Nutrient agar

For the confirmation of the typical Salmonella colonies using biochemical tests, typical and suspected colonies of Salmonella were selected from the selective plating media, streaked onto the surface of nutrient agar (Oxoid Ltd., England) plates and incubated at 37 °C for 24 h according to ISO 6579 [31 ]. All suspected non-lactose fermenting Salmonella colonies were picked from the nutrient agar and inoculated into triple sugar iron (TSI) agar, Simmon’s citrate agar, urea broth, tryptone water, methylene red and Voges-Proskauer (MR-VP) broth (Oxoid Ltd., England) and incubated for 24 at 37 °C. A colony was considered Salmonella if an alkaline slant (Red) with acid butt (yellow) on TSI with hydrogen sulfide was produced, positive for lysine (purple), negative for urea hydrolysis (red), negative for tryptophan utilization (indole test) (yellow-brown ring) and negative for Voges-Proskauer (colorless) [32 ].

The standard pour plate technique was employed to estimate TVC (total viable count), EBC (Enterobacteriaceae count), and ECC (E. coli count). A 25 g food sample was placed into 225 mL sterilised BPW (buffered peptone water) (Oxoid, Hampshire, UK). The setup was homogenised in a stomacher for 3 min. Serial dilutions up to 10⁻⁸ were prepared and plated in triplicates in nutrient agar (Oxoid, Hampshire, UK). For TVC and ECC, nutrient agar (Oxoid, Hampshire, UK) and Brilliance E. coli/coliform agar (Oxoid, Hampshire, UK) were used and plates were incubated at 37 °C for 48 h. For EBC, violet-red bile glucose agar (Oxoid, Hampshire, UK) was used and plates were incubated at 37 °C for 24 h.

Five typical colonies from each Rainbow agar O157 plate were sub-cultured on nutrient agar (Oxoid, Basingstoke, England) for biochemical confirmation by indole formation. The agar plates were incubated at 37°C for 18–24 h. One colony from the pure culture on nutrient agar was inoculated into a tube of tryptone/tryptophan medium (Oxoid, Basingstoke, England) and incubated at 37°C for 24 h. Then, 1 ml of Kovac's reagent (Oxoid, Basingstoke, England) was added and the tube allowed to stand at room temperature for 10 min. The formation of red color indicates a positive reaction (11 (link)).

The presumptive B. cereus colonies in MYP-plates isolated from non-B. thuringiensis treated spinach samples (controls and leaves sprayed with S. Montevideo alone as described in section “Post-harvest Growth of BTa ABTS-1857 and S. enterica on Spinach Cut Leaves”) and from soil samples (as described in section “Evaluation of Persistence of Vegetative Cells and Spores of BTa ABTS-1857 and S. Montevideo 1024 on Spinach Plants”) were submitted to phase-contrast microscopy (Leica, Germany) for detection of insecticidal crystals and distinction between B. cereus sensu stricto and B. thuringiensis, as recommended by the Food and Drug Administration (Tallent et al., 2001 ) described in FDA-BAM¹ and the International Organization for Standardization (ISO) (ISO 7932:2004/AMD 1:2020, 2020 ). For the detection of parasporal crystals, 100 μL of overnight cultures in BHI were transferred to strengthened Nutrient Agar (sNA) plates, containing 28 g/L Nutrient Agar (Oxoid), 0.04 g/L MgCl₂ (Sigma-Aldrich), and 0.10 g/L CaCl₂ (Sigma-Aldrich). The colonies in the sNA plates were monitored by phase-contrast microscopy until they reached the sporulation stage (approximately 24–48 h incubation). The sporulating cells were examined before lysis of the mother cells.

Overnight cultures (100 μl) of the presumptive B. cereus isolates were inoculated on strengthened Nutrient Agar (sNA; 28 g/L Nutrient Agar (Oxoid) + 0.04 g/L MgCl₂ (Sigma-Aldrich) + 0.10 g/L CaCl₂ (Sigma-Aldrich)) at 30°C for 24–48 h until sporulation was observed by phase-contrast microscopy (ZEISS Axioscope 5, Germany). Isolates with parasporal crystals (a minimum size: approximately 1/4 to 1/2 of the spore) were tentatively identified as presumptive Bt (ISO 7932:2004/Amd 1:2020).

Five typical colonies from each Rainbow agar O157 plate were subcultured on nutrient agar (Oxoid, England) for biochemical confirmation by indole formation. The agar plates were incubated at 37°C for 18–24 h. One colony from the pure culture on the nutrient agar was inoculated into a tube of tryptone/tryptophan medium (Oxoid, England) and incubated at 37°C for 24 h. Then, 1 ml of Kovack’s (Oxoid, England) was added, and the tube was allowed to stand at room temperature for 10 min. The formation of red color indicates a positive reaction (Manyi-Loh et al., 2018 (link)).