1. Scope

1.1 The Biofilm Surface Test Method (BSTM) is a high-throughput screening system used to determine the antimicrobial activity of either liquid agents or agents impregnated, coated, or incorporated into a medical device or surface, and may also be used to evaluate surface-modified devices that contain no antimicrobial agent. The BSTM incorporates key salient features to provide a simple, robust challenge of the device/surface while best simulating the real-world environment to which the device will be exposed. The assay device consists of a plastic lid that accommodates the attachment of a test article (e.g., endotracheal tube, catheter, or a coupon of relevant test material) and a corresponding receiver plate with twelve (12) individual wells that are filled with growth medium. Biofilm is established on the surface of the test device under batch conditions (i.e., no flow of nutrients into or out of an individual well) with gentle mixing, then transported to a new receiver plate for antimicrobial efficacy testing. The design allows for the simultaneous testing of replicate samples and multiple parameters, making it an efficient screening tool.

1.2 This practice mimics the host environment (e.g., by pre-conditioning the device/surface with a relevant medium, such as serum, artificial urine, or artificial mucous), the scope of organisms to which the device will be exposed (e.g., clinical isolates), and the conformation of the device as it will be used and presented to the challenge organisms in a clinical or environmental setting (e.g., if desired, testing can be limited to the extraluminal surface of a catheter by attaching it to the lid in the shape of a “U”). Corrosion, encrustation, or biofouling of devices or surfaces can also be evaluated using this practice.

1.3 This practice is versatile and can be used for growing and evaluating biofilms of many different organisms, such as those in references (1, 2, and 3). Appropriate modifications to the practice may be required when testing organisms not specified herein.

1.4 Validation of disinfectant neutralization can be included as part of the assay.

1.5 This practice describes how to sample the biofilm and quantify viable cells. Biofilm population density is recorded as log10 colony forming units (either per unit surface area or per unit volume of the recovery media). To test for any antimicrobial activity due to leaching, suspended bacterial population density is reported as log10 colony forming units per volume. Efficacy is reported as the log10 reduction of viable cells. Alternatively, or additionally, qualitative or semi-quantitative assessments can be utilized, such as colorimetric staining or turbidimetric assessment of the minimal biofilm eradication concentration (MBEC).

1.6 This practice should be performed only by those trained in microbiological techniques.

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 It is the responsibility of the investigator to determine whether Good Laboratory Practices (GLPs) are required and to follow them when appropriate.

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

Keywords

Encrustation; Corrosion; Antimicrobial; Disinfectant; Coating; Surface; Impregnated; Incorporated; Device; Catheter; Stent; Tubing; Screen; High-Throughput; Biofilm; Multi-Well; Lumen; Extraluminal

Rationale

Vegetative biofilm bacteria are phenotypically different from suspended planktonic cells of the same genotype. Biofilm is the etiological agent of many implant and device-related infections and, once established, microorganisms in biofilm can be up to 1000 times more tolerant to antibiotic therapy. Biofilm growth reactors are engineered to produce biofilms with specific characteristics. Altering either the engineered system or operating conditions will modify those characteristics. The goal in biofilm research and efficacy testing is to choose the growth reactor that generates the most relevant biofilm for the particular study. The engineering design of the BSTM allows for the simultaneous evaluation of multiple test conditions, making it an efficient screening method (e.g., for screening multiple disinfectants or multiple concentrations of the same disinfectant, for screening multiple surfaces, multiple organisms, etc.). Additional efficiency can be added by including the neutralizer controls within the assay device. Many of these advantages are shared with an existing ASTM Standard Method (E2799), which describes the MBEC screening assay. However, the device used in the MBEC assay limits the user to growing and testing biofilms exclusively on polystyrene pegs; in contrast, the BSTM utilizes actual test devices or materials attached to a multi-well lid, allowing for biofilm growth and evaluation on virtually any surface of interest. Moreover, this approach allows for a simple and efficient means of evaluating corrosion or encrustation of test devices. In addition to the throughput afforded by the use of a multi-well plate, the BSTM allows for extremely facile rinses, media changes, challenges, and recovery steps by virtue of immobilizing the test devices/surfaces to the lid of the multi-well plate. The relatively small well volume (4.5 mL) is advantageous for testing expensive disinfectants, or when only small volumes of the disinfectant are available. These advantages are in contrast to other ASTM standards, which grow more specialized biofilms in a manner less amenable to high-throughput screening, such as E3321 (meant to establish intraluminal biofilms in urinary catheters under conditions of constant media flow) or E3151 (meant to test biofilm growth on tubes, yarn, and fibers suspended in individual culture tubes). Finally, the BSTM does not require any specialized equipment, such as a CDC biofilm reactor (used in E2562, E2871, E3161), drip flow reactor (used in E2647), or rotating disk reactor (E2196).

The title and scope are in draft form and are under development within this ASTM Committee.