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1.1 This test method specifies the operational parameters required to grow a repeatable2 Pseudomonas aeruginosa biofilm close to the air/liquid interface in a reactor with a continuous flow of nutrients under low fluid shear conditions. The resulting biofilm is representative of generalized situations where biofilm exists at the air/liquid interface under low fluid shear rather than representative of one particular environment.

1.2 This test method uses the drip flow biofilm reactor. The drip flow biofilm reactor (DFR) is a plug flow reactor with laminar flow resulting in low fluid shear. The reactor is versatile and may also be used for growing and/or characterizing biofilms of different species, although this will require changing the operational parameters to optimize the method based upon the growth requirements of the new organism.

1.3 This test method describes how to sample and analyze biofilm for viable cells. Biofilm population density is recorded as log colony forming units per surface area.

1.4 Basic microbiology training is required to perform this test method.

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

1.6 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.

1.7 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.

5.1 Vegetative biofilm bacteria are phenotypically different from suspended cells of the same genotype. Biofilm growth reactors are engineered to produce biofilms with specific characteristics. Altering either the engineered system or operating conditions will modify those characteristics.

5.2 The purpose of this test method is to direct a user in how to grow, sample, and analyze a P. aeruginosa biofilm under low fluid shear and close to the air/liquid interface using the DFR. The P. aeruginosa biofilm that grows has a smooth appearance that varies across the coupon surface and is loosely attached. Microscopically, the biofilm is sheet-like with few architectural details. This laboratory biofilm could represent those found on produce sprayers, on food processing conveyor belts, on catheters, in lungs with cystic fibrosis, and oral biofilms, for example. The biofilm generated in the DFR is also suitable for efficacy testing. After the 54 h growth phase is complete, the user may add the treatment in situ or harvest the coupons and treat them individually. Research has shown that P. aeruginosa biofilms grown in the DFR were less tolerant to disinfection than biofilms grown under high shear conditions.5