Significance and Use 5.1 There are no reproducible standardized protocols for preparing specimens used to evaluate the microbicidal efficacy of non-chemical treatments such as ultraviolet (UV), highenergy electron beam, or other forms of non-chemical antimicrobial technologies. 5.2 Conventional protocols for applying bioburdens to carriers (see Test Method E2197) cause cells to stack upon one another, thereby creating multiple cell layers in which cells in layers closer to the carrier are masked by cells in overlying layers, which makes relative comparison of different non-chemical antimicrobial treatments more difficult. 5.3 Steel and other metal carriers have asperities that can shield a percentage of the applied cells from direct exposure to electromagnetic irradiation. 5.4 The combined effects of 5.2 and 5.3 confound determination of the microbicidal effect of electromagnetic irradiation on test specimens. 5.5 The practice addresses these two confounding factors by: 5.5.1 Using glass microscope slides – the surfaces of which are asperity-free – as carriers. 5.5.2 Reliably depositing bacterial cells onto the carrier as a monolayer. 5.6 The resulting specimen ensures that all microbes deposited onto the carrier are exposed equally to the irradiation source thereby ensuring that the only variables are the controlled ones – starting inoculum concentration, wavelength (λ – in nm), exposure time(s), and resulting energy dose (J).Scope 1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface. 1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet. 1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided. 1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells. 1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments. 1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies. 1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments. 1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06. 1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy. 1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions. 1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used. 1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.10 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. 1.11 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.
