Our study was conducted at the Pasteur Institute in Dakar, specifically in the Food Safety and Environmental Hygiene Laboratory.

Dehydrated VRBG medium was used to prepare agar plates for stability and microbiological performance testing. Tryptic Soy Agar (TSA) served as the reference medium for productivity tests, while Tryptone Salt (TS) medium was used to prepare bacterial suspensions and decimal dilutions. A sodium hydroxide (NaOH) solution was used to adjust the pH during the preparation of the VRBG medium.

Microbiological tests were conducted using the reference strains Escherichia coliATCC 8739, SalmonellaTyphimurium ATCC 14028, and Enterococcus faecalisATCC 19433, in accordance with the requirements of ISO 11133:2014.

Five separate batches of VRBG medium were prepared on different days in accordance with the manufacturer’s recommendations and the requirements of ISO 11133:2014 [5][9]. After weighing, the dehydrated powder was dissolved in purified water. The pH of the preparation was then adjusted to 7.4 ± 0.2 before heating in a water bath until the agar was completely dissolved. The melted medium was then distributed into five glass bottles, with one bottle dedicated to each scheduled storage point (Day 0, Day 15, Day 30, Day 45, and Day 60) which were hermetically sealed for microbiological testing, as well as into five separate aliquots used for physicochemical testing at the corresponding storage point. The various batches were stored at 5˚C ± 3˚C until evaluation.

Reference strains were subcultured on TSA one day before each control. Microbial suspensions were prepared in 9 mL of Tryptone Salt (TS) from isolated colonies and adjusted to approximately 2 McFarland (≈10⁹ CFU/mL). Serial decimal dilutions were then prepared to obtain approximately 10² CFU/mL for the productivity tests with Escherichia coli ATCC 8739 and Salmonella Typhimurium ATCC 14028, and 10⁵ CFU/mL for the selectivity test with Enterococcus faecalis ATCC 19433.

For microbiological testing, 1 mL of inoculum was used for deep inoculation. The same target inoculum was used for both VRBG and TSA.

For each microorganism and test condition, two plates were prepared, with one plate used for the reported data and the second retained as a reserve in case of a problem with the first plate.

The first layer consisted of approximately 15 mL of VRBG medium, in accordance with ISO 21528-2:2017 [4]. After solidification, approximately 5 mL of VRBG medium was added as a second layer. The covering layer was used to limit colony spreading and provide semi-anaerobic conditions during incubation.

Productivity was assessed on VRBG using TSA as the reference medium and expressed as:

Pr = Number of colonies on VRBG/Number of colonies on TSA

A Pr ≥ 0.5 was considered acceptable. Selectivity was assessed using E. faecalis ATCC 19433, with absence of growth considered satisfactory. VRBG and TSA plates were incubated at 37˚C for 24 h.

The stability of the VRBG medium was evaluated through physicochemical and microbiological tests conducted every fifteen days, including the day of preparation (D0, D15, D30, D45, and D60), over a two-month period.

At each scheduled control point, the bottle corresponding to the storage time was used for microbiological testing. The stored VRBG medium was regenerated in a water bath at 95˚C until complete regeneration and then transferred to a cooling bath at 45˚C, before inoculation.

The physicochemical tests included pH measurement as well as a visual inspection to check for color, clarity, the presence of precipitates, undissolved particles, and syneresis.

Microbiological performance was evaluated in accordance with the requirements of ISO 11133:2014 through sterility, productivity, and selectivity tests. Productivity was determined by calculating the productivity ratio (Pr), obtained by dividing the number of colonies observed on the VRBG medium by the number of colonies obtained on the reference medium (TSA). According to ISO 11133:2014, this ratio must be greater than or equal to 0.5. The acceptance criteria applied to the various tests are those defined by this standard.

For the sterility test, one uninoculated plate was examined for each batch at each scheduled storage point, and the absence of growth was considered satisfactory. For the selectivity test, complete absence of growth of Enterococcus faecalis ATCC 19433 on VRBG was considered satisfactory. Sterility and selectivity were assessed at each scheduled storage point (D0, D15, D30, D45, and D60).

The data obtained were entered and processed using Microsoft Excel. The results are presented in tables and graphs to assess changes in the physicochemical and microbiological parameters of the VRBG medium over the two-month storage period.

Changes in pH

The change in the pH of the VRBG medium over the 60-day storage period is shown in Figure 1.

Figure 1. pH trend curve for VRBG batches over 60 days.

The pH of the VRBG medium showed a slight decrease over the 60-day storage period. However, the values remained within the acceptance range specified by ISO 11133:2014 (7.2 to 7.6), thus demonstrating good physicochemical stability of the medium throughout the study period.

A slight decrease in the pH of culture media during storage was also reported by Ulisse et al. (2006), who evaluated physicochemical and microbiological parameters during the shelf-life assessment of culture media [10]. Despite this observed decrease, no impact on the microbiological performance of the VRBG medium was detected during this study.

Visual Inspection

The results of the visual examination of the VRBG medium during storage are presented in Table 1.

Table 1. Results of the visual examination of the VRBG medium during storage.

No macroscopic changes were observed during the 60-day storage period. The VRBG medium retained its color and transparency and showed no precipitates, undissolved particles, or syneresis. These observations demonstrate the physical stability of the VRBG medium and comply with the requirements of ISO 11133:2014.

The visual appearance of the VRBG medium after storage is shown in Figure 2.

Figure 2. Visual appearance of the VRBG medium after storage.

Sterility

Figure 3 shows a representative sterility test result obtained on Day 60 after 24 h of incubation.

Figure 3. Sterility test of VRBG medium after 24 hours of incubation on Day 60.

The sterility test performed on the various batches of VRBG medium showed no microbial contamination. No growth was observed in the plates incubated without inoculum after 24 hours of incubation. No growth was observed at any of the scheduled storage points. These results indicate that no detectable microbial contamination occurred during the preparation, packaging, and storage of the medium under the conditions evaluated.

Similar results were reported in guidelines and recommendations concerning the quality assurance and storage of microbiological culture media [11][12]. These recommendations emphasize the importance of appropriate preparation, storage conditions, and quality control to minimize the risk of microbial contamination during storage. In the present study, the absence of contamination up to Day 60 demonstrates that the in-house-prepared VRBG medium retained its microbiological integrity throughout the entire study period.

Productivity

The results of the productivity tests conducted with Escherichia coliand SalmonellaTyphimurium strains are presented in Table 2 and Table 3.

Table 2. Results of the productivity ratio (Pr) of the E. colistrain on VRBG medium.

Table 3. Productivity ratio (Pr) results for the S.Typhimurium strain on VRBG medium.

The change in the productivity ratio over the 60-day storage period for the E. coliand SalmonellaTyphimurium strains is illustrated in Figure 4 and Figure 5, respectively.

The results show that the productivity ratios remain above the acceptance threshold set by ISO 11133:2014 (Pr ≥ 0.5) for all batches and testing time points. These findings are consistent with previous studies evaluating the performance of VRBG and other selective media for the recovery of Enterobacteriaceae [8][13].

These fluctuations may be attributed to the biological variability of the reference strains, the preparation of the bacterial suspensions, or the seeding and

Figure 4. Curve showing changes in the productivity ratio over 60 days of storage for the E. colistrain.

Figure 5. Productivity ratio trend curve over 60 days of storage for the S. Typhimuriumstrain.

incubation conditions.

Selectivity

Figure 6 shows a representative selectivity test result obtained on Day 60 after 24 h of incubation.

Figure 6. Selectivity test of the VRBG medium after 24 h of incubation on Day 60.

The selectivity test performed with E. faecalisshowed a complete absence of growth on VRBG medium. This result was observed for all batches and at all testing time points.

The VRBG medium contains inhibitory agents, notably bile salts and crystal violet, which limit the growth of Gram-positive bacteria [14]. The absence of growth of Enterococcus faecalis, a Gram-positive bacterium, therefore supports the maintenance of the selective properties of the medium under the conditions evaluated.

These observations are consistent with previous studies evaluating the microbiological performance and selectivity of VRBG and other selective media [8][14].