Сòòò½APP

Chemical disinfectants and sanitizers that claim to reduce, destroy, or inactivate microorganisms including bacteria, fungi, yeast and viruses are regulated by the EPA. For more than three decades, Сòòò½APP has been the trusted antimicrobial laboratory partner to leading developers, manufacturers, and marketers of disinfectants and sanitizers.

Disinfectants and sanitizers can be applied to surfaces with a variety of methods. Liquid disinfectants and sanitizers can be applied via mopping or soaking, and products are also marketed with trigger/pump or aerosol spray, in addition to pre-saturated towelettes. These products can be used on hard surfaces as well as soft surfaces, and they may also contain residual properties. Sanitizers and disinfectants have also been used in antimicrobial coatings.

We have experience and expertise developing custom protocols and testing to numerous established methods. The most frequently asked for methodology has been listed here, so if you do not see the method you are interested in, connect with us.

Bactericidal and fungicidal testing

Сòòò½APP offers comprehensive bacterial and fungicidal testing to a wide range of established methods, including:

AOAC Method 961.02 Germicidal Spray Method - In this method, a series of glass slides (“carriers”) are inoculated with a representative test organism and the carriers are dried. The carriers containing the dried organism film are then sequentially treated with the spray product and are exposed for a predetermined exposure time. After exposure, the carriers are sequentially transferred to a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving test organism. The carriers are incubated and visually examined for the presence or absence of growth.

Based on the desired disinfection claim and the requirements of the regulatory agency, the product must demonstrate kill on a predetermined number of carriers inoculated with test organisms applicable to the claim. Standard disinfection organisms include but are not limited to, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. Additional pathogens of clinical, occupational or household relevance including fungi are often tested.

ASTM E2362/AOAC Method 961.02 (Modification) Pre-saturated Towelettes for Hard Surface Disinfection Test Method - In this method, a series of glass slides (“carriers”) are inoculated with a representative test organism and the carriers are dried. The carriers containing the dried organism film are then sequentially treated with the towelette product and are exposed for a predetermined exposure time (≤10 minutes). After exposure, the carriers are sequentially transferred to a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving test organism. The carriers are incubated and visually examined for the presence or absence of growth.

Based on the desired disinfection claim and the requirements of the regulatory agency, the product must demonstrate kill on a predetermined number of carriers inoculated with test organisms applicable to the claim. Standard disinfection organisms include but are not limited to: Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. Additional pathogens of clinical, occupational or household relevance including fungi are often tested.

AOAC Use Dilution Test for Liquid Disinfectant (Methods 964.02, 955.14 and 955.15) - In this method, a series of stainless-steel cylinders (“carriers”) are inoculated with a representative test organism and the carriers are dried. The carriers containing the dried organism film are then sequentially immersed into 10 mL of disinfectant and are exposed to the disinfectant for a predetermined exposure time. After exposure, the carriers are sequentially transferred to a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving test organism. The carriers are incubated and visually examined for the presence or absence of growth.

Based on the desired disinfection claim and the requirements of the regulatory agency, the product must demonstrate kill on a predetermined number of carriers inoculated with test organisms applicable to the claim. Standard disinfection organisms include but are not limited to: Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. Additional pathogens of clinical, occupational or household relevance including fungi are often tested.

AOAC Method 965.13 Disinfectant (Water) for Swimming Pools Method - In this method, a sample of test substance is inoculated with the test organism, commonly Escherichia coli and Enterococcus faecium. Over a series of time points, samples are removed, the test active is neutralized and the sample is quantitatively assayed for surviving test organism. In a similar fashion, a known concentration of a sodium hypochlorite reference standard is inoculated, exposed, neutralized and assayed for survivors. The resulting subcultures are incubated and subsequently evaluated for survivors. The test subcultures are then compared to the sodium hypochlorite control subcultures.

The lowest concentration of test substance providing results equivalent to the sodium hypochlorite control solution is considered the lowest effective for disinfection.

AOAC Method 955.16 Available Chlorine in Disinfectants - In this method, a sample of the disinfectant is inoculated with a suspension of representative test organism. After 1 minute, a sample is removed and transferred to a tube containing a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving test organism. Thirty seconds later, the same sample is re-inoculated. After an additional 1 minute of exposure, a second sample is removed and recovered. This is repeated until 10 samples have been removed. Side-by-side controls are identically evaluated using 50 ppm, 100 ppm and 200 ppm sodium hypochlorite. The subculture tubes are incubated, examined for growth, and the test subcultures are compared to the control sodium hypochlorite tubes.

For food-contact sanitizing rinses, test results should demonstrate product concentrations equivalent in activity to 50, 100, or 200 ppm of sodium hypochlorite. For hand sanitizers, test results should demonstrate product concentrations equivalent in activity to 50 ppm of sodium hypochlorite.

Typical test organisms include Staphylococcus aureus and Salmonella enterica serovar Typhi. Other foodborne pathogens are often tested as well.

Quantitative Disk Carrier Test Method (Modification of ASTM Method E2197) - In this method, a series of stainless steel disks are inoculated with the test organism. The carriers are desiccated and subsequently treated with a sample of the liquid disinfectant. After exposure, the carriers are neutralized and quantitatively assayed for surviving test organism. The resulting plates are incubated, the number of survivors is enumerated and a percent and log₁₀ reduction is determined as compared to a population control.

No regulated performance criteria have yet been established by the U.S. EPA however products may be evaluated using this method to investigate product performance. Common test organisms include Staphylococcus aureus, Enterococcus hirae and Pseudomonas aeruginosa.

AOAC Method 955.17 Fungicidal Activity Method - In this method, a sample of the disinfectant is inoculated with a suspension of fungi (Trichophyton interdigitale) and the suspension of fungi is then exposed to the disinfectant for 5 minutes, 10 minutes and 15 minutes. Following exposure, a sample of the exposed fungi is removed and is added to a tube containing a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving fungi. The subculture tubes are incubated and visually examined for the presence or absence of growth.

For disinfection claims, no growth must be observed after 10 minutes of exposure.

Mildew Fungistatic Test for Fabrics or Hard Surfaces - In this method, ceramic tile or cotton muslin carriers are treated with the product and the carriers are allowed to dry. Following drying, the carriers are inoculated with Aspergillus niger. The treated carriers are incubated in a high humidity environment and are visually rated for mold growth.

For a product to be considered an effective mildew fungistat, the treated surfaces must demonstrate no mold growth following incubation.

Laundry testing

Our consultative antimicrobial experts perform laundry testing to established standard methodology.

ASTM E2274 and E2406 Standard Test Method for the Evaluation of Laundry Disinfectants - In this method, a series of fabric swatches are inoculated with the test organism and are dried. The swatches containing the dried film of test organism are placed inside fabric-wrapped spindles and are treated in a simulated laundry apparatus. Following treatment, the swatches and a sample of the wash water are removed and are assayed for surviving test organism.

In order to successful demonstrate disinfection efficacy, no test organism must be recovered on the

treated fabric swatches or in the treated wash water. Typical test organisms include Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae.

Electrostatic sprayer testing

The EPA  specific testing to support claims for disinfectants that are to be used in electrostatic sprayer devices. Сòòò½APP’s knowledgeable antimicrobial team conducts electrostatic sprayer testing for EPA claims according to standard methodology.

Wetness Determination Using Electrostatic Sprayers - The purpose of this assay is to determine surface wetness imparted by simulated use of an electrostatic sprayer in support of label claims. Carriers are sprayed with the test substance at a minimum and maximum distance and both the gravimetric and physical wetness will be observed following the specified exposure time.

AOAC Germicidal Spray Method for Use with Electrostatic Sprayers - The purpose of this study is to determine the effectiveness of the Sponsor’s product as a disinfectant for hard surfaces following the AOAC Germicidal Spray Method and the use of an electrostatic sprayer. A film of organism cells dried on a surface of glass slide carriers is exposed to the test substance, applied with an electrostatic sprayer, for a specified exposure time.  After exposure, the carriers are transferred to vessels containing neutralizing subculture media and assayed for survivors.  Appropriate culture purity, sterility, viability, neutralization confirmation and carrier population controls are performed.

Tuberculocidal testing

Tuberculocidal testing is performed in our antimicrobial laboratories according to established methods and protocols.

AOAC Method 965.12 Tuberculocidal Activity of Disinfectants - In this method, a series of porcelain cylinders or glass slides (“carriers”) are inoculated with Mycobacterium bovis and the carriers are dried. The carriers containing the dried organism film are then sequentially treated with the disinfectant in a manner intended to simulate its intended use and are exposed for a predetermined exposure time. After exposure, the carriers are sequentially transferred to a series of liquid media specifically selected to neutralize the test substance active and subsequently recover Myocbacterium. The subculture media is incubated and visually examined for the presence or absence of growth. For tuberculocidal claims, all subcultures must demonstrate kill (no growth) of Mycobacterium bovis.

Quantitative Tuberculocidal Suspension Method - In this method, quadruplicate samples of the product are inoculated with a suspension of Mycobacterium bovis – BCG. The inoculated products are allowed to expose for a series of exposure times. After each exposure time, a sample of product is removed, neutralized and is quantitatively assayed for surviving Mycobacterium. A percent reduction is determined for the test samples, at each time point, as compared to a population control.

The U.S. EPA requires an effective product demonstrate at least a 99.99% (4 log₁₀) reduction of Mycobacterium and an average of <1 survivor at the effective time. For high-level disinfectants, the U.S. FDA requires an effective product demonstrate at least a 99.9999% (6 log₁₀) reduction of Mycobacterium and an average of <1 survivor at the effective time. When the minimum reduction is not met at any of the times utilized in the study, a survivor curve can be constructed to predict the effective time of the product.

Sporicidal testing

Сòòò½APP’s antimicrobial labs offer sporicidal testing in accordance with standard methodology.

AOAC Method 966.04 Sporicidal Activity of Disinfectants - In this method, a series of porcelain cylinders and silk or Dacron sutures (“carriers”) are inoculated with the spores of Bacillus subtilis and Clostridium sporogenes and are desiccated. The carriers containing the dried spore film are then sequentially immersed into 10 mL of disinfectant and are exposed to the disinfectant for a predetermined exposure time. After exposure, the carriers are sequentially transferred to a liquid subculture medium specifically selected to neutralize the test substance active and to recover any surviving spores. The carriers are incubated and visually examined for the presence or absence of growth.

Based on the desired claim and the requirements of the regulatory agency, the product must demonstrate kill on a predetermined number of carriers inoculated with test organisms applicable to the claim.

Re-use Stress Testing and Evaluation of Disinfectants - In this method, products are processed through a series bio-burden stressing procedures to generate a product which has undergone simulated-use. Commonly, these products are combined with an organic soil load and undergo daily simulated-use cycles by processing medical equipment into and out of the product multiple times. Bio-burden stressing is performed each day by transferring a specific amount of inoculated carriers containing vegetative and spore-forming bacteria into the product.

The products are typically stressed on a daily basis for up to 28 days to simulate the length of time the product would be re-used in a clinical setting. The final product is then evaluated for high-level disinfection efficacy using the AOAC Use Dilution, AOAC Sporicidal method and AOAC Tuberculocidal method, among others.

Biofilm testing

Biofilms are collections of microorganisms that produce slime which protects the organisms. The slime and microorganisms create a biofilm that is difficult to disinfect or remove from surfaces. Testing antimicrobial products against biofilms requires special equipment to grow the organisms in a system that mimics real world conditions. Сòòò½APP has the training, equipment, and expertise to perform the testing that is required to generate data for biofilm efficacy claims.

Single Tube Method for Determining the Efficacy of Disinfectants against Bacterial Biofilm (Pseudomonas Aeruginosa and Staphylococcus Aureus) - A biofilm is grown onto a series of coupon “carriers” and treated with a sample of product in a closed system for a specified exposure time. Following exposure, the carriers are neutralized and assayed for surviving bacteria. The resulting plates are incubated, enumerated, and the Log₁₀ reduction is determined as compared to the population control.

For biofilm claims, a 6 Log₁₀ reduction must be observed. Currently draft guidance has been published to follow U.S. EPA BEAD SOPs (MB-19 and MB-20).

MIC/MBC testing 

Our antimicrobial experts have deep expertise in Minimum Inhibitory and Minimum Bactericidal Concentration (MIC/MBC) testing to the following methods.

Minimum Inhibitory and Minimum Bactericidal Concentration Determination Macrodilution Broth Method - Determining the concentration of a test substance which shows complete inhibition of growth and/or complete kill of a test organism can be performed in a variety of ways.  The test system used in this study is a modification of the ASM Manual of Clinical Microbiology, Fifth Edition, Susceptibility Test: Macrodilution Broth Method. 

A 2X concentration of test substance is diluted in a series of two-fold dilutions within the sterile tubes.  A suspension of each test organism is added to the tubes containing the test substance dilution which dilutes the test substance two-fold to achieve the final desired concentration.  The tubes are incubated and observed for visible growth. After observation, appropriate tubes are sub cultured to solid agar plates and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) are determined. 

Minimum Inhibitory and Minimum Bactericidal Concentration Determination Microdilution Broth Method - Determining the concentration of a test substance which shows complete inhibition of growth and/or complete kill of a test organism can be performed in a variety of ways.  The test system used in this study is a modification of the ASM Manual of Clinical Microbiology, Fifth Edition, Susceptibility Test: Microdilution Broth Method. 

A 2X concentration of test substance is diluted in a series of two-fold dilutions within the wells of a microtiter plate.  A suspension of each test organism is added to the wells containing the test substance dilution which dilutes the test substance two-fold to achieve the final desired concentration.  The microtiter plate is incubated and observed for visible growth.  After observation, appropriate wells are subcultured to solid agar plates and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) are determined. 

Minimum Inhibitory Concentration – Agar Dilution Method - The test system to be used in this study will be a modification of the ASM Manual of Clinical Microbiology, Eighth Edition, Susceptibility Test:  Agar Dilution Procedure.

The test material is diluted by a series of two-fold dilutions and is mixed into a subculture agar medium within Petri dishes.  A suspension of each test organism is added to the solidified plates containing the test material within the agar.  The plates are incubated and observed for visible growth.  The lowest concentration of test substance that completely inhibits visible growth of the organism (as detected by the unaided eye) is the MIC.

Antimicrobial preservatives - effectiveness testing

A preservatives system ability to protect a product against contamination must be supported by appropriate scientific data demonstrating the efficacy of the preservative against the claimed microorganisms.  This is accomplished by treating the test material with the microorganisms under conditions which simulate as closely as possible, in the laboratory, the actual conditions under which the test material is designed to be used. In this study, incubation times and temperatures have been modified in an effort to maximize test system recovery while considering the varying recommendations between the USP and CTFA methods.

The test material is exposed to a suspension of test organism and held at the specified temperature for a specified time period. The inoculated test material will be assayed for surviving microorganisms at specified intervals. Appropriate sterility, culture purity, initial suspension population and neutralization confirmation controls are performed.

The Сòòò½APP advantage

Сòòò½APP has been the trusted partner for leading developers, manufacturers, and marketers of antimicrobial disinfectants and sanitizers for more than three decades. A leader in antimicrobial testing, our consultative scientific and regulatory experts stay up-to-date on market trends and regulatory guidance. Confidently and successfully navigate regulatory requirements and product registration with the support of Сòòò½APP.

Our team stands by the data we develop, and we will work with you and regulatory agencies to answer any questions that may be asked regarding the testing of your product. We are also prepared to defend the testing and the data developed if the need arises. If there is a method we're missing, please reach out and contact us to learn more.

For more information about Сòòò½APP’s antimicrobial disinfectant and sanitizer efficacy testing, contact us today.