**3.3 Environmental water samples**

From each sample, 100 ml was pre-enriched in 100 ml double concentrated buffered peptone water (Oxoid, Dardilly Cedex, France) at 37 °C for 24 h. Afterwards, 0.1 ml of the preenrichment sample was incubated in 9.9 ml of buffered Rappaport-Vassiliadis medium (Oxoid, Dardilly Cedex, France) and 1 ml in 9 ml of buffered selenite cystine medium for another 24 h at 37 °C; The enrichment samples were then applied onto Hecktoen and

Laboratory Typing Methods for Diagnostic of

**4. Laboratory typing methods** 

methods have been used (Riley, 2004).

**4.1 Phenotypic methods** 

*Typhi*, *Paratyphi C* or *Dublin*.

formula (Brenner et al., 2000).

the serotype was given in **Figure 2.**

**4.1.1 Serotyping** 

Minor, 1984).

Salmonella Strains, the "Old" Organism That Continued Challenges 353

The determination of the relatedness of strains within a *Salmonella* serotype is a prerequisite for the identification of the sources of infection and for tracing the routes of *Salmonella*  dissemination in outbreaks. Since biochemical analysis did not further differentiate between the bacteria assigned to the same *S. enterica* subspecies, other phenotypic and molecular

Serotyping is the initial step for routine diagnostics of *Salmonella* strains and performed with commercially available omni-, poly- and monovalent antisera. Up to date, over 2500 serotypes of *Salmonella* has been identified and classified in the Kaufmann-White scheme. This scheme differentiates between O (=somatic) antigens of the cell surface, H1 and H2 (=flagellar) antigens of the phase 1 or phase 2, respectively (Selander et al., 1996) and the Vi (=capsular) antigens which, however, may only be present in very few serotype, such as

Each *Salmonella* serogroup has a group specific O-antigen. Within each O-group, different serovars are distinguished by the combination of O- and H-antigens that are present. Each serotype has a specific antigenic formula where the O-antigens are indicated by Arabic numbers, the H1-antigens by lower case letters and the H2- antigens again by Arabic numbers. In these formulas, underlined antigens may only be expressed once the culture is lysogenised by the corresponding converting phage whereas letters or numbers in brackets indicate antigens which may be present or absent without relation to phage conversion (Le

For most of the isolates assigned to *S. enterica* and the subspecies I, antigenic formula corresponds to a serotype name. In contrast, serotypes identified after 1996 in the subspecies *salamae*, *houtenae* and *indica* and in the subspecies *bongori* are designated only by antigenic

**Serotype O-antigen(s) H1-antigen(s) H2-**

The detection of the presence of *Salmonella* O- and H- antigens were tested by slide agglutination with the commercially available antisera. One loop of appropriate antisera was dropped onto a cleaned glass slide. One loop of overnight culture grown on agar was dispersed in the drop to obtain a homogeneous and turbid suspension. The slide was rocked gently for 30 s and clumping was monitored by a magnifying glass. The scheme to obtain

*S. Enteritidis* 1, 9, 12 [f], g, m, [p] [1, 7] *S. Dublin* 1, 9, 12 [Vi] g, p - *S. Gallinarum* 1, 9, 12 - - *S. Typhimurium* 1, 4, 5, 12 i 1, 2 *S. Virchow* 6, 7 r 1, 2 *S. Infantis* 6, 7, 14 r 1, 5 Table 2. Examples for the antigenic formulas of *Salmonella enterica* subsp. *enterica* serotypes

according to Kaufmann-White scheme (Poppoff and Le Minor, 2001).

**antigen(s)** 


Kampelmacher agar. Both selective media were incubated during 24 h at 37 °C. The suspicious colonies were identified with biochemical test (as mentioned above).

Table 1. Biochemical reactions involved in API 20E (bioMérieux, Inc., France) test kits and typical *Salmonella* reactions.
