The enzymatic activities of Pseudomonas species were evaluated using certain biochemical tests, including oxidase, citrate utilization and indole tests (LaBauve and Wargo 2012 (link)). The oxidase test was performed by smearing a fresh colony of each isolate onto a sterile oxidase filter paper disc (Sigma–Aldrich, USA) soaked in distilled water. The positive oxidase activity results are indicated by the appearance of a purple colour. The capacity of Pseudomonas spp. to utilize citrate as a source of energy was carried out by streaking a fresh purified colony onto a Simmons citrate agar (Sigma–Aldrich, USA) slant and incubated at 37 °C for 5–7 successive days. Citrate utilization was indicated by the appearance of a sky-blue colour. The capability of Pseudomonas spp. to convert tryptophan into indole was tested by inoculation of the suspected organism into tryptophan broth (Sigma–Aldrich Chemie GmbH, Germany), which was then incubated at 37 °C for a couple of days. Thereafter, using gentile agitation, 0.5 ml of Kovacs reagent was added until a red–violet colour appeared, indicating positive results, while a yellow colour indicates negative results.
Simmons citrate agar
Manufactured by Merck Group
Sourced in Germany, United States
Simmons citrate agar is a microbiological growth medium used for the identification and differentiation of Gram-negative bacteria. It is designed to detect the ability of an organism to utilize citrate as the sole carbon and energy source. The medium contains sodium citrate, ammonium dihydrogen phosphate, and other salts, which provide the necessary nutrients for bacterial growth.
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Lab products found in correlation
Tryptophan broth, by Merck Group (2 mentions)
Triple sugar iron agar, by Merck Group (2 mentions)
Blood agar, by Merck Group (1 mentions)
Baird parker, by Merck Group (1 mentions)
Macconkey, by Merck Group (1 mentions)
Api staph kit, by bioMérieux (1 mentions)
Lactose broth, by Merck Group (1 mentions)
Lysine iron agar, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Macconkey agar, by Merck Group (1 mentions)
Eosin methylene blue agar, by Merck Group (1 mentions)
Xylose lysine deoxycholate agar, by Merck Group (1 mentions)
Urea agar, by Merck Group (1 mentions)
Sulfide indole motility, by Merck Group (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Sulfide indole motility medium, by Merck Group (1 mentions)
9 protocols using simmons citrate agar
1
Biochemical Tests for Pseudomonas Species
2
Biochemical Profiling of Pseudomonas Isolates
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The enzymatic activities of Pseudomonas species were evaluated using certain biochemical tests, including oxidase, citrate utilization and indole tests. The oxidase test was performed by smearing a fresh colony of each isolate onto a sterile oxidase lter paper disc (Sigma-Aldrich, USA) soaked in distilled water.
The positive oxidase activity results are indicated by the appearance of a purple colour. The capacity of Pseudomonas spp. to utilize citrate as a source of energy was carried out by streaking a fresh puri ed colony onto a Simmons citrate agar (Sigma-Aldrich, USA) slant and incubated at 37°C for 5-7 successive days. Citrate utilization was indicated by the appearance of a sky-blue colour. The capability of Pseudomonas spp. to convert tryptophan into indole was tested by inoculation of the suspected organism into tryptophan broth (Sigma-Aldrich Chemie GmbH, Germany), which was then incubated at 37°C for a couple of days. Thereafter, using gentile agitation, 0.5 ml of Kovacs reagent was added until a red-violet colour appeared, indicating positive results, while a yellow colour indicates negative results.
The positive oxidase activity results are indicated by the appearance of a purple colour. The capacity of Pseudomonas spp. to utilize citrate as a source of energy was carried out by streaking a fresh puri ed colony onto a Simmons citrate agar (Sigma-Aldrich, USA) slant and incubated at 37°C for 5-7 successive days. Citrate utilization was indicated by the appearance of a sky-blue colour. The capability of Pseudomonas spp. to convert tryptophan into indole was tested by inoculation of the suspected organism into tryptophan broth (Sigma-Aldrich Chemie GmbH, Germany), which was then incubated at 37°C for a couple of days. Thereafter, using gentile agitation, 0.5 ml of Kovacs reagent was added until a red-violet colour appeared, indicating positive results, while a yellow colour indicates negative results.
3
Bacterial Characterization of Bovine Teat Skin
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The bacteria applied in this study were isolated from the teat skin of cows within the Teagasc Moorepark research herd, by taking skin swab samples from lactating cows' teats using moistened cotton swabs, according to NMC (NMC, 2017) guidelines. The isolates were gram stained and bacterial identification was carried out using biochemical tests including lactose fermentation, motility test medium (Sigma-Aldrich, Dublin, Ireland), catalase and oxidase tests, tube coagulase (Sigma-Aldrich, Dublin, Ireland) and growth/reaction on various types of agars including blood agar (Sigma-Aldrich, Dublin, Ireland), MacConkey (Merck Millipore, Cork, Ireland), Baird Parker (Merck KGaA64271, Darmstadt, Germany) and modified Edwards agar (Oxoid 3M0027, Hampshire, UK), Simmons citrate agar (Sigma-Aldrich, Dublin, Ireland), CAMP esculin agar (Sigma-Aldrich, Dublin, Ireland) and triple sugar iron agar (Sigma-Aldrich, Dublin, Ireland). Analytical Profile Index-Staph (API-Staph Kit, bioMerieux, Marcy-l'Etoile, France) and API 20 tests (API, bioMerieux, Marcy-l'Etoile, France) were also used according to the manufacturer's instructions.
4
Salmonella Detection in Meat Samples
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Detection of Salmonella spp. was carried out by inserting 25 g of each meat sample into 225 mL of Lactose Broth (Merck 1.07661.0500 Darmstadt, Germany) and incubating it at 35°C for 24 h. Then, a 1 mL volume of suspension samples was inoculated into 9 mL of Tetrathionate Broth (TB) (Merck 1.05285.0500 Darmstadt) and was incubated at 35°C for 24 h. One TB loop was taken using an inoculating loop; it was streaked onto Bismuth Sulfite Agar (Merck 1.05418.0500 Darmstadt) and was then incubated at 35°C for 24 h. The typical Salmonella colonies were analyzed through Gram staining, lysine iron agar (LIA) media (Merck 1.11640.0500 Darmstadt), triple sugar iron agar (TSIA) media (Merck 1.03915.0500 Darmstadt), Simmons citrate agar (Merck 1.02501.0500 Darmstadt), and a urease test for biochemical identification [21 -23 ]. Salmonella spp. isolates showed positive results, as observed in the Simmons citrate agar. The LIA showed a purple slant/purple butt (alkaline), although the butt reaction may have been masked by H2S production. The TSIA showed a red slant/yellow butt (alkaline). The presence of Salmonella spp. was indicated by negative urease test results [23 ].
5
Identification of Shigella and E. coli O157:H7
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Fecal specimens were sent to referral lab in Cary-Blair transport medium (Merck, Germany), in cold boxes within 24 h. All specimens were cultured on MacConkey's agar and on more specific media: Xylose-lysine-deoxycholate, deoxycholate citrate agar, and sorbitol MacConkey agar (for isolation of E. coli O157H7) all kept in 37°C for 24 h. The Shigella isolates were identified by biochemical characterization and by culturing on differential media such as Kliger's iron agar, Simmon's citrate agar, and lysine iron agar (all media from Merck, Germany). Serotyping of Shigella isolates was carried out using group specific anti-sera (Mast, UK). Also specific anti O157 and anti H7 (Mast, UK) were used to identify E. coli O157H7.
6
Isolation and Characterization of E. coli from Diarrheal Patients
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In this cross-sectional study, 581 stool specimens were collected from patients with diarrhea referring to the teaching hospitals (Golestan and Abouzar hospitals), in Ahvaz, southwest of Iran, from September 2016 to August 2017. Patients with a history of fever, nausea, vomiting, abdominal cramps, watery, mucoidal and bloody diarrhea were included in our study. Also, they had not used the antibiotic nearly 2 weeks before. If patients had taken antibiotics in the last two weeks, they would be excluded from our study. Stool samples initially were cultured on MacConkey agar (Merck, Germany), and incubated for 24 hrs at 37°C. The lactose positive colonies were tested by standard biochemical and bacteriological tests such as Triple Sugar Iron Agar, Indole test, Methyl red and Voges-Proskauer tests, and Simmons citrate agar (Merck, Germany) for detection of E. coli isolates.13 (link) All isolates that confirmed as E. coli, were preserved in Tryptic Soy Broth (TSB) (Merck, Germany), containing glycerol (30%) at −70°C.
7
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
8
Isolation and Characterization of Stenotrophomonas maltophilia from Iranian Hospitals
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In this cross-sectional study, conducted from December 2021 to August 2022, 94 different clinical isolates of S. maltophilia were recovered from patients admitted to four tertiary-care hospitals in Iran (Tehran, Mashhad, Shiraz, and Qazvin). This study was approved by the Ethics Committee of Shahed University “IR.SHAHED.REC.1400.175.” The samples were identified using conventional microbiological and biochemical methods, including catalase and oxidase tests, DNase, urease, and reactions in media such as triple sugar iron (TSI) agar (Merck, Germany), Simmons citrate agar (Merck, Germany), and sulfide indole motility (SIM) (Merck, Germany) [15 (link)]. Stock cultures were stored in a tryptone soy broth (TSB) medium (Merck, Germany) containing 20% glycerol at −80°C until analysis. Escherichia coli (E. coli) ATCC 25922 and S. maltophilia ATCC 13637 were quality control strains.
9
Isolation and Identification of E. coli O157
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Approximately 10 g of each fecal sample was diluted in 90 mL of BPW (Oxoid) and mixed for 30 s. A 15 mL aliquot of each fecal mixture was stored for later use. Next, 1 mL of enriched fecal sample was mixed with 20 μL of magnetic beads coated with an anti-O157 antibody and IMS was performed according to the manufacturer’s instructions (Oxoid). The bead suspension (100 μL) was then plated onto CT-SMAC agar (Oxoid). The plates were incubated at 37 °C for 24 h and presumptive E. coli O157 colonies were identified as E. coli O157 that did not ferment sorbitol. These colonies were picked and inoculated into nutrient agar slants at 37 °C for 24 h and stored in a refrigerator for further biochemical analysis. These isolates were further verified using conventional biochemical tests as described by Harrigan [62 ]. Tests were performed to examine indole production and motility using Sulfide Indole Motility (SIM) medium (Merck); citrate utilization with Simmons citrate agar (Merck); and methyl red and vogues-Proskauer using MR-VP medium (Merck) [63 ].
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