Definition: The limit of detection (LOD) of an alternate microbiological method is defined as lowest number of microorganisms
in a defined volume of sample that can be detected, but not necessarily quantified, under the stated experimental conditions.
This should be conducted with the quality control organisms cited in Antimicrobial Effectiveness Testing á51ñ, Microbiological
Examination of Nonsterile Products: Microbial Enumeration Tests á61ñ, Microbiological Examination of Nonsterile Products: Tests for
Specified Microorganisms á62ñ, Mycoplasma Tests á63ñ, and Sterility Tests á71ñ as appropriate to the alternative method.
Demonstration
Method 1
• Inoculate a suitable diluent solution with a serial dilution range of each challenge microorganism, appropriate for the
intended use of the method and the technology. In most cases, the compendial media growth promotion test panel may
be sufficient.
• The level of inoculation should be adjusted to a target of 50% of the dilution samples that show growth in the
compendial test.
• Perform both the compendial and alternate tests.
• Tests should be repeated a sufficient number of times (statistically significant alpha risk: 0.05; beta risk: 0.20) for both the
compendial and alternate tests.
• Statistics: Use the chi-square test or another appropriate approach to demonstrate equivalent recovery of the
microorganism challenges.
• Alternately, use Method 2.
Method 2 (MPN Method)
• Create a dilution series of the challenge organisms in a suitable diluent solution to include at least the range of 101 cfu to
10−2 cfu (for a 10-fold series) or 5 cfu to 10−1 cfu/inocula volume (for a 2-fold dilution series).
• Perform both the compendial and alternate tests with at least 5 simultaneous replicates of each dilution from the chosen
series.
• Determine the most probable number (MPN) from three dilutions in series that provide both positive and negative growth
(or signal).
• Statistics: Use the chi-square test or another appropriate approach to demonstrate equivalent recovery of the
microorganism challenges.
