All of the bacterial isolates were reinoculated on the cysteine electrolyte deficient (CLED) agar and MacConkey agar (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated at 37 °C for 18–24 h. After the incubation period, the bacterial colonies were evaluated for growth morphology and Gram staining characteristics. The final confirmation of bacterial isolates was done using biochemical tests-based identification. The biochemical tests, including citrate, indole, oxidase and analytical profile index 20E (API 20E) (BioMérieux, Marcy-l’Etoile, France), were used. The API 20E results were evaluated using the API website (https://apiweb.biomerieux.com/login ) (accessed from 1 January 2018 to 31 December 2019).
Api 20e
Manufactured by bioMérieux
Sourced in France, United States, United Kingdom, Germany, Japan, Macao, Denmark, Italy
The API 20E is a standardized identification system for Enterobacteriaceae and other non-fastidious Gram-negative rods. It consists of 20 miniaturized biochemical tests, which allow the identification of the most frequently encountered members of the Enterobacteriaceae family as well as certain other Gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Api 20ne, by bioMérieux (40 mentions)
Api zym, by bioMérieux (21 mentions)
Macconkey agar, by Thermo Fisher Scientific (19 mentions)
Api 50ch, by bioMérieux (15 mentions)
Api staph, by bioMérieux (11 mentions)
Vitek 2, by bioMérieux (6 mentions)
Api 50 chb, by bioMérieux (5 mentions)
Orthonitrophenyl β d galactopyranoside onpg, by bioMérieux (5 mentions)
Macconkey agar, by HiMedia (4 mentions)
Vitek 2 gn card, by bioMérieux (4 mentions)
Apiweb, by bioMérieux (4 mentions)
Blood agar, by Thermo Fisher Scientific (4 mentions)
Api 20 strep, by bioMérieux (3 mentions)
Api 20ne strips, by bioMérieux (3 mentions)
Macconkey agar, by Merck Group (3 mentions)
Api lister, by bioMérieux (3 mentions)
Eosin methylene blue agar, by Liofilchem (3 mentions)
Indole, by Bio-Rad (3 mentions)
Mueller hinton agar plate, by Liofilchem (3 mentions)
Urease, by Bio-Rad (3 mentions)
333 protocols using api 20e
1
Bacterial Identification Workflow
2
Comprehensive Characterization of Novel Bacterium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gram-staining, oxidase and catalase reactions, and motility (the hanging drop method) were determined as described by Gerhardt et al. (1994) . The morphology of cells grown in MB and negatively stained with a 1% phosphotungstic acid on carbon-coated 200-mesh copper grids was examined by electronic transmission microscopy [Libra 120 FE (Carl Zeiss), provided by the Far Eastern Centre of electronic microscopy, Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences]. Hydrolysis of starch, casein, gelatin, Tween 80, DNA, L-tyrosine, chitin, nitrate reduction (sulfanilic acid/α-naphthylamine test), and growth at different salinities (0-12% NaCl), temperatures (5-45 °C), and pH values (4.5-10.0) were carried out using arti cial sea water (ASW) as described in a previous paper (Romanenko et al. 2013 ). The arti cial sea water (ASW) contained (per liter of distilled water): 30 g NaCl, 4.9 g MgCl 2 , 2.0 g MgSO 4 , 0.5 g CaCl 2 , 1.0 g KCl, 0.01 g FeSO 4 . Biochemical tests were performed using API 20E, API 20NE, API ID32 GN, and API ZYM test kits (bioMérieux, France) as described by the manufacturer except the cultures were suspended in ASW.
3
Isolation and Identification of E. coli in Fecal, Stool, and Milk Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We inoculated faecal calf samples and worker stool samples on MacConkey’s agar (Oxoid, Hampshire, UK), and the plates were incubated at 37 ℃ for 24 h. Lactose fermenter (pink in colour) colonies were then subcultured on eosin methylene blue agar (EMB; Oxoid) and were incubated under the same conditions. For milk samples, ten dilutions on Tryptone Soy broth (TSB; Oxoid) were incubated at 37 ℃ for 6 h, and then an inoculum of each sample was cultured on MacConkey’s agar, followed by EMB agar at 37 ℃ for 24 h each. Suspected E. coli colonies on EMB (green metallic sheen in colour) were biochemically confirmed by API-20E (bioMérieux, Marcy-l’Etoile, France). All EPEC isolates were serotyped using polyvalent and monovalent O-antisera sets (Denka Seiken Co., Tokyo, Japan) according to the manufacturer’s instructions.
4
Biochemical Profiling of Bacterial Isolates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The biochemical tests were carried out according to [18 ]. After performing the Gram stain test, the biochemical and physiological kit API 20E (bioMérieux, Lyon, France) was used for selected isolates. This test includes the following biochemical determinations: enzyme activity: ß-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase, gelatinase, cytochrome oxidase, citrate utilisation, production of , indole, and acetoin (Voges Proskauer); fermentation of polysaccharides: glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose. Based on these analyses, groups of isolates were identified, and then, a representative was selected to perform molecular analyses.
5
Characterization of Clinical Bacterial Isolates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The clinical strains were collected between May 2019 and April 2022 from the routine diagnostic laboratory, Mayo hospital, Lahore, Pakistan. Mayo hospital is one of the largest hospitals in South East Asia with a 3000 bed capacity. The clinical isolates were processed as given in Figure 1 . Clinical specimens were phenotypically characterized by analyzing colony morphology and Grams staining by culturing on MacConkey agar and cysteine lactose electrolyte-deficient media (Oxoid Ltd., Basingstoke, UK) for urine samples. Biochemical characterization was performed by API-20E and API-20NE (BioMerieux, Marcy-IEtoile, France).
6
Isolating E. coli from Ready-to-Eat Foods
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 252 strains of Enterobacteriaceae were isolated from 2000 RTE food samples in Guangdong, China, in 2021. To isolate Escherichia coli, 25 g of foodstuff was transferred to an aseptic bag and mixed with 225 mL of Butterfield's phosphate‐buffered solution. The sample was homogenized for 2 min at 230 rpm using a stomach machine and diluted 10‐fold. The diluent was inoculated into fermentation tubes containing lactose broth and incubated at 37°C for 24–48 h. A loopful of the suspension extracted from positive cultures (those showing lactose fermentation and gas production) was smeared onto CHROMagar E. coli agar plates and cultured at 37°C for 18–24 h. Finally, one colony from each plate was selected, analyzed, and identified using API 20E (bioMérieux) (Qinghua et al., 2018 (link)).
7
Salmonella Isolation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For pre‐incubation, the swabs were placed into non‐selective buffered peptone water (Merck) at 41.5°C for 24 hr. Afterwards, the samples were transferred into the Brilliance Salmonella medium (Oxoid). Plates were incubated at 41.5°C for 48h ± 2 hr under aerobic conditions. Afterwards, only purple colonies were considered for further examination. The final identification was performed using commercial latex agglutination tests: Salmonella test Kit (Oxoid) and API 20E (bioMérieux).
8
Isolation and Identification of Aeromonas spp. from Water
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For isolation of Aeromonas spp. from water samples, deoxycholate-hydrogen sulfate-lactose (DHL) agar medium (Nissui Pharmaceutical, Tokyo, Japan) was inoculated with 100 μL of each respective sample and incubated at 37°C overnight. For each sample, a maximum of six colonies were identified as likely Aeromonas spp. based on colony morphology on DHL agar (pink colony) and were subjected to further analyses. Specifically, the isolates were screened for oxidase production and subjected to a variety of biochemical tests (API20E; BioMérieux, Marcy l’Etoile, France). Furthermore, the identity of each strain was verified by 16S rRNA gene sequencing (Marchesi et al., 1998 ).
9
Trehalose Operon Cloning and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M sibonii has been shown to differ from M morganii subspecies morganii by assimilation of trehalose and we aimed to confirm this difference by cloning a trehalose operon from M sibonii GER-11 to M morganii subspecies morganii O86D10 and analysing the trehalose operon. 25 Whole-cell DNA was extracted using the Ultraclean Microbial DNA isolation kit (Mo Bio Laboratories, Ozyme, Saint-Quentin, France) according to the manufacturer's instructions as previously described. 26 DNA from M sibonii GER-11 was used as a template for the amplification of the trehalose operon (7517 bp) followed by cloning and expression in electrocompetent M morganii subspecies morganii O86D10 (see appendix pp 2-3 for detailed procedure). Biochemical characterisation, including trehalose assimilation of the collection, was performed using Api20E and Api50CH systems (BioMérieux, La Balme les Grottes, France) according to the manufacturer's recommendations. We also evaluated the functionality of the tetD gene, which is responsible for natural resistance to tetacycline in M sibonii, by cloning and expressing it in Escherichia coli (see appendix pp 2-3 for detailed procedure).
10
Detailed Characterization of Strain CCUG 66741T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Further characterization of strain CCUG 66741T was performed at the typing laboratory of the Culture Collection University of Gothenburg (CCUG). Colony morphology was observed after growth on Columbia Blood Agar at 30°C, aerobically, for 24 h. Cell morphology and size was determined by Gram-staining of fresh cells and observation with a transmission microscopy with a color camera using a 50X dry objective (EC Epiplan N.A. 0.75, ZEISS, Oberkochen, Germany), at the Centre for Cellular Imaging (University of Gothenburg). The microscope used was a wide-field, upright optical microscope (Axio Imager.Z2, ZEISS).
Standard biochemical tests (API® ID 32E and API® 20E, bioMérieux) were performed following the manufacturer’s instructions. The results of API® ID 32E were analyzed with the software StrainMatcher4 against the CCUG internal database. Additional biochemical tests were performed in conventional tube media (prepared by the Substrate Unit, Department of Clinical Microbiology, Sahlgrenska University Hospital), as described earlier (Bergey et al., 1994 ; Murray, 2003 ).
Standard biochemical tests (API® ID 32E and API® 20E, bioMérieux) were performed following the manufacturer’s instructions. The results of API® ID 32E were analyzed with the software StrainMatcher
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!