Cell morphology and gliding motility were observed using an Olympus BX50 light microscope with a drop of lactophenol blue added to enhance observational ability. A slide with thin SAP2 agar medium overlay was used to examine gliding motility. After placing a swarm colony on an overlay and allowing incubation, we observed gliding motility. Catalase activity was tested by bubble formation in 3 % H2O2 solution. Oxidase activity was determined by the oxidation of 1 % tetramethyl-p-phenylenediamine on filter paper. Acid production from carbohydrates was investigated using the commercial API 50 CH and API 20 E systems (BIOMERIEUX). Enzyme production was tested using the commercial API ZYM system (BIOMERIEUX), in accordance with manufacturer instructions.
Api 20e system
Manufactured by bioMérieux
Sourced in France, United States, Italy, United Kingdom
The API 20E system is a standardized identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods. It consists of a plastic strip with 20 microtubes containing dehydrated biochemical test substrates. The organism is inoculated into the strip, and the reactions that occur during the incubation period are used to identify the bacterial species.
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Lab products found in correlation
Macconkey agar, by Thermo Fisher Scientific (7 mentions)
Macconkey agar, by Merck Group (3 mentions)
Vitek 2, by bioMérieux (3 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (3 mentions)
Eosin methylene blue agar, by Thermo Fisher Scientific (2 mentions)
Rappaport vassiliadis broth, by Merck Group (2 mentions)
Api 50 ch system, by bioMérieux (2 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Vitek 2 id gnb identification card, by bioMérieux (2 mentions)
Vitek 2 automated system, by bioMérieux (2 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Bx50 light microscope, by Olympus (1 mentions)
Api 50ch, by bioMérieux (1 mentions)
Api zym system, by bioMérieux (1 mentions)
Etest technique, by bioMérieux (1 mentions)
Mueller hinton agar (mha), by Bio-Rad (1 mentions)
Api 20e test strips, by bioMérieux (1 mentions)
Cefpodoxime, by Thermo Fisher Scientific (1 mentions)
Ceftriaxone, by Thermo Fisher Scientific (1 mentions)
146 protocols using api 20e system
1
Characterization of Gliding Motility and Enzymatic Activities
2
Salmonella Prevalence in Pork Carcasses
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A total of 1728 pork specimens were obtained between April 2013 and April 2014 from one large-scale slaughterhouse, in Guangdong, China. About 40–50 randomly selected carcasses were sampled weekly before splitting. Samples of 150 g were taken from the inside of two hind legs of each carcass using a sterile knife and placed into a sterile plastic bag. All samples were placed in a low-temperature foam box and dispatched within 3 h to the laboratory. If immediate dispatch was not possible for some reason, samples were stored in a refrigerator at 4 °C until the time of dispatch. About 25 g of meat samples cut into pieces was put into 200 mL buffered peptone water (BPW), which were then incubated at 37 °C for 12 h. One milliliter aliquot of BPW cultures were transfered to 10 mL of selenite cysteine broth and incubated at 37 °C for 24 h. The enriched content was streaked on bismuth sulphite agar and xylose lysine desoxycholate agar and incubated at 37 °C for a further 24 h. Presumptive Salmonella colonies were identified with API20E systems (BioMerieux, Beijing, China) and were serotyped using Salmonella specific O and H antigens by the slide agglutination test (S&A Company, Bangkok, Thailand).
3
Isolation and Identification of Cronobacter spp.
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In accordance with the National Food Safety Standards of China document GB 4789.4-2010, the quantitative detection was prepared and the process was described in the former research (Ling et al., 2018 (link)). The most probable number (MPN) was calculated according to the schemes reported previously (Xu et al., 2015 (link)). By means of API 20E systems (BioMérieux, Marcy-l’Étoile, France), the presumable Cronobacter spp. with green or blue-green colonies in chromogenic Enterbacter sakazakii Agar Plate were characterized. Besides, fusA sequencing was manipulated to the species classification in this work.
4
Enumeration and Characterization of E. coli
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The abundance of culturable E. coli was assessed using standard membrane filtration procedures [14 ]. Briefly, 10-fold serial dilutions of collected water samples were passed through 0.45-µm nitrocellulose filters and then the filters were placed on plates containing Tryptone Bile X-Glucuronide (TBX). TBX plates were incubated at 44 °C for 24 h. Between 10–30 colonies grown on TBX plates were picked and used to inoculate API-20E systems (Biomérieux) according to manufacturer instructions to confirm their identification as E. coli. About 10–30 E. coli isolates per sample were stored in Brain Heart Infusion Broth containing 40% glycerol at −80 °C for later determinations of phenotypic resistance to antibiotics.
5
Biochemical Characterization of Isolated Bacteria
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The isolated bacteria were subjected to biochemical characterization and phenotypical analysis. Different enzymatic activity shows the ecology, physiology, or natural habitat of microorganisms. The isolates were tested on gram staining, starch hydrolysis, siderophore production, phosphate solubilization and were identified by using API 20E systems (BioMerieux, Marcy L’Etoile, France). API 20E kit is to determine the enzymatic activity of microorganisms. This system consisted of 19 enzymatic reactions for rapid detection [17 (link)].
6
Isolating Aeromonas hydrophila in Surviving Fish
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After 14 days, the survived fish was bacteriologically examined for A. hydrophila, three attempts of bacterial isolation were performed with a week-interval at days 21, 28, and 35 post-challenge. Bacterial isolation was done using randomly selected five fish from each treatment group and anaesthetized within 60 s using 50 mg/l tricaine mesylate. The fish abdomen aseptically opened (sterilized with methyl alcohol 70%), Specimens were taken from internal organs (Amlacher, 1970) and inoculated into Trypti soy roth Dif o and in u ated at 28°C for 24 hr. hen the sing e pure iso ated o onies were stored in ryovia s ontaining 20 , 30 and 50 g y ero roth at -20 C for further identification by using API®20 E systems (BioMérieux, Mar-y I"Etoi e, France) (Austin & Austin (2012) .
7
Bacterial Identification and Antibiogram Analysis
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Bacterial identification was performed using the API 20E system (bioMérieux, Marcy-l’Etoile, France). Antibiograms for 32 antibiotics (amoxicillin, amoxicillin/clavulanic acid, aztreonam, ceftazidime, cefalotine, cefmandole, cefotaxime, cefepime, cefoxitine, imipenem, meropenem, Moxalactam, pipéracilline, ticarcilline, ticarcilline/acide clavulanique, piperacilline/tazobactam, fosfomycine, colistine, rifampicine, cotrimxazole, ciprofloxacine, pefloxacine; norfloxacine, nalidixic acid, tetracycline, tigecycline, chloramphenicol, kanamycine, amikacine, netilmicine, tobramycine, gentamicine) were determined by the disc diffusion method on Mueller-Hinton agar (BioRad, Marnes-La-Coquette, France) and the susceptibility breakpoints were determined and interpreted as recommended by the Clinical and Laboratory Standards Institute [17 ]. All plates were incubated at 37 °C for 18 h. Minimum inhibitory concentrations (MICs) of given ß-lactams were determined using the Etest technique (bioMérieux, Marcy l’Etoile, France) for the following β-lactam antibiotics: amoxicillin ± clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, cefepime and imipenem. Confirmation of ESBL producers was performed by double disc synergy testing between ticarcillin/clavulanate and aztreonam and/or ceftazidime and/or cefepime [18 (link)].
8
Identification of A. baumannii Isolates
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Collection, isolation, and identification of A. baumannii isolates A total of 320 clinical samples were taken from patients of three hospitals in Erbil city, Iraq, during June 2016 to January 2017. All clinical specimens were obtained from various sites, which included tracheal aspirate, sputum, burned wound, urine, cerebrospinal fluid, and blood. Specimens were inoculated in brain-heart infusion broth (BHIB; Himedia, India) medium and then transferred to lab for culture. Samples of soil were obtained from different locations in Erbil. Tenfold serial dilutions of soil suspension were made in BHIB, and inocula (0.1 ml) from each dilution were spread with sterile glass beds onto brain-heart infusion agar (BHIA; Himedia) plates. All isolates (clinical and soil) were initially inoculated on MacConkey agar, blood agar, Herellea agar, and on the CHROMagar Acinetobacter Media (CHROMagar, France). Incubation was performed for 24 h at 37 °C. Standard laboratory methods (Gram-stain and colonial morphology) and conventional biochemical tests (catalase, oxidase, urea test, citrate test, indole test, and the reaction in triple sugar iron medium and growth in 44 °C) were used to identify A. baumannii. Bacterial species identification of the isolates was confirmed by API 20E system (bioMerieux, France) and by PCR detection of intrinsic bla OXA-51-like carbapenemase gene from these isolates.
9
Bacterial Identification Protocol
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All typical colonies were tested for oxidase and catalase enzymes, and by seeding in Kligler Iron Agar (KIA; Biolife), incubated at 30°C for 24 h. Moreover, presumptive colonies were submitted to phenotypic identification with API® 20 E system (bioMérieux, Marcy l’Etoile, France).
10
Phenotypic Identification of Pathogenic Isolate
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The phenotypic identification of the pathogenic isolate was performed by the API 20E system (Biomerieux, Lyon, France) as recommended by Abeyta et al. (2019) [40 (link)]. Briefly, the pathogenic isolate streaked on NA plates was incubated at 28 °C for 24 h, and then inoculated into the API 20E test strips (Biomerieux, Lyon, France) according to the instructions of the manufacturer. The test strips containing the isolate were incubated at 37 °C for 18 h and observed for biochemical reactions. The phenotypic characterization of A. hydrophila previously reported by Long et al. (2016) [41 ] and Ye et al. (2018) [42 ] served as a reference.
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