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.

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