The liquid suspension time-kill test is excellent for disinfectant product developers because it is a fast, relatively inexpensive, and reproducible way to measure the biocidal potential of a liquid antimicrobial formulation. It consists of directly inoculating a liquid test substance with a high concentration of test microorganisms and then determining the percentage killed over time.
The suspension-based time-kill test has been standardized by ASTM, as ASTM E2315 “Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure.”
Liquid Time-Kill Test Method Summary
- A microbial culture is prepared. For most bacteria, a 24 hour culture in nutrient broth works well. For most fungi, a spore preparation from a saline wash works well.
- Equal volumes of the test product are placed in sufficient sterile test vessels.
- A volume of microbial culture (usually 1/10 of the product volume) is placed in the test vessel and then immediately mixed.
- After the predetermined contact times, small aliquots of the mixture of bacteria and product are removed and microorganisms are enumerated.
- To measure initial microbial concentrations, a saline control vessel is spiked with the same microbial culture and then enumerated.
- Numbers of microorganisms in the reaction vessel are plotted over time.
- Neutralization controls are run as appropriate.
Strengths of the Liquid Time-Kill Test
- The impact of a disinfectant product on microorganisms over time (death curve) can be studied with relative ease using the suspension-based test method.
- Suspension-based time-kill tests are relatively inexpensive.
- The test parameters for suspension-based time-kill tests are easy to control in the laboratory setting, so comparisons can be made fairly easily between various products tested under the same conditions.
- The suspension-based time-kill test involves exposing microorganisms to excess disinfectant in a liquid setting, so the test is a fitting model system for instances in which disinfectants will be used to kill microorganisms in liquid settings (such as in a disinfectant rinse for drinking glasses in a restaurant).
- Very brief contact times can be studied with relative ease.
Weaknesses of the Test Method
- The test method is difficult to relate to disinfection of microorganisms on a surface (such as results from the AOAC use-dilution test method).
- It is somewhat of a “best case” method meaning that good percent reductions are likely to be seen if the test chemical is indeed antimicrobial and the contact times are sufficient.
- The way in which the microbial inoculum is prepared can have an impact on the test outcome, since different broths/suspensions will interact with disinfectants differently.
At Microchem Laboratory, we are proud of our tradition of collaborative, productive, antimicrobial research and development. The suspension-based time-kill test is just one of many tools we use to help companies develop disinfectants and other antimicrobial products.