5.1 The Single Tube Method is designed to evaluate the efficacy of disinfectants against biofilm grown in the CDC biofilm reactor following the procedures outlined in Practice E3161. Biofilm grown in the CDC reactor is representative of biofilm that forms under high fluid shear on surfaces conducive to biofilm formation.

5.1.1 Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm growth reactors are engineered to produce biofilm with specific characteristics (2). Altering either the engineered system or operating conditions will modify those characteristics as well as the physicochemical environment. The goal in biofilm research and testing is to choose the growth reactor and operating conditions that generate the most relevant biofilm for the particular study.

5.2 The test method was designed to determine the log₁₀ reduction in bacteria after exposure to a disinfectant in a closed system.

5.3 The test method uses 50 mL conical tubes. The conical geometry allows for disinfectant exposure to biofilm on all surfaces of the coupon. For foaming disinfectants or for disinfectants requiring a larger volume of neutralizer, 250 mL conical tubes are used which preserve the required geometry and allow for greater neutralization capacity.

5.4 Each test includes three untreated control coupons (exposed to buffered dilution water) and five treated coupons (per disinfectant/concentration/contact time combination).

1.1 This test method specifies the operational parameters required to perform a quantitative liquid disinfectant efficacy test against bacterial biofilm.

1.2 The test method was optimized and validated for a Pseudomonas aeruginosa or Staphylococcus aureus biofilm grown in the CDC Biofilm Reactor (E3161). The method is suitable for evaluating additional bacteria grown using the procedures outlined in methods with comparable coupon dimensions such as Practice E3161, Test Method E2562, or Test Method E2196.

1.3 Disinfectant preparation and contact time are used in the assessment according to the manufacturer’s instructions for use.

1.4 The test method uses a closed system to treat biofilm. A coupon is placed in a single tube for the treatment, neutralization, and harvesting steps to prevent the loss of cells.

1.5 This test method describes a harvesting and analysis procedure which includes vortexing and sonicating treated and untreated control biofilm, and recovery of culturable cells using filtration to lower the limit of detection. Biofilm population density is recorded as log₁₀ colony-forming units per coupon. Efficacy is reported as a log₁₀ reduction of culturable cells.

1.6 Basic microbiology training is required to perform this assay.

1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.