Antimicrobial Testing Methods & Procedures Developed by EPA's Microbiology Laboratory

EPA developed antimicrobial testing methods and standard operating procedures to measure the effectiveness of hard surface disinfectants against:

• Staphylococcus aureus.
• Pseudomonas aeruginosa.
• Salmonella enterica.
• Mycobacterium bovis (BCG).
• Clostridioides difficile.
• Candida auris
• Viruses.
• Biofilm.

Methods are available for the following types of antimicrobial formulations:

• Liquid.
• Spray.
• Towelette.

EPA’s standard operating procedures are strict interpretations of  AOAC International  and ASTM International  standard methods

For questions about the methods, procedures, and guidance below, please contact the OPP Microbiology Laboratory directly.

• Methods
• Procedures
• Guidance

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Methods

Method ID	Title
MB-01-10	Biosafety in the Laboratory
MB-02-09	Tracking of Test Microorganisms
MB-03-09	Screening of Polished Stainless Steel Penicylinders, Porcelain Penicylinders, and Glass Slide Carriers Used in Disinfectant Efficacy Testing
MB-05-17	AOAC Use Dilution Method for Testing Disinfectants
MB-06-10	Germicidal Spray Products as Disinfectants (GSPT): Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica
MB-07-10	Tuberculocidal Activity of Disinfectants: In Vitro Test for Determining Tuberculocidal Activity
MB-09-08	Disinfectant Towelette Test: Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica
MB-10-08	Media and Reagents: Preparation and Quality Evaluation
MB-11-07	Neutralization Confirmation Assay for Disinfectant Products Tested against Mycobacterium bovis (BCG)
MB-13-07	Handling Spills of Biohazardous Materials
MB-15-05	Standard Operating Procedure for the AOAC Sporicidal Activity of Disinfectants Test (Bacillus subtilis × porcelain carrier).
MB-16-04	Quantitative Suspension Test Method for Determining Tuberculocidal Efficacy of Disinfectants against Mycobacterium bovis (BCG)
MB-17-06	Neutralization Confirmation Procedure for Products Evaluated with the AOAC Use Dilution Method (DUM), the AOAC Germicidal Spray Products as Disinfectants Test (GSPT), and the Disinfectant Towelette Test (DTT)
MB-19-06	Preparing a Pseudomonas aeruginosa or Staphylococcus aureus Biofilm using the CDC Biofilm Reactor.
MB-20-04	Single Tube Method for Determining the Efficacy of Disinfectants against Bacterial Biofilm
MB-21-05	Quantitative Three Step Method for Measuring the Efficacy of Liquid Sporicides against Spores of Bacillus subtilis on Hard Non-porous and Porous Surfaces
MB-22-06	Preparation and Sampling Procedures for Antimicrobial Test Substances
MB-23-05	Disinfectant Towelette Test: Testing of Mycobacterium bovis (BCG)
MB-24-05	Testing of Mycobacterium bovis (BCG) Using the Germicidal Spray Products as Disinfectants Test
MB-25-05	OECD Quantitative Method for Evaluating Bactericidal and Mycobactericidal Activity of Microbicides Used on Hard, Non-Porous Surfaces
MB-26-03	Neutralization of Microbicidal Activity using the OECD Quantitative Method for Evaluating Bactericidal and Mycobactericidal Activity of Microbicides Used on Hard, Non-Porous Surfaces
MB-27-04	Germicidal and Detergent Sanitizing Action of Disinfectants Test
MB-28-08	Production and Storage of Spores of Clostridioides difficile for Use in the Efficacy Evaluation of Antimicrobial Agents
MB-29-02	Quantitative Method for Evaluating Virucidal Activity of Antimicrobial Products on Hard, Non-Porous Surfaces This SOP is currently not a recommended method to support product registration. Please consult with the agency prior to utilizing for product registration.
MB-30-03	Preparation of hard water and other diluents for preparation of antimicrobial products
MB-31-07	Quantitative Method for Testing Antimicrobial Agents Against Spores of C. difficile on Hard, Non-porous Surfaces
MB-35-03	Quantitative Method for Evaluating the Efficacy of Antimicrobial Products against Candida auris on Hard, Non-Porous Surfaces
MB-37-02	Neutralization of Microbicidal Activity against Candida auris using the Quantitative Method
MB-39-01	Standard Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces using Human Coronavirus
MB-40-00	Test Method for Evaluating the Efficacy of Antimicrobial Surface Coatings
MB-41-00	Method for the Evaluation of Antimicrobial Activity of Hard, Non-porous Copper-Containing Surface Products
N/A	Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances on Soft Surface Textiles against Viruses (pdf) (945.47 KB, August 11, 2023)
N/A	Quantitative Method for Evaluating the Efficacy of Antimicrobial Test Substances on Soft Surface Textiles Against Bacteria (pdf) (776.79 KB, August 11, 2023)
MB-42-00	Method for Testing Antimicrobial Products against Legionella pneumophila in Simulated Cooling Tower Water (LSCTW) (pdf)

Procedures

The following standard operating procedures are pertinent to specific equipment and practices used in the EPA pesticide program's Microbiology Laboratory. The SOPs describe the laboratory's standard practices for:

• Calibrating equipment (EQ).
• Performing quality control procedures (QC).
• Administrative procedures (ADM).
• Maintaining chain of custody of samples (COC).
• Assuring quality management practices (QA).

Procedure ID	Standard Operating Procedure
ADM-01-08	Preparation and Review of Product Performance Reports
ADM-02-08	Preparation and Review of Standard Operating Procedures
ADM-03-08	Maintaining, Tracking and Archiving of Records
ADM-04-06	OPP Microbiology Laboratory Personnel Training
ADM-05-06	Use and Maintenance of Laboratory Notebooks and Project Binders
ADM-06-05	Receiving and Assessing Customer Feedback
COC-01-09	Chain of Custody Procedures for Antimicrobial Samples
EQ-01-10	Calibration and Maintenance of pH Meters
EQ-02-10	Calibration and Maintenance of Thermometers and Thermometer/Hygrometers
EQ-03-10	Calibration and Maintenance of Weigh Balances
EQ-04-09	Use and Maintenance of Spectrophotometers
EQ-05-09	Calibration and Maintenance of Timers
EQ-10-05	Use and Maintenance of Incubated Orbital Shakers
EQ-11-03	Use and Maintenance of Biological Safety Cabinets
EQ-12-03	Hach’s Digital Titrator for Total Chlorine Titration
EQ-13-01	Use of the PetriSwiss PS200 Instrument
EQ-14-01	Use of Environmental Chamber (Memmert HPP11)
QC-01-09	Quality Assurance of Purified Water
QC-02-08	Monitoring of Laboratories for Airborne Contaminants
QC-03-10	Glass Washing and Detergent Residues Test
QC-05-11	Monitoring Environmental Parameters of Incubators, Refrigerators, Freezers, Laboratories and Sample Storage Rooms
QC-09-09	Establishment of Control Numbers and Tracking of Laboratory Supplies
QC-13-12	Performance Verification of Autoclaves
QC-19-11	Verification of Digital Pipettes
QC-22-06	VITEK 2 Compact: Use, Maintenance and Quality Control Procedures

Guidance

• Use Dilution Method Performance Standard Revision Document
• Sporicidal Claims against Clostridioides difficile
• Guidance for the Efficacy Evaluation of Products for Claims against Drug-Resistant Candida auris
• Methods and Guidance for Testing the Efficacy of Antimicrobial Products Against Biofilms on Hard, Non-Porous Surfaces
• Guidance for Products Adding Residual Efficacy Claims
• Guidance for Products Including or Adding Disinfectant Efficacy Claims for Use on Soft Surface Textiles in Non-Residential Settings
• Final Guidance for Efficacy Testing of Antimicrobial Products Against Planktonic Legionella pneumophila in Cooling Tower Water