AS 5013.13:2018 Food microbiology
10.5.3.2 Oxidase
Using a platinum-iridium or plastic loop (7.3), take a portion of a well-isolated colony from each individual plate (10.5.2) and streak it on to a filter paper moistened with the oxidase reagent (B.5.1); the appearance of a mauve, violet or deep blue colour within 10 s indicates a positive reaction. If a commercially available oxidase test kit is used, follow the manufacturer’s instructions.
10.5.3.3 Hydrolysis of 4 Nitrophenyl (PNP) a-D-glucopyranoside substrate
Using a loop or wire (7.3), suspend an individual colony grown on the non-selective agar such as TSA (10.5.2) in 2 ml of physiological salt solution, 0,85 % NaCl (B.5.2.4). Add 2 ml of the a-Glucosidase enzymatic assay solution (B.5.2). Incubate in a water bath at 37°C (7.12) for 4 h and measure the formation of yellow colouration in a spectrophotometer (7.9) at 405 nm. A minimal absorption of 0,3 at 405 nm after 4 h, equivalent to 16 mM PNP, can be considered positive.
10.5.3.7 MethyI red (MR) (optional)
Using a loop or wire (7.3), inoculate a tube of MR-VP broth (B.5.6) with each of the selected colonies (10.5.2) just below the surface of the medium. Incubate the tubes at 37 °C (7.2) for 48h±2 h. Add five drops of methyl red solution (B.5.6.2) to each tube. A distinct red colour indicates a positive reaction. A yellow colour indicates a negative reaction.
10.5.3.8 Voges-Proskauer (VP) (optional)
Using a loop or wire (7.3), inoculate a tube of MR-VP broth (B.5.6) with each of the selected colonies (10.5.2) just below the surface of the medium. Incubate the tubes at 37 °C (7.2) for 24h±2 h. Add 0,2 ml 40 % KOH (B.5.6.3.1) and 0,6 ml 1-naphthol solution (B.5.6.3.2). Let stand 1 h±0,5 h. An eosin pink colour around the surface of the medium indicates a positive reaction.
10.6 Interpretation of biochemical results
Cronobacter spp. are oxidase negative and are able to hydrolyse 4-Nitrophenyl a-D-glucopyranoside substrate and to produce acid from sucrose. They are unable to decarboyxlate L-lysine and to produce acid from D-sorbitol. Only very few Cronobacter strains give a positive methyl red reaction, and a negative Voges Proskauer test is also very uncommon. Cronobacter spp. can be distinguished from closely related species by the characteristics listed in Annex C.
NOTE
Cronobacter spp. refers to the descriptions found in References [2] and [11].