Time-Kill In Vitro assay tests are referred to as “suspension tests” because a measured volume of the suspension of challenge bacteria or fungi is transferred into a liquid test material, dilute or “neat,” to determine how rapidly a challenge species is killed. These same procedures are applied, in principle, to testing surface-active antimicrobials per ASTM and AATCC methods.
The basic concept of the Time-Kill Kinetic study is the establishment of the rate at which a microorganism is killed by a product as a function of survival data recorded at enough exposure time points such that a graph can be constructed modeling the decline in population over time to a point of extinction.
In general and unofficially, the 3 Log10 reductions is considered the minimum level of performance that would indicate a product has substantive killing activity versus a particular microorganism. Anything less indicates that relatively huge numbers of the microorganisms remain viable after treatment with the product – for example, a 1 Log10 reduction in a population of one million bacteria (a small number, where bacterial contamination is concerned) means that 100,000 bacteria remain.
The challenge bacteria or fungi are introduced as a suspension, generally in 0.9% saline, into the test product, which is either undiluted (99%) or as a 10% dilution. The microorganisms are exposed to the product for one or more lengths of time specified by the protocol, after which a measured quantity of the suspension containing the microorganism and product is transferred to a neutralizing fluid that has been proven effective for eliminating the antimicrobial properties of the product.
This suspension is then serially diluted (usually 1:10), and aliquots of two or more of the dilutions series are poured- or spread-plated on an agar medium to allow for enumerating the colonies of the bacteria or fungus. Key to this process is the fact that each colony represents the reproduction of a single microorganism that survived exposure to the product, and hence, the number of survivors enumerated for a given post-exposure dilution can be used to back-calculate the number that were in the original product/microorganism suspension. This number is then used in combination with the initial population number (as explained above but enumerated from dilutions of the suspension originally used to inoculate the product) to determine percent and Log10 reductions.
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