for contamination of the sample (i.e., contact of
product with sprayer's gloved hand) is reduced as compared to
positioning the spray can directly over the beaker (on biological safety
cabinet surface) and spraying down into the beaker.
10.3.3 For a towelette, if saturated, carefully express the liquid from a single
towelette by squeezing out the liquid into a sterile beaker. Use sterile
forceps to manipulate the towelette. Collect approximately 10 mL of
the liquid. More than one towelette may be required to collect a 10 mL
sample. Initiate serial dilutions from this sample (see 10.4).
If the towelette is not saturated with liquid, carefully place a folded
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towelette into 20 mL of letheen broth (38 mm x 100 mm tube) or other
suitable neutralizer. Gently agitate the tube containing the towelette.
Carefully extract the towelette with sterile forceps. Express the liquid
during the extraction. Initiate serial dilutions from the residual mixture
remaining in the tube (see 10.4).
10.3.4 A preparation number must be given to the sample. Fill in information
on a Media Preparation form as stipulated in SOP QC-15, Media and
Reagent Preparation: Assigning Prep and Sterilization Run Numbers.
In addition, fill in appropriate information on the Sterility Assessment
of Product Sample Record (see 16.1).
10.4 Preparation of Serial Dilutions:
10.4.1 Prepare the first dilution by pipetting 1 mL of the sample into a 9 mL
tube of diluent. Prepare dilutions of 1 x 10"1 through 1 x 10"5. Vortex
each dilution tube prior to a transfer. Include a tube of undiluted
sample (approx. 5 mL in a 20 mm x 150 mm tube) in the dilution set.
10.5 Inocul ati on of Culture Medi a:
10.5.1 Label the media tubes to correspond with the appropriate dilution.
Inoculate 10 mL tubes (20 mm x 150 mm) of letheen broth and fluid
thioglycollate medium in duplicate with 1 mL of each dilution; include
the undiluted sample as well.
10.5.2 Include one tube of letheen broth and fluid thioglycollate medium as
uninoculated controls. Thus, a total of 26 tubes (13 letheen broth, 13
fluid thioglycollate medium) will be inoculated per sample.
10.5.3 Incubate tubes at 36±1°C for at least 48 hours. Record (on the Sterility
Assessment of Product Sample Record) the time that tubes are placed
in the incubator. If it is observed that there is an interaction of the
disinfectant with the media as observed by cloudiness after
inoculation, it should be noted on the test form. Proceed with section
10.6.
10.5.4 Once results have been read and recorded following incubation at
36±1°C, incubate negative tubes at 55±1°C for at least 48 hours.
Record (on the Sterility Assessment of Product Sample Record) the
time that tubes are placed in the incubator. Proceed with section 10.6
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again.
10.5.4.1 Tubes which are positive (i.e., have growth) following
incubation at 36±1°C are not incubated at 55±1°C. Store
the tubes in the refrigerator. Record "N/A" for "Not
Applicable" in the results blocks of the Sterility
Assessment of Product Sample Record for tubes not
incubated at 55±1°C. Include a footnote indicating that
tubes were refrigerated rather than incubated at 55±1°C.
10.6 Results and Confirmation:
10.6.1 Record (on the Sterility Assessment of Product Sample Record; next to
"Date Recorded/Initials") the time that results are read.
10.6.2 Each tube is shaken prior to recording results to determine the
presence or absence of turbidity. Report results as + (growth) or 0 (no
growth) on the Sterility Assessment of Product Sample Record (see
16.1). A positive result is one in which the broth appears turbid as an
indication of microbial growth. A negative result is one in which the
broth appears clear.
10.6.2.1 If cloudiness was observed after inoculation, record as
"NR" (= not readable) on the form. Additionally, perform a
Gram stain on the tube to verify the presence or absence of
microbial growth. Record Gram stain results on the
Worksheet for Recording Gram stain and Acid fast
reactions (see 16.2). Attempt to identify the contaminant
by using the VITEK® 2 Compact system (see SOP QC-22,
VITEK 2 Compact: Use, Maintenance and Quality Control
Procedures).
10.6.3 Growth from 1-2 representative positive tubes (showing turbidity)
from each medium will be streaked on TSA for isolation. Gram stain
will be done for presumptive identification. Record Gram stain results
on the Worksheet for Recording Gram Stain and Acid Fast Reactions
(see 16.2). Attempt to identify the contaminant by using VITEK® 2
Compact system. Record confirmation results on the Test Microbe
Confirmation Sheet (see 16.3).
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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 the appropriate
forms. Completed forms are archived in notebooks kept in secured file cabinets in
the file room 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, Records and Archives.
13.0 QUALITY CONTROL:
13.1 For quality control purposes, the required information is documented on the
appropriate record form(s) (see 16.0).
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1 No further product testing will be initiated if product contamination is detected.
15.0 REFERENCES: None
16.0 FORMS AND DATA SHEETS:
16.1 Sterility Assessment of Product Sample Record
16.2 Worksheet for Recording Gram Stain and Acid Fast Reactions
16.3 Test Microbe Confirmation Sheet
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Sterility Assessment of Product Sample Record
OPP Microbiology Laboratory
PRODUCT INFORMATION
BACKGROUND and PREPARATION NUMBERS
Confirmed by:
Confirmed by:
EPA Reg. No.
Dale performed/initials
Name
Lclhccn broth prep. No.
Sample No.
Fluid thioglvcollalc prep. No.
Lot No.
Diluent:
Container No.
RESULTS: Date Recorded/Initials:
Dilution of
Sample
Lclhccn Broth (+/())*
Fluid Thioglvcollatc Medium (+/())*
Tube 1 (a>
36°C
Tube 1 {a>
55°C
Tubc 2 (di
36°C
Tube 2 @
55°C
Tube 1 {(i>
36°C
Tube 1 (di
55°C
Tubc 2 (di
36°C
Tubc 2 (di
55°C
Undiluted
Sample
10"1
10"2
HP1
10"4
l()-s
Uninoculated
Media Control
N/A
N/A
N/A
N/A
*+ = growth, 0 = no growth, NR=not readable, N/A=not applicable
COMMENTS
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Worksheet for Recording Gram Stain and Acid Fast Reactions
OPP Microbiology Laboratory
Slide ID:
Test:
Source/Tube No.
Source/Tube No.
Source/Tube No.
Product:
Results:
Results:
Results:
Date:
Initials:
Slide ID:
Test:
Source/Tube No.
Source/Tube No.
Source/Tube No.
Product:
Results:
Results:
Results:
Date:
Initials:
Slide ID:
Test:
Source/Tube No.
Source/Tube No.
Source/Tube No.
Product:
Results:
Results:
Results:
Date:
Initials:
AF+ =Acid fast positive; GPC =Gram positive cocci; GNR=Gram negative rod; GPR =Gram positive rod
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Test Microbe Confirmation Sheet
OPP Microbiology Laboratory
TEST INFORMATION/ Confirmed bv:
EPA Reg. No.
Test Dale
Name
Test Organism
Sample No.
Comments:
Source:
Tube/Plate
ID
Dale/ Initials
Slain
Results*
Media Information
Results
Type
Prep. No.
Inc. Time/
Temp.
Dale/Initials
Colony Characteristics
VITEK ID
(if applicable)
*Record Acid Fast or Gram Stain results as GPC = Gram positive cocci, GNR = Gram negative rods, AFR = acid fast rods, GPR = Gram positive rods.
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