2.3. Application of Standard EN 16615

The tests were carried out in accordance with the standard EN 16615:2015-06 “Chemical disinfectants and antiseptics. Quantitative test method for the evaluation of bactericidal and yeasticidal activity on non-porous surfaces with mechanical action employing wipes in the medical area (4-field test). Test method and requirements (phase 2, step 2)” [12]. This is a carrier method in which disinfected surfaces are simulated by homogeneous polyvinyl chloride (PVC) plates “Special 43 Plus E” (Tarkett, Jasło, Poland). Four fields of 25 cm² each were marked on the plates in order to assess the ability to reduce the microbe count as well as the ability to spread microbes to subsequent areas of the surface. Test organism suspensions with a density of 1.5–5.0 × 10⁹ cfu/mL (bacteria) and 1.5–5.0 × 10⁸ cfu/mL (C. albicans), were estimated by DEN-1B McFarland Densitometer, Grant Instruments Ltd., England, in the presence of a 3 g/L bovine albumin solution (Sigma, St. Louis, MO, USA), and 3 mL/L of sheep erythrocytes (Grasso, Starogard Gdański, Poland) simulating dirty conditions were applied in an amount of 0.05 mL and spread over the first field of the test surface. The test surface was dried at room temperature, which caused the microorganisms to attach to the plates. Then, a granite block weighing approximately 2.5 kg, providing adequate pressure on the surface, was covered with a 17 cm × 30 cm wipe made of low-dusting non-woven fabric (TORK, SCA, Göteborg, Sweden) soaked in 16 mL of the test disinfectant (products 1–6) or with a commercial wipe (products 7–12). Such prepared unitary weight with the soaked wipe was used for the wiping process on the surface contaminated with microorganisms beginning from 1 field up to 4 and back down to 1 field within 2 s. The wipes were weighed just before and immediately after use to determine the amount of disinfecting liquid released from the wipe (Figure 1).

Model of the markings the four test fields on the test surface. (A) Test surface; (B) Unitary weight, 1–4: Test fields 25 cm², 1: Inoculated field.

After a chosen contact time, the activity of the product was neutralized at a temperature of 20 ± 1 °C with an appropriately selected neutralizer for 15 s or (5 min for a contact time of ≥15 min). For products containing alcohols, quaternary ammonium compounds, and a chlorine compound, the Dey/Engley neutralizer (Becton Dickinson, Sparks, MD, USA) of the following composition was used: 5.0 g/L pancreatic casein extract, 2.5 g/L yeast extract, 10.0 g/L dextrose, 1.0 g/L sodium thioglycolate, 6.0 g/L sodium thiosulphate, 2.5 g/L sodium bisulphite, 805.0 g/L polysorbate, 7.0 g/L lecithin, and 0.02 g/L bromocresol purple. For the product containing hydrogen peroxide, 0.25 g/L catalase solution (Sigma, St. Louis, MO, USA) was used. The product containing glucoprotamine was neutralized with a solution of the following composition in a diluent (tryptone, 1.0 g/L pancreatic casein extract (Becton Dickinson, Sparks, MD, USA), and 8.5 g/L sodium chloride (POCH, Gliwice, Poland)): 3 g/L lecithin (AppliChem, Darmstadt, Germany), 8030 g/L polysorbate (POCH, Gliwice, Poland), 5 g/L sodium thiosulfate (Honeywell, Muskegon, MI, USA), 1 g/L L-histidine (Merck, Darmstadt, Germany), and 30 g/L saponin (AppliChem, Darmstadt, Germany).

After the wiping process was complete, the recovery procedure with the swabs was performed from each field on the test surface. The swabs were rinsed in a suitable neutralizer (5 mL), and 1 mL aliquots were inoculated in duplicate using the pour plate technique on the appropriate culture medium: trypticase soy agar (bioMerieux, Marcy l’Etoile, France) for bacteria and malt extract agar (Merck, Darmstadt, Germany) for C. albicans. The plates were incubated at 37 ± 1 °C (bacteria) for 20 to 24 h and 30 ± 1 °C (C. albicans) for 40 to 48 h. The colony-forming units were counted with the usage of Colony Counter apparatus, IUL Instruments, Spain.

The disinfectant product meets the requirements of PN-EN 16615 when the decimal log reduction of test microorganisms in the first field is at least 5 logs (bacteria) and 4 logs (yeast) and the average number of cells in the fields from 2 to 4 does not exceed 50 cfu. The following controls and validations were performed concurrently with the product activity test: control with water supplemented in 0.1% polysorbate 80 used instead of the product test solution to ensure that the test organisms are spread on the 4 fields and cells’ number reaches a certain level; drying control at time 0, just after drying (D_c0), drying control after drying time, and exposure time (D_ct) to ensure that the test organisms can survive in the test conditions, and control of neutralizer toxicity and validation of the dilution-neutralization method, to ensure that used neutralizer is suitable.

Experiments were performed three times. The arithmetic mean value and standard deviation were calculated.