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US Environmental Protection Agency

Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Determining the Presence of

Microbial Contamination in Disinfectant Products

Standard Operating Procedures (SOPs)

SOP Number: QC-21-02

Date Revised: 02-10-12

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SOP No. QC-21-02

Date Revised 02-10-12

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EPA/OPP MICROBIOLOGY LABORATORY

ESC, Ft. Meade, MD

Standard Operating Procedure

for

Determining the Presence of Microbial Contamination in Disinfectant Products

SOP Number: QC-21-02

Date Revised: 02-10-12

Initiated By: _______________________________________ Date: ___/___/___

Print Name:_____________________________

Technical Review: _______________________________________ Date: ___/___/___

Print Name:______________________________Technical Staff

QA Review: _______________________________________ Date: ___/___/___

Print Name:_____________________________QA Officer

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TABLE OF CONTENTS

Contents

Page Number

1.0 SCOPE AND APPLICATION........................................................ 3

2.0 DEFINITIONS............................................................................... 3

3.0 HEALTH AND SAFETY............................................................... 3

4.0 CAUTIONS.................................................................................... 3

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

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1.0 SCOPE AND APPLICATION:1.1 The assay provides data on the occurrence of microbial contamination of liquid

and spray antimicrobial products. This assay is consistent with methodology for

conducting the AOAC Use Dilution Method or the AOAC Germicidal Spray

Products as Disinfectants Test, but with sterile carriers.

1.2 The assay is qualitative and designed to recover and culture bacterialcontamination (i.e., spores ofBacillus sp.) from a hospital disinfectant sample. In

most instances, the assay will be deemed necessary due to historical evidence ofbacterial contamination associated with the sample itself, a previously-collected

sample of the same product, or a registrants product line.

1.3 Products that show no evidence of product-borne contamination when tested bythis procedure are considered adequate for efficacy testing; however, samples

with confirmed contamination will be deemed unsuitable for conducting efficacytests.

2.0 DEFINITIONS:2.1

AOAC = AOAC INTERNATIONAL

2.2 UDM = AOAC Use Dilution Method

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and adjusted accordingly.

4.3 Strict adherence to the protocol is necessary for the validity of the test results.4.4 Follow appropriate chain-of-custody guidelines during testing as stipulated in

SOP-COC-01, Sample Log-in and Tracking. Sample handling and custody

procedures are considered the same as for efficacy testing.

4.5 To avoid introduction of contaminating organisms into the test system and cross-contamination from tube to tube during the assay, it is recommended that hooks

be autoclaved prior to use, that one hook be used for a predetermined number oftubes and then be replaced with an autoclaved, unused hook, that there is

sufficient time between transfers (from disinfectant to neutralizer and neutralizer

to subculture medium) to thoroughly flame (to red-hot) the wire hook, and that theuse of a water bath be avoided (see section 10.1.3). In addition, the ultraviolet

light (UV) in the biological safety cabinet should be left on overnight prior to

performing the assay (see section 10.1.4).

5.0 INTERFERENCES:5.1 Exposing the product to external contaminants during opening and dispensing,

and the use of non-sterile laboratory supplies may interfere with the outcome ofthis analysis.

5.2 Quality control measures for media and reagents used in this evaluation must be

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number: EPA-1112-151CLSR).

7.3 Appropriate Spray Disinfectant Apparatus.7.4 Glassware. For disinfectant (UDM), use autoclavable 25 100 mm tubes

(Chemglass Life Sciences, Vineland, NJ). For neutralizer and subculture media,

use autoclavable reusable or disposable 20 150 mm tubes. For the GSPT, use38 mm 100 mm tubes (Bellco Glass Inc., Vineland NJ or Chemglass Life

Sciences, Vineland NJ) for neutralizer and subculture media. Sterilize all

glassware in hot air oven at 180C or steam sterilize for a minimum of 20 minutes

at 121C with drying cycle.

7.5 Test tube racks. Any convenient style.7.6 Wire hook. For carrier transfer. Make 3 mm right angle bend at end of 50-75 mm

nichrome wire No. 18 B&S gage. Place other end in suitable holder.

7.7 Positive displacement pipet(to deliver 10l to glass slides) and sterile tips.7.8 Timer. For managing timed activities, any certified timer that can display time in

seconds.

7.9 Gram stain kit.7.10 VITEK 2 Compact. For the automated identification of microorganisms.

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specify otherwise.

9.3 Prepare disinfectant sample per SOP MB-22, Disinfectant Sample Preparation.10.0 PROCEDURE AND ANALYSIS:

10.1 To conduct the assay, follow OPP Microbiology Laboratory SOP MB-05, AOACUse Dilution Method for Testing Disinfectants for liquid products, and SOP MB-

06, Germicidal Spray Products as Disinfectants Test for spray products, including

liquid products to be applied as a spray. The following exceptions/comments will

apply:

10.1.1 No inoculum will be generated as test microbes are not used in thisassay.

10.1.2 If test parameters call for an organic soil load, disregard thisrequirement. However, see section 10.1.8 of this SOP for arecommendation to apply an organic substance to glass slides in the

event that the spray product beads up and flows off of a glass slide in

the AOAC Germicidal Spray Products as Disinfectants Test. The

addition of an organic substance (e.g., 5% horse serum in sterile

deionized water, letheen broth) in this case is to increase dispersion ofthe spray product across the entire slide.

10.1.3 For liquid products, the recirculating chiller and water bath specified

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incorporated in this assay. In the future, the laboratory may decide touse only primary subculture tubes for efficacy evaluation (SOPs MB-

05 and MB-06). However, the change to efficacy test method SOPs

will not impact this SOP.

10.1.7 For liquid products: Sterile stainless steel carriers are removed fromthe water, placed onto filter paper in sterile glass Petri dishes, anddried for 40 minutes per SOP MB-05. Once drying is complete, the

AOAC Use Dilution Method is conducted using the dried,

uninoculated carriers according to the test parameters (i.e., product

dilution, neutralizer, subculture media, contact time). Alternately, thedrying step may be eliminated, and sterile stainless steel carriers may

be transferred from the tube of sterile water directly to tubes of

disinfectant according to the test timing scheme (e.g., 30 second dropinterval).

10.1.8 For spray products: Conduct the AOAC Germicidal Spray Products asDisinfectants Test method using the sterile glass carriers according to

the test parameters (i.e., product dilution, neutralizer, subculture

media, contact time).

Recommendation: Before conducting the spray test, the analyst must practice

applying the spray product to sterile glass slides to determinethe products level of dispersion.

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product dilution, neutralizer, subculture media, contact time).

10.1.9 Carrier counts are not to be performed.10.1.10 Report results as (+) for microbial growth or (0) for no growth on the

results sheet. In the Comments section of the results form, describe

the growth in positive tubes (e.g., uniformly turbid, string-like, etc.).Note: Shaking tubes prior to recording results may disrupt the unique

physical appearance of typicalBacillus sp. growing in liquid media

(i.e., string-like, fibrous, not producing uniformly turbid media).

10.1.11 A minimum of three positive (showing microbial growth) carrier sets,if available, should be confirmed using Gram staining, growth on

trypticase soy agar (TSA; for initial identification and isolation), andVITEK. If there are less than three positive carrier sets, then each

carrier set is confirmed. If both tubes are positive in a carrier set, only

one tube is selected for confirmatory testing (i.e., tube with carrier).

10.1.12 For a test with greater than 20 positive carrier sets, confirm at least20% by Gram staining. Up to 4 representative positive carrier sets will

be assayed for confirmation using Gram staining, growth on TSA, and

VITEK analysis. Again, if both tubes are positive in a carrier set, onlyone tube is selected for confirmatory.

11.0 DATA ANALYSIS/CALCULATIONS: None

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14.0 NONCONFORMANCE AND CORRECTIVE ACTION:14.1 If a product is confirmed to be contaminated, no further efficacy testing will be

initiated.

14.2 Strict adherence to the protocol is necessary for the validity of the test results.Any deviation from the standard protocol must be brought to the Study Directorsattention and recorded in the raw data along with an explanation for the deviation

given. The deviation and reason for it must be documented on the GLP

Compliance form in the final report.

15.0 REFERENCES:15.1 Official Methods of Analysis. 2009. 18th Ed., AOAC INTERNATIONAL,

Gaithersburg, MD, (Methods 955.15, and 964.02).

15.2 Official Methods of Analysis. 2006. 18th Ed., AOAC INTERNATIONAL,Gaithersburg, MD, (Method 955.14).

15.3 Official Methods of Analysis. 2009. 18th Ed., AOAC INTERNATIONAL,Gaithersburg, MD, (Method 961.02).

16.0 FORMS AND DATA SHEETS:16.1 Determining Microbial Contamination in Liquid Disinfectants: Time Recording

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16.8 Determining Microbial Contamination in Spray Disinfectants: Results Sheet16.9 Test Microbe Confirmation Sheet

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16.1Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier

Inoculation StepsOPP Microbiology Laboratory

TEST INFORMATION/Confirmed by: _______

Test Date

Product Reg. No.

Product Name

Sample No(s).

Organism Not Applicable

Initials/Date Test ID

Carrier Dry Time*

Start Time End Time

UDT

* Recorded from laboratory clock/and timer.

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16.2Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier TransfersOPP Microbiology Laboratory

TEST INFORMATION/Confirmed by: _______

Test Date

Product Reg. No.

Product Name

Sample No(s).

Organism Not Applicable

Initials/Date Set

Drop

Interval

Carrier Drop Start Time (into the

disinfectant)

Carrier Drop End Time (into the

primary subculture/neutralizer media)

Carrier Transfer (into secondary

subculture)

Clock Timer Clock Timer Start Time1

Comments:

1Carrier transfer into secondary subculture (time elapsed after last carrier dropped in primary); taken from clock

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16.3

Determining Microbial Contamination in Liquid Disinfectants: Information

SheetOPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:_______EPA Reg. No. SOP

Name Test Date

Sample No. Comments: Assay will be performed without test

microbes.Lot No.

Expiration Date

TEST PARAMETERS/Confirmed by:_______

H2O Hardness (CaCO3) ppm Specified Titrated (Buret)/Date/Init. HACH/Date/Init.

Use Dilution Specified As Prepared/Date/Init.

Neutralizer Specified

Contact Time Specified As Tested

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16.4

Determining Microbial Contamination in Liquid Disinfectants: Results SheetOPP Microbiology Laboratory

PRODUCT INFORMATION/Confirmed by:_______

EPA Reg. No. Test Date

Name Test Organism Not Applicable

Sample No.

CARRIER INFORMATION (to be completed by Analyst)

Carrier Drop Time Interval Carrier Set Analyst

TEST RESULTS

Date Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)

1 2 3 4 5 6 7 8 9 10

/ / / / / / / / / /

11 12 13 14 15 16 17 18 19 20

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16.5Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier

Inoculation StepsOPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:_______

Test Date

Product Reg. No.

Product Name

Sample No(s).

Test Organism Not Applicable

Initials/Date Test ID

Carrier Dry Time*

Start Time End Time

GSPT

* Recorded from laboratory clock/and timer.

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16.6

Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier TransfersOPP Microbiology Laboratory


Test Date

Product Reg. No.

Product Name

Sample No(s).

Test Organism Not Applicable

Initials/date SetSpray

Interval

Carrier Spray Start Time (into the

disinfectant)

Carrier Spray End Time (into the

neutralizer/primary subculture)1

Carrier Transfer (into

secondary subculture)

Clock Timer Clock Timer Start Time

Comments: Carriers sprayed by: ______. Carriers transferred by: ______.

1Carrier transfer into secondary subculture; taken from clock.

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16.7

Determining Microbial Contamination in Spray Disinfectants: Information

SheetOPP Microbiology Laboratory

TEST INFORMATION/Confirmed by:_______EPA Reg. No. SOP

Name Test Date

Sample No. Comments: Assay will be performed without test

microbes.Lot No.

Expiration Date

TEST PARAMETERS/Confirmed by:_______

H2O Hardness (CaCO3) ppm Specified Titrated (Buret)/Date/Init. HACH/Date/Init.

Use Dilution Specified As Prepared/Date/Init.

Neutralizer Specified

Contact Time Specified As Tested

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16.8

Determining Microbial Contamination in Spray Products: Results SheetOPP Microbiology Laboratory

PRODUCT INFORMATION/Confirmed by:_______



Sample No.

TEST RESULTS

Date Recorded/Initials

Primary Subculture / Secondary Subculture (carrier)1 2 3 4 5 6 7 8 9 10

/ / / / / / / / / /

11 12 13 14 15 16 17 18 19 20

/ / / / / / / / / /

21 22 23 24 25 26 27 28 29 30

/ / / / / / / / / /

31 32 33 34 35 36 37 38 39 40

/ / / / / / / / / /

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16.9

Test Microbe Confirmation SheetOPP Microbiology Laboratory




Sample No. Comments

Source:

Tube/Plate ID

Date/

Initials

Stain

Results*

Media Information Results

Name Prep. No.Inc. Time/

Temp.

Date/

InitialsColony Characteristics

VITEK ID**

(if applicable)

* GPC=Gram positive cocci, GNR=Gram negative rods, GPR=Gram positive rods ** VITEK numerical profile number

qc-21-02

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