Xylose lysine deoxycholate agar xld

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.

All samples (10 mL) were inoculated with 0.1 mL Salmonella spp. cocktail to obtain an initial density of 3.5 ± 0.5 log CFU/mL. The inoculated samples were stored at 5, 10, 15, 20, 25, 30, and 37 °C under isothermal conditions. To measure bacterial cell counts, each sample was taken at different time intervals for each temperature, and 10 mL sterilized saline was added to each sample and homogenized via vortexer. Ten-fold dilutions of samples were plated in duplicate onto Xylose Lysine Deoxycholate Agar (XLD, Oxoid, Basingstoke, UK), and the agar plates were incubated at 37 °C for 24 h. Typical colonies were counted and converted to log CFU/mL.

Samples were homogenized using a laboratory blender (Interscience^TM BagMixer^® 400, Saint-Nom-la-Bretèche, France). Subsequently, Salmonella, Escherichia coli, total coliform bacteria, and total yeast and molds enumeration were performed.
The protocol to isolate Salmonella was performed according to the Bacteriological Analytical Manual (BAM) method: Salmonella, Chapter 5 (FDA). Suspicious colonies on solid media were confirmed with the VITEK 2 system (Biomérieux, Durham, NC, USA) following the manufacturer’s instructions.
For the quantification of Salmonella, the samples were homogenized with 90 mL of 0.1 g/100 mL Sterile Peptone Water (PW; Oxoid Ltd., Basingstoke, UK) using a stomacher blender; additional decimal dilutions were applied to this original suspension using PW-containing tubes, and a volume of 100 µL of proper dilutions were used to inoculate Tryptic Soy Agar (TSA; Oxoid Ltd., Basingstoke, UK) and Xylose Lysine Deoxycholate Agar (XLD; Oxoid Ltd., Basingstoke, UK) plates. Agar plates were incubated at 35 °C for 24 h, and typical Salmonella colonies were quantified to obtain the population from each sample (log CFU g⁻¹).
Coliform bacteria were analyzed following APHA/CMMEF methods 9.91–9.94 based on an MPN technique.
The enumeration of yeast and molds was analyzed following the respective method of the Bacteriological Analytical Manual [32 ].

Prior to subjecting the sample to culture media, physical analysis of the stool sample was performed in order to check whether the sample was diarrheal or not, presence of blood, pus and mucus. Children with diarrhea, blood, mucus were included. Diarrhea stool specimen was inoculated using sterile wire loop on MacConkey Agar (Oxoid, Ltd), and Xylose lysine- Deoxycholate agar (XLD) (Oxoid Ltd). The inoculated plates ware incubated at 37 °C for 24 h (SOPs) [12 ]. After 24 h incubation, the plates were checked for colony characteristics of Salmonella and Shigella species. Furthermore, colorless to yellow colonies on MacConkey Agar and pink to red colonies Xylose Lysine Deoxycholate (XLD) (Oxoid Ltd) [12 ] agar were typically considered as Non-lactose fomenters. In addition, the colonies considered as non-lactose fomenters were further identified by biochemical tests to confirm the identification to genus level of the pathogens Biochemical tests such as klingler iron agar (KIA), motility indole, lysine medium, simon’s citrate agar, and unease test were used [13 ].

At the time of the experiment, whole carcasses were transferred into sterile plastic bags containing 400 ml of buffered peptone water (BPE; Liofilchem, Teramo, Italy). Bags were vigorously massaged and shaken for 1 min at room temperature. Rinse solutions were transferred to bottles and incubated at 37°C for 24 hr. After incubation, a 0.1 ml aliquot was transferred to 10 ml of Rappaport‐Vassiliadis Broth (RV: Oxoid, Basingstoke, UK) and incubated for 24 hr at 42°C. A loopful (10 μl) was then plated on xylose‐lysine‐deoxycholate agar (XLD; Oxoid) and incubated at 37°C for 24 hr.