All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).
Lysine iron agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Lysine iron agar is a microbiological culture medium used for the isolation and differentiation of certain enteric bacteria. It is designed to detect the production of hydrogen sulfide and lysine decarboxylase, which are characteristic of certain pathogenic bacteria. The medium contains lysine, iron salts, and pH indicators to facilitate the identification of target organisms.
Automatically generated - may contain errors
Lab products found in correlation
Triple sugar iron agar, by Thermo Fisher Scientific (4 mentions)
Simmons citrate agar, by Thermo Fisher Scientific (3 mentions)
Mobility indole ornithine agar, by Thermo Fisher Scientific (2 mentions)
Polyvalent and monovalent antisera, by Denka Seiken (2 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (2 mentions)
Salmonella shigella agar, by Thermo Fisher Scientific (2 mentions)
Urease agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Sulfide indole motility agar, by Thermo Fisher Scientific (1 mentions)
Simmons citrate agar, by HiMedia (1 mentions)
Xylose lysine deoxycholate xld, by Thermo Fisher Scientific (1 mentions)
Selenite f, by Thermo Fisher Scientific (1 mentions)
Kligler iron agar, by Thermo Fisher Scientific (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
6 protocols using lysine iron agar
1
Shigella Identification Protocol
2
Identification of Enterobacteriaceae from Stool Samples
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Cultural observation. Preliminary identification of bacteria was done based on colony characteristics of the organisms. Some colony characteristics like (size, shape, color, pigmentation, texture, elevation, and edge [30 ].
Microbiological analysis of stool specimen. Enterobacteriaceae were identified by inoculating /streaking of stool samples on MacConkey agar (Oxoid Ltd, Basingstoke, UK) based on their color morphology after an incubation time of 18–24 hours at 37°C. The smear was prepared from each different colony observed on the plates and Gram staining was performed. The Results such as gram reaction (gram-negative), arrangements, and shape of bacteria are seen from the examinations using a microscope [31 ].
Biochemical examination. Biochemical tests were performed on colonies from pure cultures for the identification of the isolates. Triple sugar iron agar (Oxoid Ltd, Basingstoke (for gas production, lactose fermentation, and hydrogen sulfide production), UK), indole test (for tryptophan utilization, Simon’s citrate agar (Oxoid Ltd, Basingstoke, UK) (citrate utilization test), urease agar (Oxoid Ltd, Basingstoke, UK) (urease production test), lysine iron agar (Oxoid Ltd, Basingstoke, UK) (lysine decarboxylase test), and Motility medium (Oxoid Ltd, Basingstoke, UK) (motility test) were included in the biochemical tests for species identification [32 ].
Microbiological analysis of stool specimen. Enterobacteriaceae were identified by inoculating /streaking of stool samples on MacConkey agar (Oxoid Ltd, Basingstoke, UK) based on their color morphology after an incubation time of 18–24 hours at 37°C. The smear was prepared from each different colony observed on the plates and Gram staining was performed. The Results such as gram reaction (gram-negative), arrangements, and shape of bacteria are seen from the examinations using a microscope [31 ].
Biochemical examination. Biochemical tests were performed on colonies from pure cultures for the identification of the isolates. Triple sugar iron agar (Oxoid Ltd, Basingstoke (for gas production, lactose fermentation, and hydrogen sulfide production), UK), indole test (for tryptophan utilization, Simon’s citrate agar (Oxoid Ltd, Basingstoke, UK) (citrate utilization test), urease agar (Oxoid Ltd, Basingstoke, UK) (urease production test), lysine iron agar (Oxoid Ltd, Basingstoke, UK) (lysine decarboxylase test), and Motility medium (Oxoid Ltd, Basingstoke, UK) (motility test) were included in the biochemical tests for species identification [32 ].
3
Biochemical Identification of Suspected Salmonella
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Suspected Salmonella colonies from nutrient agar (Oxoid, England CM0003-500G) were picked up and its biochemical characteristics were determined using triple sugar iron agar (Oxoid, England CM277-500G), lysine iron agar (Oxoid, England CM0381-500G), Simmon’s citrate agar (Himedia, India CM0129-500G), urea slant (Himedia, India M111A-500G), and sulfide indole motility agar (Oxoid, England S12-500G).3 (link)
4
Shigella Identification Protocol
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All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).
5
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).
6
Salmonella Isolation from Food Samples
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Pre-enrichment of the samples was conducted in buffered peptone water at 1% (w/v) (BPW, Oxoid Ltd., Basingstoke, UK), which was 25 g of solid sample added to 225 mL of BPW, sponges were added to 90 mL of BPW, and 200 mL of the water samples were centrifuged at 3500× g for 15 min and the pellet was added to 100 mL of BPW incubated at 35 °C for 18 to 24 h. Salmonella isolation was conducted according to the protocol described by USDA [39 ]. Colonies with typical Salmonella characteristics on triple sugar agar and lysine iron agar (Oxoid) were subjected to a serological test by using polyvalent somatic and flagellar antisera (Probac do Brasil, São Paulo, SP, Brazil), and further biochemical characterization by the following tests: urease, indole, methyl red, Voges-Proskauer, citrate, motility, and malonate [39 ]. Isolates identified as Salmonella spp. (n = 210) were serotyped by slide agglutination (Kauffmann–White–Le Minor scheme) using diverse somatic and flagellar antisera in the Bacteriology Section of the Instituto Adolfo Lutz (São Paulo, SP, Brazil) and Enterobacteriaceae Laboratory from the Fundação Oswaldo Cruz (Rio de Janeiro, RJ, Brazil). Isolates were kept stored in trypticase soya broth (TSB, Oxoid) added to glycerol at 10% at −20 °C.
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