The isolation and characterization of Salmonella and Shigella spp. were performed based on the standard procedure described by Cheesbrough,30 with some modifications. Briefly, a mixture of a stool sample (1 mL) was transferred from the Cary Blair medium into a tube containing 9 mL of Selenite F broth (Oxoid, Ltd) and incubated at 37°C for 24 hours to enrich the bacteria. An inoculum from Selenite F broth was subcultured onto deoxycholate agar (DCA) and xylose lysine deoxycholate (XLD) agar (Oxoid, Ltd). After overnight incubation at 37°C the growth of Salmonella and Shigella was differentiated by their colony characteristic appearance on XLD agar (Shigella: red colonies, Salmonella red with a black center) and DCA (Shigella: pale colonies, Salmonella black center pale colonies). Further identification was done biochemically using analytical profile index 20E (API 20E; bioMérieux SA, France) and Gram stain. Pure colonies with or without black centered on DCA or XLD was picked and suspended in sterile normal saline (0.85% NaCl). API 20E microtubes were filled up to the edge with the suspension. Sterile oil was added into the arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide, and urease production test compartments to create anaerobiosis. Reading of the result was done as per the manufacturer’s instruction after overnight incubation at 37°C.
Selenite f broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Selenite F broth is a selective culture medium used for the isolation and enrichment of Salmonella species from food and clinical samples. It contains sodium selenite, which inhibits the growth of many other bacteria, allowing for the preferential growth of Salmonella organisms.
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Lab products found in correlation
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (3 mentions)
Macconkey agar, by Thermo Fisher Scientific (2 mentions)
Xylose lysine deoxycholate agar xld, by Thermo Fisher Scientific (2 mentions)
Xylose lysine deoxycholate (xld), by Thermo Fisher Scientific (2 mentions)
Campygen, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Merck Group (1 mentions)
Hektoen agar, by Thermo Fisher Scientific (1 mentions)
Bactec blood culture system, by BD (1 mentions)
Cystine lactose electrolyte deficient agar, by Thermo Fisher Scientific (1 mentions)
Blood agar and macconkey agar plates, by Thermo Fisher Scientific (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Api 20e, by bioMérieux (1 mentions)
Deoxycholate citrate agar, by Thermo Fisher Scientific (1 mentions)
Vitek 2 c15 automated system for bacterial identification, by bioMérieux (1 mentions)
Nutrient broth, by Thermo Fisher Scientific (1 mentions)
Sheep blood agar, by Thermo Fisher Scientific (1 mentions)
Triple sugar iron agar, by Thermo Fisher Scientific (1 mentions)
Urea agar, by Thermo Fisher Scientific (1 mentions)
Api 10s, by bioMérieux (1 mentions)
Macconkey agar plate, by Thermo Fisher Scientific (1 mentions)
20 protocols using selenite f broth
1
Salmonella and Shigella Isolation and Identification
2
Salmonella and Shigella Detection from Stool
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Stool samples preserved in the Cary-Blair transport media were transferred to Selenite F broth (Oxoid) and incubated for 18 h at 37°C. Following the incubation of Selenite F broth, a loop full of samples was streaked to XLD (Oxoid) and MacConkey agar (MAC) (Oxoid) was incubated at 37°C for 24 h. The growth of Salmonella species and Shigella species were detected by their characteristic appearance on XLD agar (Shigella: red/pink colonies, Salmonella: red with some times black centre [33 , 34 (link)]. For confirmation, at least 1–3 presumptive colonies were selected and purified by streaking on to MAC (Oxoid) plates and incubated at 37°C for 24 h. Pure colonies with white/colourless colonies that grow on MAC were used for biochemical tests. The biochemical tests used for final identification were Klinger iron agar (KIA), lysine iron agar (LIA), Simmons citrate agar, sulphide indole motility test (motility, H2S production, indole), citrate utilization, and urease production test [33 , 34 (link)].
3
Salmonella Isolation and Identification
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Salmonella was isolated according to24 . Briefly, the internal organs from each bird were pooled together and considered one sample. One gram of each sample was added to 9 ml of nutrient broth (Oxoid, UK) and incubated at 37 °C for 18 h. After pre-enrichment, 1 ml of each broth culture was transferred to 9 ml of Selenite F broth (Oxoid, UK) and incubated at 37 °C for 18 h. A loop-full of culture from Selenite F broth was streaked into plates of XLD (Oxoid, UK). The plates were incubated at 37 °C for 18 h and checked for growth of typical colonies of Salmonella spp. Conventional biochemical methods including the urase test, triple sugar iron (TSI), lysine decarboxylase test, ornithin decarboxylase test, indole test, citrate utilization test and xylose, sucrose, rhamnose and arabinose fermentation tests were performed according to25 . According to the Kauffman-White scheme26 , the serological identification of Salmonella isolates for the detection of somatic and flagellar antigens was performed.
4
Isolation and Characterization of Enteric Pathogens
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A single stool sample was collected from each study participant using a sterile and disinfectant-free container. Stool samples were cultured on MacConkey (MAC) and Xylose Lysine Deoxychocolate (XLD) agar media after enrichment on Selenite-F Broth (Oxoid, United Kingdom). Further, blood free Campylobacter selective agar with Cefoperazone, Amphotericin B and Teicoplanin (CAT) selective supplement (Himedia, Ltd) was inoculated for isolation of Campylobacter species XLD and MAC agar plates were incubated aerobically at 37 °C for 24 h. Agar plates for isolation of Campylobacter species were incubated under microaerophilic conditions (5–10% O2 and 10% CO2 concentration) at 37 °C for 48 h. To create microaerophilic condition, the inoculated agar plates were kept in a gas jar containing gas generating kit (Campy Gen™, Oxoid, United Kingdom). Isolates were characterized using biochemical tests. Serotyping was performed for Salmonella, Shigella and E. coli using commercial anti-sera.
5
Isolation and Identification of Shigella
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We isolated and identified Shigella as per the standard bacteriological methods [9 , 10 ]. Samples were aseptically inoculated into selenite F broth (Oxoid, UK) and incubated aerobically at 37°C for 18–24 hours; subsequently, a loopful of the culture was streaked on MacConkey agar (Merck, Germany) and xylose lysine deoxycholate (XLD) agar (Oxoid, UK) and incubated aerobically at 37°C for 18–24 hours. Biochemical tests including carbohydrates fermentation (triple sugar iron agar), urea test, indole test, and motility test were then used to identify the resultant colonies. Shigella serogroups were determined using a commercially available serological kit (Denka Seiken, Tokyo, Japan).
6
Isolation and Identification of Gastrointestinal Pathogens
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All stool specimens were inoculated into Selenite F broth (Oxoid, Basingstoke, UK) and then incubated at 37°C for 24 hours. After the pre-enrichment period, the broth was subcultured onto MacConkey and xylose lysine deoxycholate agar media and incubated under aerobic conditions at 37°C for 24 hours. Growth of Salmonella and Shigella sp. was detected by their characteristic appearance on MacConkey and xylose lysine deoxycholate agar. Suspected colonies were further tested by a series of biochemical analyses to identify Salmonella and Shigella sp. [26 ]. Corresponding American Type Culture Collection strains were used as reference standards to validate the biochemical identification of Salmonella and Shigella. For the isolation of Campylobacter sp., campylobacter agar base with 10% sterile defibrinated sheep blood and rehydrated contents of Campylobacter Supplement-I (Blaser-Wang) (FD006) were used. Agar plates were incubated under microaerophilic conditions (5%–10% O2 and 10% CO2 concentrations) at 42°C for 24–48 hours. Gram staining and biochemical tests were performed to identify Campylobacter [26 ]. A standard reference strain of Campylobacter jejuni (ATCC 700819) was used as the quality control.
7
Comprehensive Microbial Identification and Susceptibility Testing
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Stool samples were routinely cultured on MacConkey agar, xylose lysine deoxycholate agar, and Hektoen agar (Oxoid Ltd, Basingstoke, UK). Stool samples were also inoculated into selenite F broth (Oxoid Ltd, Basingstoke, UK). Blood samples were collected in blood culture bottles and incubated in the BD BACTEC™ blood culture system (Becton, Dickinson and Company, Franklin Lakes, USA). A blood culture sample flagged as positive by the system was inoculated on blood agar, chocolate agar, and MacConkey agar. Identification of microbes from urine samples was performed by plating the sample onto MacConkey agar and cystine lactose electrolyte deficient (CLED) agar (Oxoid Ltd, Basingstoke, UK). Samples, other than stool or blood, e.g. urine, abdominal abscess, and surgical wound swabs, were cultivated as per standard operating procedures followed at the microbiology laboratory at KFHU.
Identification and antimicrobial susceptibility testing of all bacterial isolates was performed by the Vitek2 automated card system (bioMérieux Vitek Inc., Hazelwood, MO, USA). When required, an antimicrobial susceptibility test was performed manually using the disc diffusion method following the Clinical and Laboratory Standards Institute guidelines.20
Salmonella serogrouping was performed using the BD Salmonella antisera (Becton, Dickinson and Company, Franklin Lakes, USA).
Identification and antimicrobial susceptibility testing of all bacterial isolates was performed by the Vitek2 automated card system (bioMérieux Vitek Inc., Hazelwood, MO, USA). When required, an antimicrobial susceptibility test was performed manually using the disc diffusion method following the Clinical and Laboratory Standards Institute guidelines.20
Salmonella serogrouping was performed using the BD Salmonella antisera (Becton, Dickinson and Company, Franklin Lakes, USA).
8
Isolation and Identification of Salmonella and Shigella
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The collected samples were immediately processed for bacteriological analysis. Stool swabs were inoculated in to Selenite F broth (Oxoid) and incubated for 24 h at 37 °C followed by sub culturing on Xylose lysine deoxycholate agar plates (Oxoid) and re incubated for 24 h at 37 °C. Examination of the plates for significant colonies of Salmonella and Shigella species was done. An inoculum of each stool specimen was made on Blood agar and MacConkey agar plates (Oxoid) using cotton swab and then streaked with sterile inoculation loop. The plates were incubated at 37 °C for 24–48 h. Preliminary identification of pathogens was based on colony characteristics of the organisms and motility tests. Biochemical tests (Oxoid) were also carried out by inoculation of Kliger iron agar, Lysine iron agar, simmons citrate agar and media for indole and urease production for final identification.
9
Stool Sample Processing and Bacterial Identification
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One-gram stool sample was collected from each patient using sterile screw capped tubes containing transport media [9 mL buffered peptone water (Oxoid, UK)] and transported to Jimma University, Research and Postgraduate Laboratory for microbial analysis. After 24 hr incubation at 37°C, 1 mL of the sample was transferred into 10 mL selenite F broth (Oxoid, UK) and incubated. A loopful of culture was transferred onto xylose-lysine-deoxycholate agar (XLD) (Oxoid, UK) and incubated. The typical colonies were then further characterized based on colony morphology (Shigella appears as pink to red colonies on XLD, while Salmonella appears as red with black center). The cell morphology of pure culture was assessed by Gram staining. The morphological study includes cell shape, cell arrangement, presence or absence of endospore, and motility. Results of the isolates Gram reaction test were further confirmed with the rapid KOH method [13 (link)]. The isolates capability of catalase production, hence formation of bubbles, was checked using a 3% H2O2 solution.
For identification of Shigella and Salmonella spp., all suspected colonies were inoculated into appropriate biochemical media including Triple Sugar Iron Agar (TSIA), Lysine Iron Agar (LIA), Urea Agar (UA), Simmon's Citrate Agar (SCA), and Sulfide Indole Motility (SIM) medium.
For identification of Shigella and Salmonella spp., all suspected colonies were inoculated into appropriate biochemical media including Triple Sugar Iron Agar (TSIA), Lysine Iron Agar (LIA), Urea Agar (UA), Simmon's Citrate Agar (SCA), and Sulfide Indole Motility (SIM) medium.
10
Isolation and Characterization of Shigella
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Isolation and characterization of Shigella species were performed based on the practical guideline and handbook of clinical microbiology procedures [15 (link), 16 ]. In order to enrich the bacteria, a mixture of a feces sample (1 mL) was added to a tube containing 9 mL of Selenite F broth (Oxoid Ltd., UK) and incubated at 37°C for 24 hours. Then, an inoculum was transferred from Selenite F broth into MacConkey agar (MAC) and xylose lysine deoxycholate (XLD) agar plates. Shigella growth was distinguished from other lactose-negative suspected colonies on MAC by their distinctive colony morphology on XLD agar after subculture and overnight incubation at 37°C. Suspected Shigella colonies' further identification was performed biochemically using oxidase, Kligler iron agar (KIA), urea, indole, citrate, SIM media, and lysine iron agar (lysine decarboxylase (LDC)) tests. After incubation for 18–24 h at 37°C, the test media were read for characteristic Shigella species reactions.
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