ASTM E2871:2019 pdf download.Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown inthe cDC Biofilm Reactor Using the Single Tube Method.
9.7 Dilute and Recover Disaggregated Biofilm Samples:
9.7.1 Dilute and recover treated samples followed by control samples. Initiate dilutions within 30 mm of neutralization.
9.7.2 Serially dilute the disaggregated biofilni from control samples in buffered water. Test coupons may be serially diluted, if necessary, to achieve countable filters in the target range of 20-200 CFU.
9.7.3 For treated coupons, tiller at least 25 % of the total volume of neutralizer + disinfectant from the iO° reaction tube through 0.45 pm PES membrane filters. Initiate filtration within 30 mm of making dilutions.
9.7.3.1 Vortex the reaction or dilution tube prior to filtration.
9.7.3.2 Pre-wet the membrane filter with approximately 10 mL dilution buffer.
9.7.3.3 Filter the appropriate volume. Liquid should pass through the filter quickly (for example, within approximately I mm of addition) with limited pooling of liquid in the filter apparatus. If necessary to lessen this occurrence, use multiple membrane filters per sample. Separate filters but the same filtration unit can be used for a given coupon provided the dilutions are filtered in order starting with the most dilute.
9.7.3.4 If filtering the entire contents of a tube, rinse the tube with approximately 10 mL dilution buffer, vortex, and filter the ri n sate.
9.7.3.5 Rinse the sides of the filter funnel with additional dilution buffer (for example, approximately 40 mL) and transfer the membrane filter to the recovery medium. Gently roll the filter onto the surface of the agar to prevent trapping air bubbles between the agar and the membrane; use sterile forceps to reposition the filter if necessary.
9.7.4 For control coupons, briefly vortex each tube and spread-plate aliquots of the appropriate dilutions in duplicate on the recovery medium.
9.7.5 Incubate plates from control coupons at 36 ± 2 °C for 48 ± 4 h. Incubate plates with filters from treated coupons for 72 ±4 h.
9.7.5.1 If necessary, monitor recovery media for growth and assess the number of visible colonies beginning at 24 h.
9.7.6 Count the appropriate number of CFU according to the recovery method used (for example, up to 200 CFU for filters and tip to 300 CFU for plating); use CFU to calculate log reduction. Log reduction is used to determine disinfectant effectiveness.
9.7.7 Inspect the growth on the plates and filters for purity and typical characteristics of the test microbe.
9.8 Coupon, Reactoi; and Splash guard Reuse:
9.8.1 After use in the reactor, place contaminated coupons in an appropriate vessel, cover with liquid (for example, waler), and autoclave with the other paris of the contaminated reactor system (including splashguards).
9.8.2 After sterilization, clean the reactor components and splashguards with a 1:100 dilution of detergent and tap water. After washing, rinse all components with deionized water.
9.8.3 Clean and rescreen the coupons per Practice E3161.
10. Data Analysis
10.1 Calculate biofllm density for control coupons.
ASTM E2871:19 pdf download
