Standard Time-Kill Testing

Introduction

A Standard Time-Kill, often referred to as STK, Suspension Test, or Time-Kill Kinetics Assay, is an in vitro test which measures the changes in a population of aerobic microorganisms in a pre-determined sampling time while antimicrobial properties are present.  This test method is often used to measure antimicrobial activity in hand sanitizers, antiseptic wound gels, mouth washes, and other products that are expected to have microbiocidal properties [4]. Standard Time-Kill methods are based on the American Society for Testing and Materials method, Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure (E2315-16) [1].

When performing this technique, it is critical that variables associated in the test such as sampling time(s), test organism, and temperature(s) are kept standarized throughout testing.  Generally, a 3-log₁₀ reduction is considered the minimal level that would indicate a product has a significant killing activity against a particular test microorganism. [3]

Test Organism

The test organism selected may be representative of the microbial flora challenged by the test article, or a standardized strain. The test organism selected should be pure and easily identified in the case of contamination in test article. 

EQUIPMENT AND REAGENTS

Equipment

  • Sterile Vials 
  • Dilution Tubes
  • Calibrated Digital Timer 
  • Calibrated Water Bath or Controlled Temperature Chamber
  • Manual or Automatic Colony Counter 
  • Sterile Petri Dishes
  • Calibrated Incubator
  • Calibrated Autoclave 
  • Vortex Mixer
  • Sterile Calibrated Pipettes (10µl, 100µl, 1000µl)
  • Sterile Pipette Tips 
  • Sterile Positive Displacement Pipette Tips (optional if working with viscous materials)

Reagents

  • Dilution Fluid (sterile water, 0.9% (w/v) saline, sterile butterfield's phosphate diluent, sterile phosphate buffered saline, or equivalent)
  • Neutralizer (optional if diluent does not quench antimicrobial properties. Examples include D/E neutralizing broth, letheen broth, buffer peptone water)
  • Broth Growth Medium 
  • Solid Growth/Plating Medium
  • Sterile Deionized water

Procedure

Preparation of Test Microorganism

  1. Transfer cultures from a known stock in appropriate growth medium suitable for the selected organism. A second transfer can be made in order to have a sufficient  volume to be used during testing and controls.
  2. To prepare inoculum suspension for testing, 1:10 dilutions of the suspension in may be performed to reduce the concentration to one suitable for testing.  
  3. The inoculum suspension should be plated using standard microbiological procedures at the start and completion of testing.  The initial and final titer should be within ± 0.5 log₁₀ for a valid test. 

Preparation of Test Article

  1. Select the concentration of test material need to perform the experiment. Each concentration should be tested in duplicate, however more replicates can be used if needed. Each recovery sample associated with each replicate should be plated in either duplicate or triplicate.
  2. Prepare each test concentration by dilution using sterile distilled water or other appropriate diluents. Some test materials may require heating to become dispersed in solution. Allow each solution to equilibrate to 25 ± 2°C. (additional test temperatures can be considered depending on intended use. For example, 22 ± 2°C for room temperature; 30 ± 2°C for temperature of human skin; and 38 ± 2°C for temperature of “warm” water [1] ) 
  3. Select contact times associated with the minimum time period needed for test article. (10s, 15s, 30s, 60s, etc.) Other time points may be selected depending on the test article or if construction of a curve is desired. 

PREPARATION OF TEST SETUP

  1. To minimize buffer interference and reduction of antimicrobial activity during testing, the volume of inoculum suspension should be less than or equal to 5% of the total test volume. The microbial population should achieve a minimum of 10^6 CFU/ mL per test.
  2. Prior to testing, inoculum should be uniformly mixing. Ensure uniform mixing throughout the test for repeatability.
  3. Begin mixing test sample and transfer suspension sample and control blanks. If applicable, mix with care to avoid foam formation that can cause anomalous results.  
  4. At the predetermined intervals, remove an aliquot (1 mL, or appropriate volume) and diluent either directly into a neutralizer based diluent, or add the aliquot to a known volume of neutralizer and then silent appropriately for plating. 

Plating and INCUBATION of TEST ARTICLES and Sterilities

  1. Recover viable organisms from dilution tubes by culturing and plating. (either spread, pour-plating, microbial filtration, spiral plating, or other viable recovery methods.)
  2. Appropriate sterility controls should be conducted including an isolation streak of test strain to verify culture purity, sterility controls of all reagents used during testing(diluents, growth media, neutralizer, etc.),  and if applicable, a standard plate count to verify any microbial contamination in the test article. 
  3. Plates should be incubated at specified temperatures for optimal growth ± 2°C for 24-48 hours or as appropriate depending on the test organism. Incubation of plates should allow for growth of surviving organisms while preventing overgrowth.
  4. After Incubation, determine the surviving organisms by counting colonies (automatic or manual) and record raw data as CFU/plate. Average duplicate counts and multiply by the dilution factor to arrive at CFU/mL. This count should be converted to log₁₀ scale in order to calculate microbial reduction. 

Calculating Microbial log₁₀ reduction

Screen Shot 2018-06-08 at 1.15.57 PM.png

References

[1] ASTM E2315-16, Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure, ASTM International, West Conshohocken, PA, 2016, www.astm.org

[2] Foerster, Sunniva et al. “Time-Kill Curve Analysis and Pharmacodynamic Modelling for in Vitro Evaluation of Antimicrobials against Neisseria Gonorrhoeae.” BMC Microbiology 16 (2016): 216. PMC. Web. 8 June 2018.

[3] “Time-Kill Kinetics Assay (Antimicrobial Efficacy Testing).” Microbiology and Cell Biology, Medicinal Chemistry - Emery Pharma, emerypharma.com/biology/time-kill-kinetics-assay/.

[4] “Time Kill Test.” Nelson Labs - Microbiology Testing, www.nelsonlabs.com/Test/Time-Kill-Test.