Cm0003b

The rectal swabs inoculated into Rappaport-Vassiliadis enrichment broth were incubated aerobically at 37°C for 24 h. Subcultures from Rappaport-Vassiliadis enrichment broth were made onto brilliant green agar (Oxoid^®, CM0263B) and incubated at 37°C for 24 h aerobically. The agar plates were examined for the growth of colonies after 24 h. From each of the selective agar plates, presumptive Salmonella colonies were selected for microscopy.
Non-lactose fermenting (pink) colonies that were Gram-negative coccobacilli, oxidase-negative, and catalase-positive were tentatively identified as Salmonella organisms and were streak-purified on nutrient agar (Oxoid^®, CM0003B). Confirmation of these isolates was done by latex agglutination technique using Oxoid Salmonella Test Kit^® (DR1108A). Isolates that showed agglutination in less than 1 min were identified as Salmonella organisms. The Oxoid Salmonella Test Kit detects the majority of common Salmonella serotypes, including Salmonella typhimurium and Salmonella enteritidis.

The plate method was used to conduct the microbiological assessment per the recognized food microbiology guidelines. Total viable count (TVC) was assessed on nutritional agar (CM0003B, Oxoid) following ISO 4833-1 (2013) . On MacConkey agar (CM0115, Oxoid), the Enterobacteriaceae count (ENT) was determined following ISO 21528-2 (2017) . Each analysis was done in triplicate, and standardized plate count methodologies were used to calculate plate counts and convert them to log₁₀ CFU values (Vanderzant and Splittstoesser, 1992 ).

Susceptibility testing of honey sample was carried out using nutrient agar (CM0003B, Oxoid) plates through Kirby–Bauer disc diffusion technique following Kassim et al. (2016) (link). The composition of nutrient agar is given in Table 2. Each plate was properly inoculated with each test organism by streak-plate method. Wells were made using a sterile cork borer and each well was filled with prepared concentrations (25, 50, 75 and 100 %) of the honey. A safer distance was maintained from the edges of the plates to prevent overlapping of the inhibition zones. The inoculated plates were incubated overnight at 35 °C. After successful incubation, the plates were examined and the diameter (mm) of the inhibition zones was measured in triplicate for each isolate.Table 2

Composition of nutrient agar.

Chemical constituent	Quantity (g/L)
Agar	15.0
‘Lab-Lemco’ powder	1.0
Peptone	5.0
Sodium chloride	5.0
Yeast extract	2.0
pH: 7.4 ± 0.2 at room temperature