Time-Kill Kinetics Assay – Antimicrobial Efficacy Test

Time-Kill Kinetics Test – Antimicrobial Efficacy Testing

The Time-kill kinetics assay is used to study the activity of an antimicrobial agent against a bacterial strain and can determine the bactericidal or bacteriostatic activity of an agent over time.

Basics of the Time-Kill Kinetics

Bactericidal activity is defined as greater than 3 log10 -fold decrease in colony forming units (surviving bacteria), which is equivalent to 99.9% killing of the inoculum. The time kill analysis can monitor the effect of various concentrations of an antimicrobial agent over time in relation to the stages of the growth of the bacteria (lag, exponential, stationary phase). Time-kill kinetics assays for agents such as antiseptics require a shorter time-kill kinetics study and follow different methodology. In contrast to the multiple time points in a time-kill kinetics assay, the minimal bactericidal concentration (MBC) test is defined as a 99.9% or greater killing efficacy at a specified time.

  • Time Kill Kinetics Assay for antimicrobial agents are performed using CLSI guidelines (see illustration)
  • Time Kill Kinetics Assays for antimicrobial agents that require shorter time analysis (seconds/minutes) are performed using ASTM guidelines

Figure Legend. A sample time-kill kinetics assay is depicted. Three different test compounds, Compound 1, Compound 2, and Compound 3 are added to media containing a starting culture of bacteria. A vehicle only control and a growth control are included as negative controls. The log CFU/mL for all groups is determined at time 0 and at subsequent time points up to 24 hours, depending on the bacteria strain and the media used. In this example, Compound 1 exhibited bactericidal effect, reducing the starting log CFU/mL by greater than 3 logs. Compound 2 exhibited bacteriostatic effect, as the log CFU/mL over time remained roughly the same as the starting log CFU/mL concentration. Compound 3 exhibited little antimicrobial effect, as the bacteria in the presence of this compound grew over time to a level similar to the vehicle only control.

Emery Pharma has successfully performed several Time-Kill studies for multiple clients in pharmaceutical industry. All testing follows the guidelines set by the Clinical & Laboratory Standards Institute (CLSI).


CLSI Methods of Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline, M26, V. 19, No. 18, September 1999.

ASTM Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure. Method E2315 – 16.