Culture, morphophysiology and biochemical characters of the microbial isolates were studied following standard methods (Ventosa et al., 1998 (link)). The isolates were checked for colony characters, motility, shape/size of the organism, and Gram and endospore staining. Biochemical tests, hydrolysis to carbohydrates and its metabolism as well as tolerance to NaCl were performed. Colony characters of isolates were observed on HiCrome Bacillus agar, rapid HiEnterococci agar, rapid HiColiform agar, Enterococcus confirmatory agar, Kligler iron agar, Chapman stone agar, Pikovskaya's agar, KG agar, Pseudomonas isolation agar, Hifluoro Pseudomonas agar base phenylalanine agar, triple sugar-iron agar, coagulase mannitol agar and HiCrome staph agar base for characterization of the bacteria (HiMedia Pvt Ltd, Mumbai).
Triple sugar iron agar
Manufactured by HiMedia
Sourced in India
Triple sugar iron agar is a culture medium used for the differentiation and identification of enteric bacteria. It contains three sugars (glucose, lactose, and sucrose) and an indicator system to detect carbohydrate fermentation and hydrogen sulfide production.
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Lab products found in correlation
Pseudomonas isolation agar, by HiMedia (3 mentions)
Eosin methylene blue agar, by HiMedia (3 mentions)
Macconkey agar, by HiMedia (2 mentions)
Simmons citrate agar, by HiMedia (2 mentions)
Hicrome bacillus agar, by HiMedia (1 mentions)
Hicrome staph agar, by HiMedia (1 mentions)
Salmonella shigella agar, by HiMedia (1 mentions)
Xylose lysine deoxycholate medium, by HiMedia (1 mentions)
Brilliant green agar, by HiMedia (1 mentions)
Plate count agar, by HiMedia (1 mentions)
Tetramethylmurexide, by Merck Group (1 mentions)
1 1 3 3 tetramethoxypropane, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by HiMedia (1 mentions)
Brij l23, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Peptone water, by Thermo Fisher Scientific (1 mentions)
Citrate agar, by Thermo Fisher Scientific (1 mentions)
M ller hinton agar, by Thermo Fisher Scientific (1 mentions)
10 protocols using triple sugar iron agar
1
Comprehensive Microbial Characterization Protocol
2
Characterization of Bacterial Isolates
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Characterization of bacterial isolates was carried out using colonial morphology, microscopic techniques and biochemical tests including gram’s reaction, coagulase test, oxides test, Oxidation–Fermentation test, catalase test and 3 % KOH tests. Highly selective media like Edwards Medium (HiMedia, India), Manitol Salt Agar(HiMedia, India), MacConkey agar (HiMedia, India), Eosin Methylene Blue agar (HiMedia, India), Xylose-lysine-deoxycholate medium (HiMedia, India), Brilliant Green Agar (HiMedia, India) and Salmonella Shigella Agar (HiMedia, India) were used. Triple Sugar Iron agar (HiMedia, India) was also used for differentiation of coliforms based on their ability of fermenting sugar and H2S production.
3
Analytical Characterization of Biochemical Compounds
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All chemicals used were of analytical grade. Thiobarbituric acid (TBA), trichloroacetic acid (TCA), 1,1,3,3-tetramethoxypropane, Brij-L23, tetramethylmurexide, and L-β-(3,4 dihydroxyphenyl) alanine (L-DOPA) were procured from Sigma Aldrich (St. Louis, MO, USA). Potassium carbonate and copper (II) sulfate were purchased from Thermo Fisher Scientific (Auckland, New Zealand). The microbial media (eosin methylene blue agar (EMB), Pseudomonas isolation agar (PIA), plate count agar (PCA), triple sugar iron agar, and Mueller–Hinton broth (MHB) were acquired from Himedia (Mumbai, India).
4
Microbial Identification and Antimicrobial Testing
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Culture media used were Peptone water, Nutrient Agar, Blood Agar, MacConkey Agar, Plate Count Agar and Sabouraud Dextrose Agar (all from Oxoid Limited, Thermo Fisher Scientific Inc., UK). These culture media were prepared as per the manufacturer's instruction leaflet and under standard sterile conditions in order to prevent external contaminations. Other media such as the Tryptone Soy Agar (TSA) (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was used for storing pure isolates for further evaluation in the study. Mannitol Salt Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was used for the identification of Staphyloccocus aureus. Shigella-Salmonella Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) were used for the identification of Shigella. Triple sugar iron Agar (HiMedia Laboratories Pvt. Ltd., India) and Citrate Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) were also used in the biochemical assay of the various microbes found. Müller Hinton Agar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) was also used in the culture and sensitivity testing. All these were prepared as directed by the manufacturer's leaflet.
5
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)).
6
Microbial Enumeration and Isolation
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All chemicals used were of analytical grade. Plate count agar was procured from Difco (Maryland, USA). Eosin methylene blue (EMP) agar, triple sugar iron agar, and Pseudomonas isolation agar were obtained from Himedia (Mumbai, India).
7
Soil Microbial Diversity in Nepal
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This study was carried out in Central Campus of Technology Hattisar Dharan and Regional Agricultural Research Station (RARS) Tarhara from October 2018 to March 2019. Soil samples were collected from different altitudes ranging from Itahari (374 ft.), Tarhara (418 ft.), Dharan (1272 ft.) and Vedetar (5140 ft.) of Koshi Zone, Nepal. From each geographical region 25 different soil samples were collected and in total 100 different soil samples from four regions were collected. Soil samples weighing 10gm was collected in sterile plastic bags from 3-5cm depth and transported into the laboratory at 4°C. Materials used in this experiment were Sodium acetate (HiMedia, India), Lauria Bertani (LB) broth (HiMedia, India). Nutrient Agar (HiMedia, India), Coomassie brilliant blue (HiMedia, India), Phosphate buffer saline (HiMedia, India), Grams staining reagents (HiMedia, India), MR-VP medium and reagents (HiMedia, India), Indole medium (HiMedia, India), Citrate agar medium (HiMedia, India), Gelatin agar medium (HiMedia, India), Starch agar medium (HiMedia, India), Egg yolk agar medium (HiMedia, India), Blood agar medium (HiMedia, India), Carbohydrate fermentation broth (HiMedia, India), SIM medium (HiMedia, India), Triple sugar iron agar (HiMedia, India).
8
Characterization of Ligninolytic Bacterium JD0705
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The bacterium isolate JD0705 was indicated as the most active ligninolytic bacterium obtained from the previous experiment. It was grown and characterized by culture in Tryptone Soya Broth under different conditions, including pH of 4.0-11.0, a temperature between 20-55°C and NaCl concentration of 0-7.0% (w/v). All cultures were incubated for 24 h on the shaking incubator at 150 rpm and measured the bacterial growth by spectrophotometric method at 600 nm (OD600) after incubation. The number of the "+" symbol represents the bacterial concentration based on the OD600 value. "+" represents OD600 of 0.001-0.300, while "++" represents OD600 of 0.301-0.800, and "+++" represents OD600 more than 0.800. All experiments were assayed in triplicates.
The anaerobic test was determined by Difco TM Anaerobic Agar (Becton, Dickinson and Company, USA) in an anaerobic jar (Becton, Dickinson and Company, USA) supplemented with Anaerocult ® A (Merck, Germany). The bacterial abilities to ferment sugars and produce hydrogen sulfide were analyzed by Triple Sugar Iron Agar (Himedia, India). Twelve biochemical characterizations were determined by using a KB013 identification kit (HiMedia, India), including malonate utilization, acetoin production, citrate utilization, galactosidase determination, nitrate reduction, catalase activity determination, arginine utilization and five carbohydrate utilization.
The anaerobic test was determined by Difco TM Anaerobic Agar (Becton, Dickinson and Company, USA) in an anaerobic jar (Becton, Dickinson and Company, USA) supplemented with Anaerocult ® A (Merck, Germany). The bacterial abilities to ferment sugars and produce hydrogen sulfide were analyzed by Triple Sugar Iron Agar (Himedia, India). Twelve biochemical characterizations were determined by using a KB013 identification kit (HiMedia, India), including malonate utilization, acetoin production, citrate utilization, galactosidase determination, nitrate reduction, catalase activity determination, arginine utilization and five carbohydrate utilization.
9
Isolation and Identification of E. coli
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Isolation of E. coli was performed using standard bacteriological methods [15 ]. Organ samples were crushed by gentle maceration, mixed separately with buffered peptone water (BPW) and incubated at 37 °C for overnight. A loopful of the culture suspension was streaked onto MacConkey agar (HiMedia, Pvt. Ltd., India) and incubated for 24 h at 37 °C aerobically. The next day those pink coloured presumptive E. coli colonies were sub-cultured onto nutrient agar to get a pure colony, followed by sub-culture on Eosin Methylene Blue (EMB) agar (HiMedia, Pvt. Ltd., India). Colonies with metallic green sheen on EMB were later characterized microscopically using Gram’s stain. Putative E. coli colonies were then transferred onto nutrient agar for further identification using biochemical tests. Triple sugar iron (TSI) agar (HiMedia, Pvt. Ltd., India) was used for further characterization. Observation of yellow slant, yellow butt, presence of gas bubbles, and absence of black precipitate in the butt was considered as potentially E. coli isolate. Then the isolates were subjected to different biochemical tests such as indole production, methyl-red, Voges- Proskauer, citrate utilization (IMViC) and motility tests as per Quinn et al. [15 ]. E. coli ATCC 35218 (obtained from Ethiopian public health institute) was used as a reference organism.
10
Identification of Presumptive Salmonella Isolates
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A loop full of inoculums from each RVB and SFB cultures were plated onto Xylose lysine desoxycholate (XLD) (Oxoid) and Brilliant green (BGA) (Oxoid) agar plates and incubated at 37°C for 24 hours for identification. Characteristic red colonies with black centers on XLD and pink colonies on BGA plates were examined and considered as presumptive Salmonella colonies. Five typical colonies from both XLD and BGA were streaked onto the surface of pre-dried nutrient agar plates (Oxoid) and incubated at 37°C for 24 hours.
Then, typical colonies from nutrient agar were inoculated into the following biochemical tests for further identification as per ISO-6579 guidelines12 and incubated for 24 hours at 37°C. The biochemical tests conducted include triple sugar iron (TSI) agar (Hi Media), lysine iron agar (Hi Media), Simmon’s citrate agar (Hi Media), urea broth (Hi Media), and indole reaction (motility indole ornithine, MIO) medium (Pronadisa)
Then, typical colonies from nutrient agar were inoculated into the following biochemical tests for further identification as per ISO-6579 guidelines12 and incubated for 24 hours at 37°C. The biochemical tests conducted include triple sugar iron (TSI) agar (Hi Media), lysine iron agar (Hi Media), Simmon’s citrate agar (Hi Media), urea broth (Hi Media), and indole reaction (motility indole ornithine, MIO) medium (Pronadisa)
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