**6.2 Biochemical identification**

Biochemical identification of the colonies tried characteristics is done in two steps:

1. The search for characters of the family: often, the Gram stain, the presence of catalase, the absence of cytochrome oxidase, mobility, respiratory type, the mainstream culture

finding a pathogenic bacteria specie in very small proportion of a product heavily contaminated with the bacteria most various conventional methods of analysis, sampling and isolation have been proposed. Several standards govern the detection of Salmonella in food hygiene (Humbert et al., 1998): the horizontal standards applicable to all types of products (ISO6579 December 1993) at international level and industry standards specific to one type of product (NF V59 - 109 for edible gelatin). The detection of Salmonella in a food according to ISO 6579 has four key steps: The pre-enrichment; Enrichment; Isolation and

It's a non-selective phase that uses a rich medium in which the sample is diluted to one tenth (1 / 10) and for which the incubation period is about twenty hours at 35 ° C or 37 ° C (Humbert et al ., 1998). The pre-enrichment allows bacteria to sublethal to recover all of their potential at the end of incubation. The media used are liquids, most often using buffered peptone water or lactose broth (Humbert et al., 1998). For dairy products can be used

Enrichment is designed to minimize the growth of other bacteria associated with the collection and continue the selective breeding of Salmonella. 0.1 ml or 1 ml of the solution pre-enrichment was transferred to one or more enrichment media (10 ml of medium). The

This is a phase that uses selective solid media cast in Petri dishes. The isolation media contains a variety of combination of selective factors (Humbert et al., 1998). *Salmonella* colonies appear as features in their form, color and morphology. Solid media used for isolation are: Rambach medium; Hektoen medium; Salmonella agar - Shigella (SS agar); brilliant green agar and phenol red (VB-RP); xylose lysine Tergitol medium (XLT); Compass mid Salmonella; mannitol lysine, crystal violet, brilliant green; deoxycholate citrate agar lactose sucrose (DCLS); xylose lysine deoxycholate agar (XLD) and Bismuth

With conventional bacteriological diagnostic methods, other unconventional techniques can be used. These are among others: sensitivity to phage 01, and Felix and Callow and standardized systems (API 20 E, 20 E RAPID, Enterotube Rocks, MIS *Enterobacteriaceae*). The lysis by phage 01 (Felix and Callow) that can be used as a confirmatory test for membership of Salmonella

1. The search for characters of the family: often, the Gram stain, the presence of catalase, the absence of cytochrome oxidase, mobility, respiratory type, the mainstream culture

that do not provide positive results with all strains (Gledel and Corbion, 1991).

Biochemical identification of the colonies tried characteristics is done in two steps:

enrichment media are classified into three families (Humbert et al., 1998):

 broths containing tetrathionate (Muller Kauffmann broth) and broth containing malachite green and magnesium chloride.

Biochemical and serological identification.

Ringer's solution or phosphate buffer solution.

**6.1.1 The pre-enrichment** 

**6.1.2 Enrichment** 

selenite broth;

**6.1.3 Isolation** 

sulfite agar.

**6.2 Biochemical identification** 

and fermentation of glucose are sufficient to encourage the search for differential characters.

2. The search for differential characters requires pure cultures. Used for this purpose, the reduced rack of le Minor which is a set of five settings: the mid-Hajna Klinger; the Simmons citrate medium; the lysine iron or Taylor medium; the tryptophan-urea medium and mannitol nitrate mobility medium.

**The mid-Hajna Kliger** (solid medium): The mid-Hajna Kliger has a carmine red color. This medium is only valid for the fermentative bacteria. It is part of the study of carbohydrate metabolism. **The lysine iron medium** or mid-Taylor (purple), enriched with L-lysine and contains low concentrations of glucose. **The Simmons citrate** medium (green), contains citrate as the sole carbon source. Bacteria that are able to use this carbon source, will grow on this agar and cause a pH change at the origin of the middle turn blue. Remains green agar for strains citrate (-). **The urea-indole medium** (liquid medium), the urea -indole or urea -tryptophan medium, is a medium orange, made up of urea and tryptophan. It allows for three enzymatic activities of protein metabolism, including urease, tryptophanase and tryptophan deaminase. **The medium with glycerol** (liquid medium), this medium is green; it is very often added to other settings of Le Minor rack. It helps to distinguish Citrobacter and Salmonella genus. Citrobacter degrades glycerol by causing acidification of the medium (turns to yellow) and Salmonella do not degrade it, the green color is maintained. In summary, the general biochemical characteristics of most serotypes isolated from humans and warm-blooded animals are:


It should be noted that there are important exceptions. The serotype Typhi does not decarboxylated ornithine, does not grow on a medium composed of Simmons citrate, it is agazogene and produces only trace amounts of H2S. Serotype Paratyphi A does not decarboxylated lysine and does not grow on Simmons citrate medium. Finally, Salmonella Paratyphi A, Choleraesuis, and Gallinarum do not produce H2S. In this case, the settlements will not have black centers on isolation media consisting of iron citrate and sodium thiosulfate (eg, XLD, Hektoen, SS).
