Standard Microbiological Methods for Antimicrobial Products in North America

Standard Microbiological Methods for Antimicrobial Products in North America

Scott Steinagel

Elaine Black

Products used for the reduction and elimination of microorganisms, known broadly as antimicrobial products, have become part of our daily lives. They are used across the health care, food production, food service, and hospitality industries as well as in private households across the globe.1,2,3,4,5 In order to measure the effectiveness of new and existing antimicrobial products, a set of repeatable and reproducible standard methods must exist. Standard test methods are vital, science-based, regulatory, and research tools that help ensure antimicrobial products perform as expected.6,7,8 Standard microbiological methods are modeled on typical use conditions for the products being tested, and for this reason, a range of methods exists to mimic immersion, spray, wipe, and other product applications.7,8 Other use conditions that can affect the efficacy or performance of a product can be incorporated and, in some cases, altered depending on the nature of the product and its intended use. Conditions such as product diluent (water hardness level) and the presence of interfering substances (eg, organic soil), which model bodily fluids or environmental soils, are often part of standard methods.6,9

The basic premise of an antimicrobial test method is the exposure of a test microorganism (such as vegetative bacteria, bacterial spores, fungal spores, or viruses) to the test product for a defined period of time (contact time), neutralization of the biocidal activity, and subsequent determination of efficacy (enumeration of survivors).6,7,8 Standard microbial test methods in North America are generally developed, adapted, and maintained by standard setting bodies such as AOAC International (formerly Association of Official Analytical Chemists) and ASTM International (formerly American Society for Testing and Materials). The membership of these organizations include academic, industrial, and governmental stakeholders. Methods are developed, tested, or improved through collaborative trials involving multiple stakeholder laboratories and are reviewed and voted on by members of these bodies.

The following chapter describes many, although not necessarily all, of the test methods used by producers of antimicrobial products to substantiate efficacy and product label claims of typical antimicrobial products. All information is current at time of publication but is subject to change. The opinions expressed in this chapter are those of the authors and not of the agencies referenced. The information within should not be used in lieu of the most current regulatory guidance or consultation with the relevant regulatory authority.


In the United States, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) establishes the authority of the US Environmental Protection Agency (EPA) over the registration, labeling, sale, distribution, and use of pesticidal products.10 The FIFRA’s definition of a “pest” includes but is not exclusive to any fungus, bacterium, virus, or other microorganism that is injurious to health or the environment. The act defines a “pesticide” as “any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest.” By definition, the word pesticide is a misnomer because it includes modes of action that are not technically “cidal.” In the United States, disinfectants, sanitizers, and sporicides are regarded as pesticides for regulatory purposes. Some antimicrobials, however, do not fall under the jurisdiction of the EPA. Under the Federal Food Drug and Cosmetics Act,11 the US Food and Drug Administration (FDA) has authority over high-level disinfectants (HLDs) and sterilants used for critical and semicritical medical devices and for food additives, including but not limited to antimicrobials used as processing aids and food additives used for the reduction of pathogenic bacteria in food. Consequently, EPA and FDA have dual jurisdiction over antimicrobials for the
treatment of raw agricultural products and for the treatment of process water in food processing facilities.

In Canada, antimicrobial products are regulated mainly by Health Canada’s Pest Management Regulatory Agency (PMRA) or the Natural and Nonprescription Health Products Directorate (NNHPD). Other agencies or directorates are involved for specific product types and are discussed elsewhere in the chapter. Through the Food and Drugs Act, NNHPD is the regulatory body responsible for disinfectants used on environmental surfaces or inanimate objects.12 These products are referred to as “disinfectant drugs” by NNHPD and require a drug identification number (DIN). The PMRA, however, is given authority over some antimicrobial products such as soft surface sanitizers, laundry antimicrobials, bacteriostatic surface treatments, and nonfood contact sanitizers without disinfection claims through the Pest Control Products Act.13 Canada and the United States are closely aligned with respect to efficacy standards and require data be generated by many of the same methods. With the exception of some minor nomenclature differences, the contents of this chapter reflect the typical methods needed for registration of pesticides/antimicrobial agents used on inanimate surfaces under both EPA and Health Canada jurisdictions. This chapter does not necessarily reference every standard test method that can be used and excludes those antimicrobials used as food tissue treatments and antiseptics.

Antimicrobial Product Efficacy Guidelines in North America

In order to assess the safety and effectiveness or efficacy of antimicrobial products, the EPA has published a series of testing guidelines that serve as testing recommendations. The 810 series Product Performance Test Guidelines describe the testing requirements used to substantiate the efficacy of antimicrobial products that bear public health claims—claims against organisms that pose threat to human health, on inanimate or environmental surfaces.14 A series of documents authored by Health Canada are Canada’s equivalent to the 810 series15,16,17,18 and cover disinfectant drugs, disinfectant sanitizers, and HLDs/sterilants for critical and semicritical medical devices. The FDA testing requirements for HLDs used for medical devices can be found in the Guidance for Industry and FDA Reviewers: Content and Format of Premarket Notification [510(k)] Submissions for Liquid Chemical Sterilants/High Level Disinfectants.19

Within these guidance documents, antimicrobial products can be classified as follows:

  • Sanitizer—a substance, or mixture of substances, that reduces the bacterial population on environmental surfaces and inanimate objects by significant numbers (eg, a minimum of 3 log10 reduction) due to the antimicrobial action of the active ingredient(s) but which does not destroy or eliminate all bacteria

  • Disinfectant-sanitizer (Canada only)—a chemical product represented for use as a sanitizer on hard nonporous environmental surfaces and inanimate objects which is also represented for use as a hard surface disinfectant

  • Disinfectant—a substance, or mixture of substances, capable of destroying or irreversibly inactivating pathogenic (disease causing) and potentially pathogenic (opportunistic) microorganisms (eg, bacteria, fungi, and viruses) but not necessarily bacterial spores, present on environmental surfaces and inanimate objects due to the antimicrobial action of the active ingredient(s)

  • Sporicide—a substance, or mixture of substances, capable of destroying or irreversibly inactivating bacterial spores present on environmental surfaces and inanimate objects

  • HLD—a substance, or mixture of substances, capable of destroying or irreversibly inactivating all microbial pathogens, but not necessarily large numbers of bacterial spores

  • Sterilant—a substance, or mixture of substances, capable of destroying or irreversibly inactivating all forms of microbial life present on inanimate objects, including all forms of vegetative bacteria, bacterial spores, fungi, fungal spores, and viruses


Efficacy claims for pesticides are substantiated in the laboratory by standardized test methods. The methods can be classified as either qualitative or quantitative test methods and as suspension-based or carrier-based test methods.8

Qualitative test methods—Qualitative test methods provide results classified by the presence or absence of the microorganism following treatment rather than a numerical count of survivors (eg, colony-forming units [CFUs] or plaque-forming units). In qualitative methods, a series of coupons used to represent the intended treatment surface, called “carriers,” are inoculated with the test system or a suspension of the microorganism is treated with the antimicrobial product. The treated carrier or a sample of the treated suspension is then transferred to a neutralizing growth medium, incubated, and examined for presence or absence of the test system as indicated by turbidity or other visual end point. This positive/negative end point is the hallmark of a qualitative method. Although these methods have a qualitative end point, the advent of more advanced statistical methods and extensive replication has allowed for “semiquantitative” conclusions to be made in some methods.20

Quantitative test methods—Quantitative test methods provide quantifiable results that allow for the calculation of log10 or percentage reductions following treatment. In these methods, an inoculated and subsequently treated coupon
or treated suspension of the test system is neutralized and quantitatively assayed for survivors using standard enumeration techniques (eg, standard plate count for bacteria/fungi or cell culture recovery of viruses). The survivors are enumerated as compared to an analogous untreated control and microorganism reductions can be calculated.

Suspension-based test methods—In a suspension-based test method, a sample of test system is introduced directly into the antimicrobial solution and contact with the antimicrobial begins immediately. A sample of the test system/antimicrobial solution mixture is removed and is neutralized once the desired contact time has been reached. Survivors may then be enumerated as compared to an analogous untreated control suspension. Alternatively, assessments of survival following treatment may be made qualitatively. These methods do not rely on the specific use of a treatment surface material, or carrier, and instead involve direct treatment of the test system with the antimicrobial.

Carrier-based test methods—In a carrier-based test method, the test system is first placed onto a coupon or carrier intended to represent the surface to be treated. The test system is then dried to the carrier surface. The coupon is then introduced to or treated by the antimicrobial product. Carrier-based test methods differ from suspension-based test methods in that the antimicrobial product must penetrate a dried organism or virus film to inactivate the test system. The treated surface is then neutralized once the desired contact time has been reached. The neutralized material can either be evaluated quantitatively or qualitatively to evaluate the efficacy of the treatment procedure.

In addition to the mechanism of action process followed in the test method (suspension versus carrier), some key use conditions must also be considered when developing antimicrobial products and evaluating their efficacy. These include contact time and temperature, the intended diluent for concentrated antimicrobial products, and the organic burden (soiling) that might be present under claimed use conditions. For spray products, the spray time and distance traveled by the chemistry can impact efficacy. For towelette products, the folding and wiping procedure can impact efficacy. The following provides more detail regarding intended diluent and organic soiling.

Intended diluent (hard water)—Antimicrobial products can be produced in the form of ready-to-use products or concentrated liquids/solids for dilution in water at the use site. In North America, concentrated antimicrobials to be diluted in water are required to be tested under conditions that demonstrate efficacy in the presence of the water type listed on the label such as hard water.14 Synthetic hard water introduces minerals into the diluted product including magnesium and calcium. This inorganic burden may interfere with the activity of the product, particularly those actives sensitive to cationic binding.21 As such, efficacy testing should be conducted in the presence of hard water to confirm the efficacy of the product in use. Two routinely used efficacy test methods give instruction on the preparation of synthetic hard water for use in testing.22,23

Organic burden—In North America, disinfectants may be identified as one-step cleaner disinfectants or one-step cleaner sanitizers, which provide efficacy claims in the presence of light to moderate levels of organic burden.14 This is an important consideration because organic burden can have a negative impact on the activity of various chemicals, particularly those sensitive to the presence of protein.9 Because of this, an organic soil load should be included in testing to simulate these use conditions in order to substantiate one-step cleaner-disinfectant/sanitizer claims. For many methods, this involves the introduction of a minimum 5% organic soil such as blood serum into the test system prior to treatment. In other methods, as in the case for efficacy claims against Clostridium difficile spores, a three-part soil load including bovine serum albumin, yeast extract, and mucin is required to simulate the environment these spores are likely to be found.24 Once substantiated, these one-step treatment claims provide labor-saving value to the end user of these products by limiting the effort required to effectively disinfect or sanitize surfaces or materials. It should be noted that in all cases where heavy soil is present, the use directions must require precleaning prior to antimicrobial product treatment.


The term sanitizer is not a globally used term but is widely used in North America.14 Generally speaking, sanitizers have lower levels of efficacy than disinfectants and are used in areas where complete kill of microorganisms is not necessary or the bioburden in these use sites is lower. There are many types of sanitizer treatments including food contact and nonfood contact sanitization of hard surfaces, soft surface sanitization, toilet sanitization, laundry sanitization, carpet sanitization, and residual hard surface sanitization. Sanitizers play an important role in protecting public health as well as protecting the safety of the food supply in North America.

Classification of Sanitizers

In North America, hard surface sanitizers can be broken into two main categories: food contact surface sanitizers and nonfood contact surface sanitizers.

Food Contact Surface Sanitizers

Food manufacturing/food service industries rely heavily on food contact sanitizers to prevent foodborne illness and spoilage/quality issues in foods. Food contact sanitizers are used to treat the surfaces which food may come into contact and require a higher bactericidal performance than those required by nonfood contact sanitizers. Food contact sanitizers are most often used as part of a broader sanitation process that includes a cleaning step to
remove visible soil from the surface.25 Proper cleaning is essential to remove any soils that may protect microorganisms or neutralize the activity of the sanitizer. The selection of a sanitizer can be based on a number of factors other than efficacy that include cost, stability, safety, and material compatibility.25

In 2015, the US Food Safety Modernization Act established a number of rules related to the production and transportation of human and animal food.26 In the final rule on current good manufacturing and risk-based preventative controls, the FDA requires that food manufacturers adhere to controls that prevent hazards in the US food system including sanitation controls to ensure a facility is maintained in sanitary condition.27 The US Federal Food Code outlines model guidance on food safety sanitation that can be uniformly adopted for restaurants and food retail establishments. The Food Code includes detailed practices for the use of food contact sanitizers.28 These laws and guidance underline the importance of sanitizers for maintenance of a safe food supply. Although FDA and the United States Department of Agriculture (USDA) regulate the production of food in the United States, sanitizers used in the food industry are classified as pesticides and are therefore regulated under FIFRA. Standard test methods for the measurement of the efficacy of sanitizers incorporate test organisms that are representative of those found on surfaces to be sanitized.29

Food contact sanitizers come in direct contact with the food we eat, are classified as Indirect Food Additives in the United States, and are regulated under 21 Code of Federal Regulations (CFR) 178.1010 because they are reasonably expected to become incorporated into food through contact with treated processing equipment or utensils. Sanitizers must therefore be shown to be safe prior to their use.30 A manufacturer of a sanitizer must provide assurance to the governing regulatory body that the product does no harm to the public by estimating the sanitizer residue following use and establishment of the probable daily intake of the substance.

In Canada, food contact sanitizers are assessed by Health Canada’s Bureau of Chemical Safety (BCS) and the Food Directorate. The BCS determines whether or not the level of sanitizer product residues that could remain on the surface of food is acceptable in terms of human safety and Health Canada BCS issues a No Objection Letter for these products. This allows a registrant to add a “no rinse required” statement to the label, and the product can remain on the surface prior to actual food contact, saving time and labor in processing environments. This “no rinse” paradigm is not accepted globally, making these use directions very North America-centric.

Sanitizer uses include (1) use in public eating establishments to sanitize eating utensils, (2) use on processing equipment and utensils in dairy plants, and (3) use on processing equipment and utensils in other (ie, nondairy) food-processing plants.31 The storage, dispensing, and use of sanitizers are often strictly controlled by the food processing company following internal Sanitation Standard Operating Procedures (SSOPs), which follow EPA labeling on concentration and contact time.25 Food contact surface sanitizers used in food service and private homes are often used on food preparation areas such as counter tops and on eating utensils in manual and machine dishwashing systems. Application of sanitizers in these use sites rely heavily on the EPA label and use instructions.

For the purpose of efficacy testing, food contact surface sanitizers are further broken into halide and nonhalide product types. Halide chemical products include those formulated with iodophors, mixed halides, and chlorine-bearing chemicals.29 Nonhalide products include but are not limited to quaternary ammonium compounds, acids, and peroxygens.

Nonfood Contact Surface Sanitizers

Nonfood contact surface sanitizers are products used on surfaces that will not come into contact with foods or beverages. These sanitizers have a lower hurdle for kill and allow for longer contact times, which is indicative of the less critical nature of their use compared to food contact surface sanitizers. Nonfood contact sanitizers are used routinely on floors, walls, and other hard surfaces not directly used for food production.

Hard-Surface Sanitizer Test Methods

The EPA Product Performance Test Guidelines Office of Chemical Safety and Pollution Prevention (OCSPP) 810.2300 describes the efficacy data requirements and testing recommendations for sanitizers used on hard surfaces.29 According to the Guideline, the following sanitizer classifications exist:

  • Food contact surface sanitizers (halide based)

  • Food contact surface sanitizers (nonhalide based)

  • Nonfood contact surface sanitizers

Halide-Based Food Contact Sanitizers: AOAC Chlorine (Available) in Disinfectants (955.16)

The AOAC Chlorine (Available) in Disinfectants describes a qualitative, suspension-based test method used to evaluate the germicidal properties of halide-based antimicrobial products.32 In this method, 10 mL of the sanitizing product, diluted to its use-solution in AOAC hard water, if applicable, is inoculated with 50 µL of the test organism suspension. The solution is mixed and allowed to stand for 1 minute. After mixing, a 10 µL loopful of the suspension is transferred to 10 mL of neutralizing growth medium. Thirty seconds later, the same tube of sanitizing product is inoculated with 50 µL of test organism again. One minute later, the suspension is mixed and 10 µL is transferred (subcultured) to a second tube containing 10 mL of neutralizing growth medium. The inoculation
and subculturing procedure is repeated until 10 tubes of growth medium have been subcultured. The test procedure is repeated for three known and verified concentrations of available chlorine (50 parts per million [ppm], 100 ppm, and 200 ppm) to be used as reference solutions. The subculture tubes are incubated and subsequently examined for the presence or absence of test organism growth as determined by the presence or absence of turbidity. The number of consecutive tubes showing growth for the sanitizing product is compared to the number of consecutive tubes showing growth for the three available chlorine reference solutions, allowing the analyst to compare the germicidal efficacy of the test product to the three concentrations of available chlorine. Refer to Table 62.1 for the test organisms and performance standard associated with this method.

TABLE 62.1 Sanitizer efficacy data requirements

Product Type

Test Methods

Test Organisms

Evaluation of Success

Halide-based food contact sanitizers (water-soluble powders/liquids)

AOAC Chlorine (Available) in Disinfectants (955.16)

Staphylococcus aureus (ATCC 6538) or Salmonella enterica (ATCC 6539)

Test results should demonstrate product concentrations equivalent to 50, 100, or 200 ppm of available chlorine.

Nonhalide-based food contact sanitizers (water-soluble powders/liquids)

AOAC Germicidal and Detergent Sanitizing Action of Disinfectants (960.09)

S aureus (ATCC 6538) and Escherichia coli (ATCC 11229) The control counts must demonstrate 7.0-8.0 logs.

A 99.999% reduction in the number of each test organism within 30 s

Nonfood contact sanitizersa

ASTM Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous, Nonfood Contact Surfaces (E1153)

S aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352) or Klebsiella (formerly Enterobacter) aerogenes (ATCC 13048)

The control counts for the organisms listed earlier must demonstrate a minimum average of 7.5 × 105 CFUs per carrier.

A 99.9% reduction over parallel control counts within 5 minutes

Laundry sanitizers (in wash)

ASTM E2274 or ASTM E2406 (for high efficiency)

S aureus (ATCC 6538)b

K pneumoniae (ATCC 4352)b

Pseudomonas aeruginosa (ATCC 15442)b

A minimum of 1 × 104 CFUs per carrier and 1 × 104 CFUs per mL must be recovered on the control carriers and in the wash water control, respectively.

A minimum 99.9% (3 log10) reduction in bacteria over the control count for both the treated swatches and treatment solution wash water

Abbreviations: AOAC, Association of Official Analytical Chemists; ATCC, American Type Culture Collection; CFUs, colony-forming units; ppm, parts per million.

a This method may also be used to support laundry presoak sanitizing treatment claims. A 5% organic soil load must be included in the organism suspension.

b For broad-spectrum laundry sanitizers, test S aureus and K pneumoniae. For laundry sanitizers used in health care facilities, test S aureus, K pneumoniae, and P aeruginosa.

Nonhalide-Based Food Contact Sanitizers: AOAC Germicidal and Detergent Sanitizing Action of Disinfectants (960.09)

The AOAC Germicidal and Detergent Sanitizing Action of Disinfectants method describes a quantitative, suspensionbased test method used to evaluate the germicidal properties of nonhalide-based food contact sanitizers.22 In this method, the sanitizer is diluted to its use-solution in AOAC synthetic hard water, if applicable, and a 99-mL sample of the prepared sanitizer solution is equilibrated to 25°C. A standardized suspension of the test organism is prepared, and a 1-mL sample of the organism is added to the sanitizer solution. The solution is mixed and the organism is exposed to the sanitizer for 30 seconds. After exposure, a 1-mL sample is removed and transferred to 9 mL of a neutralizer medium. The neutralized
contents are mixed and enumerated by plating onto an appropriate agar. Following incubation, the surviving bacteria are enumerated and bacterial reduction is calculated as compared to the starting population of bacteria used in the test. The method includes sterility controls and a numbers control intended to enumerate the population of organism added to the sanitizer as well as a confirmation that the neutralizer used was suitable. The performance standard for the method can be found in Table 62.1.

ASTM Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Nonfood Contact Surfaces (E1153)

The ASTM Method E1153 describes a carrier-based, quantitative test method used to evaluate the efficacy of nonfood contact sanitizers.33 In this method, glass, stainless steel, or other appropriate surface material 25 mm × 25 mm coupons are inoculated with 10 to 30 µL of test organism. The carriers are placed in an incubator to dry and are sequentially treated with 5 mL of the sanitizer at its use-solution. After the contact time (≤5 min), the treated carriers are neutralized with 20 mL of neutralizer. The neutralized carriers are mixed and enumerated by plating onto an appropriate agar. Following incubation, the surviving bacteria are enumerated and the bacterial reduction is calculated compared to a control treatment. The method includes controls to enumerate the number of viable organisms present on the carriers (carrier counts) as well as a confirmation that the neutralizer used was suitable for the sanitizer. The performance requirements for the method can be found in Table 62.1.


In the United States, disinfectants are regarded as products that provide a high level of public health protection when used according to label instructions. In health care environments, disinfectants are extremely important in making and keeping surfaces safe in the clinical environment by killing bacteria, viruses, and fungi. They reduce the likelihood of microbial cross-contamination from inanimate objects like bedrails and equipment to patients and staff, thus reducing the spread of infection in hospitals.4 Disinfectants are similarly used in workplaces, hotels, and homes to reduce the spread of infectious agents. Disinfectants are most commonly applied by mopping, wiping, or spraying onto a hard surface but can, in some cases, be fogged, misted, or applied as a foam.

Classification of Disinfectants

The EPA Product Performance Test Guideline OCSPP 810.2200 describes the efficacy data requirements and testing recommendations for disinfectants used on hard surfaces.34 According to the guideline, the following disinfectant classifications exist:

  • Limited-spectrum (bactericidal) disinfectants

  • Broad-spectrum (bactericidal) disinfectants

  • Hospital or health care (bactericidal) disinfectants

  • Fungicidal disinfectants

  • Virucidal disinfectants

  • Tuberculocidal disinfectants

Limited-Spectrum Disinfectants

Limited-spectrum disinfectants are classified as possessing disinfection efficacy against either the model gram-positive bacterium, Staphylococcus aureus American Type Culture Collection (ATCC) 6538, or the model gram-negative bacterium, Salmonella enterica ATCC 10708. Any additional bactericidal claims that can be made must match the gram stain type (positive or negative) base claim. Virucidal, fungicidal, and tuberculocidal claims cannot be made for limited-spectrum disinfectants.

Broad-Spectrum Disinfectants

Broad-spectrum disinfectants are classified as possessing disinfection efficacy against both gram-positive and gram-negative bacteria and thus must demonstrate efficacy against both S aureus ATCC 6538 and S enterica ATCC 10708 or Pseudomonas aeruginosa ATCC 15442. Additional bactericidal, virucidal, fungicidal, and tuberculocidal claims can be made, where substantiated.

Hospital or Health Care Disinfectants

Health care disinfectants used in hospitals, clinics, dental offices, nursing homes, or any other health care-related facility are classified as possessing disinfection efficacy against both gram-positive bacterium, S aureus ATCC 6538, and a clinically significant and difficult to kill gram-negative bacterium, P aeruginosa ATCC 15442. Additional bactericidal, virucidal, fungicidal, and tuberculocidal claims can be made, where substantiated.

Fungicidal Disinfectants

Broad-spectrum or hospital disinfectants may possess claims against pathogenic fungi to be classified as a fungicidal disinfectant. Disinfectant efficacy must be demonstrated against a clinically relevant fungus: Trichophyton interdigitale ATCC 9533 (formerly known as Trichophyton mentagrophytes), the fungus associated with athlete’s foot. Once efficacy against T interdigitale is established, claims against fungi can be made. For example, mildewcidal disinfectant claims can be made where efficacy is demonstrated against Aspergillus niger, a mildew-causing black mold.

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May 9, 2021 | Posted by in MICROBIOLOGY | Comments Off on Standard Microbiological Methods for Antimicrobial Products in North America
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