*2.6.1 Orientation of the diagnosis by observing particularly discriminating features of the Enterobacteriaceae*

Characteristics such as pigmentation and mucoid of colonies, invasion of solid media by colonies, appearance of small colonies were observed directly on the culture medium after incubation at 37°C for 24 h. For mobility, 20 L of a suspension from a colony dissolved in 1 mL of peptone water and incubated at 37°C for 30 minutes was placed on a slide using a 50 L micropipette and covered with a coverslip. This mount was viewed under the 40X objective of the microscope to observe the movement of the bacteria. Suspicion of enterobacteria was made when the bacteria were either immobile or showed peritrich-like mobility.

For Gram staining, a colony from the culture medium was placed in a thin layer on the slide using a platinum loop. The layer formed was fixed by the flame maintained by the Bunsen burner. This layer was covered with gentian violet for 45 seconds, rinsed with water and then covered again with Lugol's. This Lugol's was cleaned after 45 seconds with 95° alcohol in a wash bottle and then rinsed with water. The washed slide was then covered with Fuchsin for 45 seconds, rinsed again with water, dried and read under a 100X microscope objective. The presence of an enterobacterium was confirmed if a Gram-negative bacillus was observed with bipolar staining.

#### *2.6.2 Revelation of the biochemical characteristics that characterise their metabolism*

The biochemical characteristics of the metabolism of the enterobacteria retained after diagnostic orientation were obtained using the API 20E gallery. Firstly, the tubes were moistened by introducing 10 mL of sterile distilled water. A bacterial suspension for each species was prepared by diluting the colonies from MH in 5 mL of sterile distilled water. Each tube in the gallery was inoculated with the corresponding suspension using a sterile Pasteur pipette. They were filled by pressing the Pasteur pipette inwards and to the side to avoid bubbles. The wells for citrate (CIT), Voges Prauskauer (VP), gelatinase (GEL) traits were filled completely (tube and cup) for aerobic conditions. For the Arginine dehydrogenase (ADH), Lysine decarboxylase (LDC), Ornitine decarboxylase (ODC), Hydrogen sulphide (H2S) and Urease (URE) wells, the filling was done only at the level of the tube and the well was filled with paraffin oil to create anaerobic conditions. The whole set was incubated at 37°C in the incubator for 22 hours and then a drop of developer was introduced in some wells. These were FeCl3 in the Tryptophan deaminase (TDA) well, Kovacs reagent in the Indole (IND) well, naphthol and NaOH in the VP well and Nit1, Nit 2 in the BNit well. The staining obtained in each well provided guidance on the positivity or negativity of the reaction.
