## 2 Materials and Methods
using turbidimetry (around 0.5 McFarland units) (DEN-1B densitometer, Grant Instruments, Cambridgeshire, UK), from which serial dilutions were prepared using the same diluent. For the productivity assays, serial dilutions were prepared to inoculate 50-120 CFU per plate. For the selectivity assays (_S. aureus_ and _E. faecalis_ species), serial dilutions were prepared to inoculate \(\geq\)10\({}^{4}\) CFU per plate, according to ISO 11133:2014/amended 1:2018 [19]. Productivity assays were performed using the pour plate method, according to the corresponding ISO (ISO 4833-1:2014 [21]; ISO 21149:2017 [22]; ISO 16212:217 [23]; ISO 21528-2:2004 [24]) and European Pharmacopoeia (Ph. Eur., 10 th ed.) [3], with the culture media required for each strain (Table 1). A microbial suspension (1 mL) containing 50-120 CFU (with a precision limit of \(\pm\)20 CFU) was plated on sterile Petri dishes, and then the culture medium was poured onto the Petri dish and mixed with the inoculum. In the productivity assays, to ensure the exact CFU were inoculated by the pour plate method in each Petri dish, a microbial suspension containing 50-120 CFU (100 \(\mu\)L) was also inoculated in the TSA or SDA, using the spiral plate method (Eddyjet, IULmicro, Barcelona, Spain), as a control. Selectivity assays for VRBG were performed using the pour plate technique, according to ISO 6579 [25], ISO 21528-2:2017 [24], and ISO 11133:2014/amended 1: 2018 [19], with _S. aureus_ (ATCC, equivalent to WDCM 00032) and _E. faecalis_ (ATCC 19433, equivalent to WDCM 0009), inoculated at \(\geq\)10\({}^{4}\) CFU (1 mL). Seeding method \begin{table}
\begin{tabular}{c c c c c} \hline
**Culture Media** & **Assay** & **Micro-Organism** & **ATCC 1** & **WDCM 2** \\ \hline \multirow{4}{*}{TSB} & & _Bacillus subtilis_ & 6633 & 00003 \\  & & _Pseudomonas aeruginosa_ & 9027 & 00026 \\  & Qualitative & _Staphylococcus aureus_ & 6538 & 00032 \\  & assays & _Escherichia coli_ & 8739 & 00012 \\  & & _Salmonella enterica_ subsp. & & \\  & & _ enterica_ ser. & 14028 & 00031 \\  & & Typhimurium & & \\ \hline \multirow{4}{*}{SDS} & Productivity & _Apergillus brasiliensis_ & 16404 & 00053 \\  & assays & _Candida albicans_ & 10231 & 00054 \\  & & _Saccharomyces cerevisiae_ & 9763 & 00058 \\ \hline \multirow{4}{*}{Sabouraud dextrose broth} & Qualitative & _Candida albicans_ & 10231 & 00054 \\  & assays & _Escherichia coli_ & 8739 & 00012 \\  & & _Apergillus brasiliensis_ & 16404 & 00053 \\ \hline \multirow{4}{*}{VRBG} & & _Escherichia coli_ & 8739 & 00012 \\  & & _Salmonella enterica_ subsp. & & \\  & & _ enterica_ ser. & 14028 & 00031 \\  & assays & Typhimurium & & \\  & & _Pseudomonas aeruginosa_ & 9027 & 00026 \\  & & _Escherichia coli_ & 25922 & 00013 \\ \cline{2-5}  & Selectivity & _Enterococcus faecalis_ & 19433 & 00009 \\  & assays & _Staphylococcus aureus_ & 6538 & 00032 \\ \hline \end{tabular} 1  
\end{table} 
Table 1: _Cont._Two different assays, quantitative (solid media) and qualitative (liquid media), were performed to compare the quality of the media sterilized using both methods with different micro-organisms (Table 1). In the quantitative assays, samples of 1 mL were plated in triplicate onto sterile Petri dishes, using the pour plate method. Then, the medium (20-25 mL), after melting in a boiling bath, was added once it was cooled to 40-45 \({}^{\circ}\)C, in both sterilization methods. In the qualitative assays, 1 mL (50-120 CFU) was inoculated in triplicate in broth bottles (TSB and Sabouraud dextrose broth, 250 mL) and then incubated for the required time and temperature. The TSA plates and TSB bottles were incubated at 32.5 \(\pm\) 2.5 \({}^{\circ}\)C for 24-48 h, the SDA plates and Sabouraud dextrose broth at 22.5 \(\pm\) 2.5 \({}^{\circ}\)C for 2-5 days, and the VRBG plates and broth at 37 \(\pm\) 2.5 \({}^{\circ}\)C for 24-48 h. For each medium and strain, inoculation was repeated in triplicate.