E. coli O157:H7
and B. cereus strains were plated individually on Brain Heart Infusion
(BHI) agar [37 g BHI broth (Oxoid, Basingstoke, UK) and 12 g agar bacteriological
(Oxoid), in 1 L of distilled water]. C. jejuni strains were plated
individually on blood agar [40 g blood agar base (Oxoid) and 7%
sterile blood in 1 L of distilled water]. BHI agar plates with either E.
coli, L. monocytogenes or Salmonella spp. were incubated aerobically at
37 °C for 24 h. Plates with B. cereus were incubated aerobically at 30 °
C for 24 h. Plates with C. jejuni were incubated microaerobically at
41.5 °C for 48 h. To prepare the inoculum, a colony of each strain was
picked from a respective plate and inoculated into BHI broth, with
heart infusion (HI) broth (Oxoid) being used for each C. jejuni strain. Incubation
was done in stationary incubators for 24 h, reaching a bacterial
broth culture concentration of approximately 9 log cfu/mL. A cocktail of
five strains of each species in suspension was produced by mixing equal
portions (2 mL) of each strain in a sterile test tube. The three strains of
Salmonella Enteritidis, a strain each of Salmonella Paratyphi B and Salmonella
Typhimurium were mixed together in equal portions (2 mL) of
each strain in a sterile test tube. Each pathogenic bacterial cocktail was
then ready for inoculation into mutandabota. In total 5 pathogenic bacterial
cocktails were produced.