Jump to main content.

Questions for the SAP on Phenol Resistance


What scientific direction should be taken in regard to the lack of standard and uniform resistance levels to phenol of the test cultures used in the existing AOAC test methods?


"The ability to maintain and propagate selected test cultures at standard and uniform resistance levels to phenols is a fundamental necessity for the correct evaluation of chemical germicides by the AOAC Phenol Coefficient Method. This method prescribes the preparation and maintenance procedures. It occasionally happens that test cultures prepared in this manner are found to possess levels of resistance outside the limits prescribed by the official test. This results not only in invalidating the results secured in the particular tests in question, but also disrupts the entire testing program until a procedure is worked out to re-establish the test cultures at the resistance levels desired."

"The normal variations in cultures of E. Typhosa and S. aureus 209 when maintained and propagated according to the prescribed procedure have been recorded.......The resistance to phenol dilutions varied with S.aureus from 1-50 to 1-75, and with E.typhosa from 1-65 to 1-110. These resistance levels are somewhat wider than the 1-60 to 1-65 dilutions specified for S.aureus and the 1-85 to 1-95 dilutions specified for E.typhosa in the test."

"From the results secured in this study, it appears that the procedure described for maintaining and propagating test cultures in the official AOAC methods will provide test cultures of the prescribed resistance in at least 64 out of 100 cases for E.typhosa and 70 out of 100 cases of S.aureus."

The above three paragraphs are quotes from a scientific article entitled: Variations in Resistance in E. typhosa and S. aureus When Maintained As Specified According To The Official Phenol Coefficient Method, Journal of the Association of Official Agricultural Chemists, May 1949. The phenol resistance variability problems cited in this study were reported by Ortenzio, Friedl, and Stuart, (the forefathers of the current Antimicrobial Program) to the AOAC at the annual meeting of 1948.

The inability to consistently obtain the required phenol resistance patterns for test organisms to be used in effectiveness testing of antimicrobial products has been known to exist for almost 50 years. In fact, the same problem was addressed by Dr. George Reddish and reported to the Society of American Bacteriologists in December, 1924, which is 73 years ago.

From a historical perspective, it appears that the ability to maintain and propagate selected test cultures at standard and uniform resistance levels to phenol is a scientific/technical problem that has defied resolution for at least 70 years, since the development of the test procedure itself. Therefore, it does not appear that loss of resistance of the required test microorganisms is a recent problem as has been claimed by various groups.


The phenol resistance testing procedures and requirements appear in the AOAC Phenol Coefficient Method, and are referenced/cited in other AOAC methods for testing disinfectants and sanitizers. The following deficiencies; lack of clarity; inconsistencies; and lack of information in regard to resistance testing should be noted:

In summarization, the technical problems/issues discussed above must be considered in any attempt to resolve the standardization of phenol resistance patterns for the AOAC methods for testing disinfectants and sanitizers. There is no indication that any research is underway, or will be undertaken, to resolve phenol resistance problems. The Agency seeks scientific/technical direction in this matter.


The EPA's Office of Pesticide Programs (OPP) Cincinnati Microbiology Laboratory, and reportedly, commercial, industry, academic, private, and other government laboratories all experience difficulty in maintaining specified phenol resistance patterns. The OPP Laboratory reports failure to meet the specified resistance for the microorganisms used in the AOAC Use Dilution Method 25% of the time, and failure to meet the specified resistance in the AOAC Tuberculocidal Activity Method 50% of the time. Failure at the 25% level for the Use Dilution Method is fairly consistent with the 1948 report of 30-36% failure rate for the phenol resistance assays.

Procedures to restore lost resistance to the required resistance patterns have been unsuccessful. Such procedures have included passage through Dnase Agar and culturing in AOAC Synthetic Broth, as well as use of horse serum.

Failure to obtain the required phenol resistance patterns in the AOAC methods results in repeat of the entire test method, including phenol resistance, over and over again until the proper, required resistance is met. The loss of productivity under such circumstances has a significant impact on registration and regulatory activities.


OPP microbiologists have attempted to identify alternatives to the current situation involving lack of standard and uniform resistance levels to phenol of the test cultures used in existing AOAC test methods. The following three alternatives are offered for consideration by the Scientific Advisory Panel:

  • Totally eliminate the phenol resistance requirement.

  • Modify the required phenol resistance patterns to provide a broader range of acceptable resistance.

  • Replace the phenol resistance requirements with some other procedure that assures hardiness and equivalence of test cultures, such as standard, quantitative inoculum level.

The Agency requests that the Scientific Advisory Panel consider these and other options which they may identify, and provide specific guidance on a feasible approach/direction to resolve the phenol resistance problems experienced in association with the AOAC efficacy test methods.

Local Navigation

Jump to main content.