In a cross-sectional study from April to July 2012, a total of 115 E. coli isolates were obtained from patients with UTI in Nemazee Hospital, Shiraz, Iran. The bacteria were identified and confirmed using standard methods. After gross and microscopic examinations, the urine was cultured on MacConkey agar and Xylose lysine deoxycholate agar (Merck, Germany). The cultured plates were incubated at 37°C for 24 hours until the occurrence of growth. Up to 5 lactose-fermenting colonies were selected separately and subjected to routine biochemical tests. The samples were then stored and sub-cultured for further analysis.
Xylose lysine deoxycholate agar
Manufactured by Merck Group
Sourced in Germany, United Kingdom, Switzerland
Xylose lysine deoxycholate agar is a microbiological culture medium used for the selective isolation and differentiation of Salmonella and Shigella species from clinical samples and other sources. It contains xylose, lysine, and deoxycholate as selective agents that inhibit the growth of most Gram-positive bacteria and some Gram-negative bacteria, while allowing the growth of Salmonella and Shigella species.
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Lab products found in correlation
Nutrient agar, by Merck Group (3 mentions)
Buffered peptone water (bpw), by Merck Group (3 mentions)
Eosin methylene blue agar, by Merck Group (2 mentions)
Urea agar, by Merck Group (2 mentions)
Tryptose soy broth, by Merck Group (2 mentions)
Pbs solution, by Merck Group (2 mentions)
Macconkey agar, by Merck Group (1 mentions)
Hektoen enteric agar, by Merck Group (1 mentions)
Xld agar, by Merck Group (1 mentions)
Brilliance salmonella agar, by Thermo Fisher Scientific (1 mentions)
Mkttn, by Merck Group (1 mentions)
Triple sugar iron agar, by Merck Group (1 mentions)
Simmons citrate agar, by Merck Group (1 mentions)
Trypticase soy agar, by Thermo Fisher Scientific (1 mentions)
Nutrient agar, by HiMedia (1 mentions)
Triple sugar iron agar, by HiMedia (1 mentions)
Tetrathionate broth, by HiMedia (1 mentions)
Salmonella shigella agar, by BD (1 mentions)
Sim medium, by BD (1 mentions)
Simmons citrate agar, by HiMedia (1 mentions)
18 protocols using xylose lysine deoxycholate agar
1
Isolation and Identification of E. coli from UTI Patients
2
Isolation and Identification of Shigella in Diarrheal Samples
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The present research was performed in Ardabil, an ancient city in northwestern Iran. From January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea who had been referred to the laboratory of Bu Ali Hospital at Ardabil University, belonging to the Iranian health system in Ardabil, Iran. The fecal samples were cultured on MacConkey agar and selenite F medium, and all plates were incubated overnight at 37 °C. After incubation time, all colonies were transferred to Hektoen enteric agar (HE) and xylose-lysine deoxycholate agar (XLD agar), (Merck, Hamburg, Germany) and were incubated at 37 °C for 18 to 24 h. Specimens with green colonies on HE medium, colorless colonies on XLD medium, and non-lactose fermenting colonies on MacConkey agar were suspected of Shigella species. The final identification of Shigella species was conducted using conventional biochemical tests such as triple sugar iron (TSI), SIM medium (sulfide indole motility medium), ornithine decarboxylase (ODC), lysine iron agar (LIA), Simmons citrate, and urea agar. In the next step, all confirmed bacteria were stored at −80 °C in 10% glycerol (Fig. 1 ).![]()
A flowchart of method steps
3
Isolation and Identification of Salmonella spp.
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The cloacal swabs were pre-enriched in buffered peptone water (BPW Oxoid, Biolab, South Africa) at 37°C for 24 hours. A loopful of the bacterial cells in buffered peptone water was streaked onto xylose-lysine-deoxycholate agar (Merck, Wadeville, South Africa) and Brilliant Green agar (Scharlau Chemie S.A. Barcelona, Spain). The streaked plates were then incubated at 37°C for 24 hours. The colonies were examined for their morphological appearance on the plate (colonies with or without black centers, colorless, or opaque-white colonies surrounded by pink or red zones on XLD). The suspected Salmonella spp. colonies, those with glossy large black centers or almost black colonies, were examined for pure culture isolation on BGA. Between three and five colonies were selected and purified on nutrient agar (Merck, Wadeville, South Africa) and incubated at 37°C for 18 to 24 hours.
4
Salmonella Detection and Identification Protocol
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To carry out non-selective enrichment, 25 g of the sample was transferred into a
container with 225 mL BPW (Merck) and incubated at 37 ± 1°C for 16-20 h. Next 1 mL of the
sample was inoculated into 10 mL of Muller-Kauffmann tetrathionate-novobiocin broth
(MKTTn; Merck,) and incubated at 37 ± 1°C for 24 ± 3 h to carry out selective enrichment.
Subsequently, 0.1 mL of the same sample was placed into 10 mL of Rappaport Vasiliadis Soy
(RVS; Merck) broth and incubated at 41.5 ± 1°C for 24 ± 3 h. Incubated MKTTn and RVS broth
were streaked on xylose lysine deoxycholate agar (Merck) and Brilliance Salmonella agar
(Oxoid, England) and further incubated at 37 ± 1°C for 24 ± 3 h and 36 ± 1°C for 48 h,
respectively. The suspected Salmonella spp. was confirmed biochemically
using Microgen GN-ID A+B system in accordance with the manufacturer’s14 ) instruction (Microgen
Bioproducts, Camberley, UK).
container with 225 mL BPW (Merck) and incubated at 37 ± 1°C for 16-20 h. Next 1 mL of the
sample was inoculated into 10 mL of Muller-Kauffmann tetrathionate-novobiocin broth
(MKTTn; Merck,) and incubated at 37 ± 1°C for 24 ± 3 h to carry out selective enrichment.
Subsequently, 0.1 mL of the same sample was placed into 10 mL of Rappaport Vasiliadis Soy
(RVS; Merck) broth and incubated at 41.5 ± 1°C for 24 ± 3 h. Incubated MKTTn and RVS broth
were streaked on xylose lysine deoxycholate agar (Merck) and Brilliance Salmonella agar
(Oxoid, England) and further incubated at 37 ± 1°C for 24 ± 3 h and 36 ± 1°C for 48 h,
respectively. The suspected Salmonella spp. was confirmed biochemically
using Microgen GN-ID A+B system in accordance with the manufacturer’s14 ) instruction (Microgen
Bioproducts, Camberley, UK).
5
Isolation and Identification of Shigella spp. from Pediatric Diarrhea
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We collected 3,254 stool culture of children <16 years of age with diarrheal infections referred to Golestan and Abuzar Hospitals during a period of 10 months from August 2016 to June 2017. The study design was approved by the Research Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Iran (IR.AJUMS.REC.1395.427). These samples were inoculated into Gram-negative (GN) Broth tubes as an enrichment medium as a part of the routine hospital laboratory procedure and then immediately transferred to the Laboratory of Microbiology Department of Medicine School of Ahvaz, Iran. From each patient, only one Shigella isolate in the diarrheal phase was included in this study. The GN Broth tubes were incubated at 37°C for 4–6 hrs and then streaked on Xylose Lysine Deoxycholate Agar and Eosin Methylene Blue Agar (Merck-Germany). All plates were incubated at 37°C for 24–48 hrs. The suspected colonies were biochemically identified as Shigellaspp. using Lysine Decarboxylase Agar, Sulfide-indole-motility, Triple-sugar iron Agar, Urea Agar and Simmons citrate Agar (Merck-Germany).11
6
Bacterial Survival Evaluation under Alkaline pH
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In order to evaluate the effect of alkaline pH on the bacterial survival after elution, the pellets were suspended in 100 μl of sterile normal saline, plated on the general and selective media, incubated at 37°C for 24–48 h and finally the colonies were counted. The used Media were; nutrient agar [Himedia, India (NA), trypticase soy agar [Oxoid, UK (TSA)], sorbitol-MacConky agar [Merck, Germany (SMAK)] for E. coli O157:H7; XLD agar [xylose lysine deoxycholate agar (Merck, Germany)] for S. enterica serovar Typhimurium; and PALCAM Listeria selective agar [Sigma-Aldrich, Germany (PALCAM)] for L. monocytogenes].
7
Isolation and Identification of Salmonella
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The samples for Salmonella isolation and identification were tested using the ISO 6579 protocol. After incubation, the pre‐enriched media (TSB) were cultured in a ratio of 1:10 on tetrathionate broth (HiMedia, LOT no. M032) and in a ratio of 1:100 on Rappaport Vassiliadis medium (HiMedia, LOT no. M880), followed by incubation at 37 and 42°C, respectively. After the incubation period, the cultures were streaked on xylose–lysine–deoxycholate agar (Merck, LOT no. 105287) and Salmonella‐Shigella agar (BD, LOT no. 274500) media. Suspected colonies for Salmonella spp. (H2S‐producing and non‐lactose fermenter colonies) were selected for confirmatory biochemical tests. Isolates were cultured on Triple Sugar–Iron agar (HiMedia, LOT no. 211825), SIM medium (BD, LOT no. 211578), urea agar (Merck, LOT no. 108492), lysine iron agar (BD, LOT no. 211363), Simmons citrate agar (HiMedia, LOT no. M099) and Methyl Red‐Voges Proskauer broth (HiMedia, LOT no. GM070) for further confirmation of Salmonella spp. ONPG disks (HiMedia, LOT no. DD008) were used to detect the beta‐galactosidase activity of late lactose fermenters (Bahramianfard et al., 2021 (link)).
8
Fecal Microbiota Enumeration Protocol
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On day 28 of the experiment, fecal samples (about 50 g) of one male and one female per pen were collected directly from rectum, transferred to sterile falcons, and immediately placed on ice in an insulation box for a maximum of 1 h transportation to the laboratory for further analysis. At the laboratory level, 0.01 g of fresh fecal sample of each animal was taken and placed into Eppendorf tubes supplemented with 990 mL 1× phosphate-buffered saline and 6-fold dilutions were prepared. The samples were plated on agar plates. Bacteria were quantified based on colony-forming units (cfu) counting on the culturing plates (log10 cfu g−1). E. coli were enumerated on eosin methylene blue agar (Merck, Germany) after aerobic incubation at 37°C for 1 day. Salmonella species were enumerated on xylose lysine deoxycholate agar (Merck, Germany) after aerobic incubation at 37°C for 1 day. Clostridium perfringens were enumerated on Tryptose Sulfite Cycloserine agar (Merck, Germany) after anaerobic incubation at 37°C for 1 day. Total anaerobic bacteria were enumerated on nutrient agar (Merck, Germany) after anaerobic incubation at 37°C for 1-2 days. Before statistical analysis, fecal microbiota concentrations were transformed (log).
9
Salmonella Typhimurium Quantification in Decontaminated Chicken
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Microbiological analysis of S. Typhimurium in the decontaminated chicken carcasses at the different application times was analyzed using conventional culture methods.
For this purpose, freshly processed broiler carcasses were rinsed, and the rinses were serially diluted 10-fold with 0.1% peptone water (Oxoid, England). The samples were then spread on xylose lysine deoxycholate agar (Merck) and brilliant-green phenol-red lactose sucrose agar (Merck). Colonies were inoculated into triple sugar iron (Merck) agar and lysine iron agar (Merck). The isolates were then tested with Salmonella antiserum (Difco 2264-47-2) (Mulder et al., 1987; (link)Andrews et al., 2019) .
For this purpose, freshly processed broiler carcasses were rinsed, and the rinses were serially diluted 10-fold with 0.1% peptone water (Oxoid, England). The samples were then spread on xylose lysine deoxycholate agar (Merck) and brilliant-green phenol-red lactose sucrose agar (Merck). Colonies were inoculated into triple sugar iron (Merck) agar and lysine iron agar (Merck). The isolates were then tested with Salmonella antiserum (Difco 2264-47-2) (Mulder et al., 1987; (link)Andrews et al., 2019) .
10
Salmonella Isolation and Enrichment Protocol
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From each of the enriched samples, 0.1 mL was aseptically transferred into 10 mL of Rappaport Vassiliadis (RV) broth (Sigma-Aldrich, India) and incubated for 24 hrs at 42°C. RV is a selective medium that is enriched with malachite green which inhibits the growth of microorganisms other than Salmonella. A previously identified and confirmed Salmonella enterica was used as a positive control.51 (link) Bacterial isolation was performed on Xylose-Lysine-Deoxycholate (XLD) agar (Sigma-Aldrich, Switzerland) by aseptically streaking a loopful of the culture from RV broth onto the plates. S. enterica is differentiated from Escherichia coli and Shigella spp. by producing red colonies with black centers on XLD agar. After 24 hrs of incubation at 35°C, the plates were observed for the growth of the expected colonies. Representative single colonies of the correct morphology were randomly picked from each plate and transferred into tubes containing 10 mL of tryptose soy broth (Merck, South Africa) and incubated at 37°C for 18–24 hrs. A 2 mL of the culture was used for DNA extraction. Equal amounts of 0.5 mL each of 60% glycerol and Salmonella pure culture were mixed in 1.5 mL cryotubes and stored at −80°C for future use.
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