Preparation of antiseptic solutions
Octenidin-dihydrochloride (Octenisept®; OCT, 0.1%; Schülke & Mayr GmbH, Norderstedt, Germany) and povidone-iodine (Betaisodona®; PVP-I, 10%; Mundipharma GmbH, Limburg, Germany) were used as readily available customary products of everyday clinical use (concentrations as indicated by manufacturer).
Polyhexamethylene-biguanide (PHMB, 20%; Bonding, Shanghai, China) and chlorhexidine (CHX, 2%; Carl Roth, Karlsruhe, Germany) were prepared in distilled water to reach desired final test concentrations of 0.02% (v/v; PHMB) and 0.2% (v/v; CHX) in compliance with previously published studies and available wound cleansing products.
Cetylpyridinium-chloride (CPC; Sigma-Aldrich, Schnelldorf, Germany) and Miramistin (MST; Farmhim, Shostka, Ukraine) were purchased as powders and also prepared in distilled water to reach best suited final test concentrations of 0.5% (w/v; CPC) and 0.05% (w/v; MST) according to previous experiments . An overview as well as specifications for the tested products/substances is provided in table 1.
Test organisms and nutrient solutions
In this work, Pseudomonas aeruginosa (DSM-939), Staphylococcus aureus (DSM-799), Escherichia coli (DSM-11250), Enterococcus faecium (DSM-2146) and Candida albicans (DSM-1386; all DSMZ, Braunschweig, Germany) were used as bacterial test strains. As nutrient solution sterile casein/soy peptone broth (CSB) was prepared consisting of 15 mg ml-1 casein peptone, 5 mg ml-1 soy peptone and 5 mg ml-1 sodium chloride diluted in distilled water. The pH value was adjusted to 7.2 using 5N sodium hydroxide (all AppliChem, Darmstadt, Germany). One fresh colony of each bacterial strain was added to 50 ml CSB and incubated over night at 37°C under aerobic conditions and agitation. Bacterial test solutions were adjusted using a spectrophotometer to result in initial counts of ~ 1.5 – 3.0 x 108 CFU ml-1. Fungal test solutions were prepared in the same manner using malt/soy peptone broth (MEB) and malt agar (MEA). Initial microbial CFU ml-1 counts were controlled by spreading serial dilutions of untreated microbial test solutions of each experiment onto agar plates allowing exact calculations of reduction rates.
Challenge and neutralisation solutions
To simulate a challenging wound environment and determine possible decrease in efficacy of the tested antiseptics especially under high-protein conditions, bovine albumin (Carl Roth, Karlsruhe, Germany) was added to the experimental setup (as recommended in the standard DIN EN 13727 by the german institute for standardization (DIN)).  Challenge solutions contained either 3 mg ml-1 (0.3%) or 30 mg ml-1 (3%) albumin and were prepared using an autoclaved casein/natriumchloride solution dissolved in distilled water. The challenge solution was subsequently aliquoted and stored at -18°C until usage.
To prevent further antimicrobial activity beyond sampling time points, a neutralisation solution was used in accordance with DIN EN 13727, comprising 3 g l-1 sodium thiosulfate, 30 g l-1 saponine, 30 g l-1 polysorbate 80 (Tween 80), 3 g l-1 lecithin, 1 g l-1 L-histidine and 1 g l-1 L-cysteine (all Carl Roth, Karlsruhe, Germany) diluted in distilled water and autoclaved for sterility. Both, challenge and neutralization solution, were individually validated regarding their purpose and showed no antimicrobial effects of its own.
Quantitative suspension method
The test method for evaluation of antimicrobial efficacy was based on DIN EN 13727 and slightly adjusted to fit the purpose.  Briefly, 1 ml microbial and 1 ml challenge solution (0.3% or 3% bovine albumin) were carefully mixed for two minutes and 8 ml of antimicrobial test solution was added. After 0.5 , 1, 3, 5 and 10 min of exposure 1 ml of the resulting solution was transferred into the prepared neutralisation solution (8 ml neutralizer and 1 ml distilled water) and continuously agitated. After 10 sec, 0.5 ml was again transferred into 4.5 ml neutralisation solution to thoroughly block further antimicrobial activity. Subsequently, this sample was serially tenfold diluted in either CSB (for bacteria) or MEB (for yeast), 25 l of every dilution step were seeded on either CSA or MEA plates and incubated at 37°C under aerobic conditions overnight. Surviving microorganisms (in CFU ml-1) were counted. Experiments were performed threefold at different times and in duplicates for each tested antiseptic and microorganism challenged with either 3 mg ml-1 or 30 mg ml-1 albumin solution.
Reduction rates were calculated for all tested antimicrobials (in Δlog10 CFU ml-1). For bacteria, a high antimicrobial efficacy (reducing at least 99.999% of initial bacterial counts) is indicated by a reduction of at least 5 log10 reduction steps within 1 min as specified in DIN EN 13727.  For yeast the cut-off for a high efficacy is considered at least 4 log10 reduction steps as specified in DIN EN 13624. Mean values and SEM were calculated and differences considered statistically significant at p < 0.05.
Statistical evaluations of the antimicrobial efficacy were performed using two-way repeated measures ANOVA with Tukey’s HSD test as post-hoc analysis for multiple comparisons. The statistic package GraphPad PRISM (GraphPad Software, Inc., La Jolla, United States of America) was used for statistical analysis.