The isolation of Salmonella spp. was carried out in accordance with ISO 6579–1:2017. After pre‐enrichment in BPW for 24 h at 37°C, 1 and 0.1 ml of each pre‐enrichment solution was inoculated into 10 ml of Selenite Cystine Broth base (CM 0699, Oxoid) and 10 ml of Rappaport‐Vassiliadis Broth (CM 669 B, Oxoid), respectively, and then incubated at either 37°C (Selenite Cystine Broth) or 41℃ (Rappaport‐Vassiliadis Broth) for 24 h and plated onto selective Xylose Lysine Deoxycholate (XLD) Agar (CM 0469, Oxoid) and Hektoen Enteric Agar (HEA) (CM 0419, Oxoid). Following 24 h incubation, suspect colonies of Salmonella spp. were tested by inoculation into Kligler iron agar (CM0033, Oxoid).
Kligler iron agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Kligler Iron Agar is a laboratory culture medium used for the differentiation and identification of Gram-negative bacteria. It is designed to determine the ability of bacteria to ferment glucose and lactose, as well as to detect hydrogen sulfide production.
Automatically generated - may contain errors
Lab products found in correlation
Hektoen enteric agar, by Thermo Fisher Scientific (1 mentions)
Rappaport vassiliadis broth, by Thermo Fisher Scientific (1 mentions)
Selenite cystine broth, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Urea agar, by Thermo Fisher Scientific (1 mentions)
Lysine agar, by Thermo Fisher Scientific (1 mentions)
Simmon citrate agar, by Thermo Fisher Scientific (1 mentions)
Xylose lysine deosxycholate agar, by Thermo Fisher Scientific (1 mentions)
Selenite f enrichment broth, by Thermo Fisher Scientific (1 mentions)
Lysine iron agar, by Thermo Fisher Scientific (1 mentions)
Simmons citrate agar, by Thermo Fisher Scientific (1 mentions)
Xylose lysine deoxycholate xld, by Thermo Fisher Scientific (1 mentions)
Selenite f, by Thermo Fisher Scientific (1 mentions)
4 protocols using kligler iron agar
1
Isolation and Identification of Salmonella spp.
2
Isolation and Identification of Shigella
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MacConkey agar, Xylose Lysine Deosxycholate agar and Selenite F enrichment broth (Oxoid, England) were used for isolation of Shigella. Culture negative specimens on primary solid media were sub-cultured from the enrichment broth to primary solid media to improve recovery of the isolates. All inoculated media were incubated at 37 °C for 18–24 h. After overnight incubation, non-lactose fermenters were further identified by biochemical tests using appropriate media namely: Kligler Iron Agar for carbohydrate fermentation test, Urea agar for the urea utilization test, tryptophan broth for Indole test, Simmon Citrate agar for citrate utilization, Motility agar for motility test, Lysine agar for lysine utilization test (all Oxoid, England).
3
Stool Sample Isolation and Identification of Salmonella and Shigella
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Two grams of semi-formed stool or 2 mL watery stool sample was collected from
the study participants using a coded disposable plastic cup. First stool
samples were inoculated onto selenite F (Oxoid, Hampshire, UK) broth and
incubated at 37°C. Then subcultured onto Xylose Lysine Deoxycholate (XLD;
Oxoid) agar and incubated at 35°C–37°C for 18–24 h. After 24 h of
incubation, the culture media was evaluated for the presence of bacterial
growth. The identity of bacteria was confirmed using a panel of biochemical
tests recommended for enteric bacteria.10 Typical colonies were then further characterized based on colony
morphology (Salmonella appears as pink-red colonies with a
black center, while Shigella appears as pink-red colonies
on XLD). For identification of Salmonella serovars and
Shigella species, all suspected colonies were
inoculated onto appropriate biochemical media (Oxoid) including Kligler iron
agar, Lysine iron agar, Simmon’s citrate agar, sulfide indole motility
medium, and urea. For identification of Salmonellaserovars, Wellcolex Color Salmonella (Remel Inc., Lenexa,
KS, USA) was used. Shigella species was confirmed by slide
agglutination using commercially available, absorbed rabbit antisera (Biotec
Laboratories, Radstock, UK).
the study participants using a coded disposable plastic cup. First stool
samples were inoculated onto selenite F (Oxoid, Hampshire, UK) broth and
incubated at 37°C. Then subcultured onto Xylose Lysine Deoxycholate (XLD;
Oxoid) agar and incubated at 35°C–37°C for 18–24 h. After 24 h of
incubation, the culture media was evaluated for the presence of bacterial
growth. The identity of bacteria was confirmed using a panel of biochemical
tests recommended for enteric bacteria.10 Typical colonies were then further characterized based on colony
morphology (Salmonella appears as pink-red colonies with a
black center, while Shigella appears as pink-red colonies
on XLD). For identification of Salmonella serovars and
Shigella species, all suspected colonies were
inoculated onto appropriate biochemical media (Oxoid) including Kligler iron
agar, Lysine iron agar, Simmon’s citrate agar, sulfide indole motility
medium, and urea. For identification of Salmonellaserovars, Wellcolex Color Salmonella (Remel Inc., Lenexa,
KS, USA) was used. Shigella species was confirmed by slide
agglutination using commercially available, absorbed rabbit antisera (Biotec
Laboratories, Radstock, UK).
4
Vibrio sp. Screening Protocols
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The following were utilised as screening tests: Gram-stain and cell morphology determination; oxidase production (OXIBIOSWAB, Biolife); glucose fermentation, utilisation of lactose and H2S (hydrogen sulfide) production on Kligler Iron Agar (Oxoid); motility and indole production on SIM (sulphide-indole-motility) medium (Oxoid); reduction of nitrate (Zen-Yoji et al., 1973) . When non-halophilic Vibrio sp. was suspected, growth on TCBS (Thiosulfate-Citrate-Bile salts-Sucrose) Agar was also tested.
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