Significance and Use
This test method is fully quantitative and it also avoids any loss of viable organisms through wash off, making it possible to produce statistically valid data using many fewer test and control carriers than other quantitative methods based on most probable numbers (MPN).
The design of the carriers makes it possible to place into each a precisely measured volume of the test suspension. The use of the threaded stir bars allows for efficient recovery of the inoculum even after its exposure for several hours to strong fixatives such as glutaraldehyde.
The membrane filtration step allows processing of the entire eluate from the test carriers and therefore the capture and subsequent detection of even low numbers of viable organisms that may be present.
This test can be performed with or without a soil load to determine the effect of such loading on microbicide performance. Consult the target regulatory agency on the need, type(s), and acceptable level(s) of soil load prior to testing. One type of soil load (Quantitative Disk Carrier Test Method E2197) to consider for this test is a mixture of three types of proteins (high molecular weight proteins, low molecular weight peptides, and mucous material) to represent the body secretions, excretions, or other extraneous substances that chemical microbicides may encounter under field conditions. It is suitable for working with the various test organisms included here. The components of the soil load are readily available and subject to much less variability than animal sera.
If distilled water or other diluent is not to be specified on the product label, the diluent for the test substance is assumed to be tap water. Since the quality of tap water varies considerably both geographically and temporally, this test method incorporates the use of water with a specified and documented level of hardness to prepare use-dilutions of test substance that require dilution in water before use. Consult the target regulatory agency regarding the use and level of water hardness prior to testing.
1.1 This test method is designed for use in product development and for the generation of product potency data. This test method permits the loading of each carrier with a known volume of the test organism. The incorporation of controls can also determine the initial load of colony forming units (CFU) of organisms on the test carriers and any loss in CFU after the mandatory drying of the inoculum.
1.2 This test method is designed to have survivors and also to be used with a performance standard. The surviving microorganisms on each test carrier are compared to the mean of no less than three control carriers to determine if the performance standard has been met. To allow proper statistical evaluation of results, the size of the test inoculum should be sufficiently large to take into account both the performance standard and the experimental variation in the results. For example, if an arbitrary performance standard of 6-log10 reduction in the viability titer of the test organism is used, and an inoculum size of 107 CFU, then theoretically a maximum of ten survivors per carrier is permitted; however, because of experimental variability, the exact target may need to be higher than 106 CFU/carrier, thus fewer survivors would be permitted.
1.3 This test method should be performed by persons with training in microbiology and in facilities designed and equipped for work with infectious agents at the appropriate biosafety level (3).
1.4 In this test method, SI units are used for all applications, except for distance, in which case inches are used and SI units follow.
1.5 It is the responsibility of the investigator to determine whether Good Laboratory Practice Regulations (GLPs) are required and to follow them where appropriate (40 CFR Part 160 for EPA submissions and 21 CFR Part 58 for FDA submissions).
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
E1054 Test Methods for Evaluation of Inactivators of Antimicrobial Agents
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
21CFR Part 58
Bacillus subtilis spores; bactericides; chemical microbicides; Clostridium sporogenes spores; eluate; eluent; environmental surfaces; fungicides; germicides; log10 reductions; medical devices; membrane filtration; mycobactericides; Mycobacterium terrae; Pseudomonas aeruginosa; quantitative carrier test; soil load; sporicides; Staphylococcus aureus; standard hard water; surrogate; Trichophyton mentagrophytes conidia;
ICS Number Code 71.040.50 (Physicochemical methods of analysis)
ASTM International is a member of CrossRef.
Citing ASTM Standards
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