Carriers, discs (1 cm in diameter) made of AISI Type 304 Stainless Steel with 150 grit unidirectional finish on one side. Thickness is 22 gauge. Passivate by soaking in 7% citric acid for 20 to 30 minutes according to ASTM A967 before cleaning. Clean with a non-ionic detergent, rinse with water and sterilize. Use the screened, brushed surface of the carrier as the testing surface. Carriers are single-use.
Centrifuge with rotor capable of achieving 5000g.
Centrifuge tubes, conical, 15 mL, for centrifugation of test cultures.
Culture tubes, 20 mm × 150 mm glass, with Morton closures, for test culture preparation.
Desiccation unit with gauge to measure vacuum and maintain chamber pressure of 0.068 to 0.085 MPa. Use of color indicator desiccant recommended. Sufficient size to hold at least three 100 cm diameter Petri dishes; used for drying of inoculated carriers.
Filter sterilization units (with polyethersulfone membrane, 0.2 µm pore size), use to filter sterilize soil components.
Forceps, straight or curved, with smooth flat tips to handle membrane filters, and to transfer inoculated carriers into vials.
Filter membranes (polyethersulfone) for recovery of test microbe, 47 mm diameter and 0.2 µm pore size.
Micropipettes, calibrated (e.g., 200 µL, 1 mL) with 10-100 or 20-200 µL tips, use for deposition of antimicrobial test substance or control substance on carriers and preparing dilutions.
Petri plates, 150 mm diameter, sterile.
Pipette, calibrated 10 µL positive displacement, with corresponding 10 µL tips, for carrier inoculation.
Timer, for tracking of timed events and intervals.
Vials with lids, plastic, that are sterile, flat-bottomed, wide-mouthed (at least 25 mm diameter), for holding inoculated carriers and for accommodating neutralizer.
Vortex-style mixer, for vortex-mixing of various solutions.
Bovine Serum Albumin (BSA) stock. Add 0.5 g BSA (radio immunoassay (RIA) grade or equivalent, CAS # 9048-46-8) to 10 mL of PBS, mix to dissolve completely and pass through a 0.2 µm pore size (polyethersulfone) membrane filter, aliquot, and store at -20°C ± 2 °C.
Detergent, non-ionic (1% solution), use to clean carriers.
Dextrose solution, 10%(w/v). Filter sterilize the solution using a 0.2 µm filter. Added to SB prior to use.
Mucin stock. Add 0.04 g mucin (from bovine submaxillary gland, CAS # 84195-52-8) to 10 mL of PBS, stir or vortex-mix until thoroughly dissolved, and pass through a 0.2 µm pore size (polyethersulfone) membrane filter, aliquot, and store at -20°C ± 2°C. To facilitate dissolving the mucin, add the PBS to a small vessel with a stir bar and place on a stir plate to create a strong vortex. Slowly sprinkle the mucin into the liquid vortex and stir for at least 20 minutes or until completely dissolved.
Neutralizer; varies based on the test substance. Confirm adequate neutralization by adding 20-200 CFU of the test organism to mixtures of the test substance and the neutralizing agent (e.g., use Letheen broth with 0.5% Tween® 80 and 0.07% Lecithin for quaternary ammonium compounds) and then plating survivors per the protocol. Confirm lack of neutralizer toxicity to the test organism in a similar manner.
Phosphate buffered saline (PBS), pH of 7.0 ± 0.5.
Synthetic broth (SB), growth medium for test cultures.
Tryptic soy agar (TSA). This medium is available commercially in either pre-poured plates or dehydrated form to prepare according to manufacturer’s instructions.
Tryptic Soy Broth (TSB), use to rehydrate lyophilized cultures.
Tryptic Soy broth (TSB) with 15% (v/v) glycerol, use as a cryoprotectant. Suspend 7.5 g tryptic soy broth in 212.5 mL de-ionized water. Add 37.5 mL glycerol and stir, warm slightly to dissolve. Dispense into bottles and autoclave for 15 min at 121°C.
Water, use reagent-grade water free of substances that interfere with analytical methods. Any method of preparation of reagent-grade water is acceptable provided that the requisite water quality can be met.
Yeast Extract stock. Add 0.5 g yeast extract to 10 mL of PBS, mix to dissolve completely, and pass through a 0.2 µm pore size (polyethersulfone) membrane filter, aliquot, and store at -20°C ± 2 °C.