Officially called the Chlorine (available) in Disinfectants Germicidal Equivalent Concentration, AOAC method 955.16 is typically used to test the efficacy of halogens such as chlorine, bromine, iodine. It is a rather unique method among disinfectant and sanitizer test methods, designed to identify the amount of a test product that is equivalent to a known-to-be-germicidal concentration of free chlorine.

Below, you will find a summary of the AOAC Chlorine Equivalency Method, along with some of its strengths and weaknesses.

Note: The information below is presented for educational purposes only. Current, detailed AOAC Methods can be obtained from AOAC.


Summary of the AOAC Chlorine Equivalency Method

  • Standard chlorine solutions are prepared at 200, 100, and 50 parts per million (ppm) concentrations for comparative use against the test substance.
  • A timer is started upon addition of test microorganism to the 200 ppm chlorine standard solution.
  • After 1 minute a transfer of the solution is made to an appropriate subculture tube using a sterile 4 mm loop.
  • After 1.5 minutes an additional inoculum of test microorganism is added to the standard solution.
  • At the 2.5 minute time point a transfer of the solution is made to a subculture tube.
  • This pattern of culture addition and subculturing of the solution is carried out a total of 10 times.
  • The method is then carried out in a similar manner using the remaining standard chlorine solutions.
  • The test method is then carried out for the test solution in a similar manner as the standard solutions.
  • All Subculture tubes are then incubated at 36°C for 48 hours.
  • To determine chlorine activity equivalent of test substance to standard solutions, the subculture tubes are analyzed for growth and must show similar growth of test microorganism in consecutive tubes as is observed in the standard solutions. 

Strengths of the AOAC Chlorine Equivalency Method

  • The method is capable of determining chlorine equivalents of test substances over a broad range of available chlorine concentrations.
  • It is the EPA-preferred method for substantiating food contact sanitization claims for halide chemicals.
  • The study sponsor is free to choose the test substance preparation method and concentration tested.

Weaknesses of the AOAC Chlorine Equivalency Method

  • Considerable knowledge of chemistry is required to accurately prepare the chlorine standard solutions. Improperly prepared chlorine solutions can increase variability from test to test.
  • The method requires generation of free chlorine performance data each time it is run, which is burdensome to the laboratory and costly to the Study Sponsor.

Microchem Laboratory generally runs the AOAC Chlorine Equivalency Test Method after some initial screening using a method such as the suspension time-kill test indicates success is likely. It is typically run under GLP test conditions, for submission to EPA.