All swab samples were enriched in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) and incubated at 42 °C overnight. A loopful of each of the enrichment cultures was then plated on sheep blood agar (DifcoTM Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland), and again incubated at 42 °C overnight. The colonies were subsequently washed off with 2 mL 0.85% NaCl solution. Of this colony suspension, an aliquot (100 μL) was combined with 200 μL Gram-negative lysis buffer and heated at 60 °C for 50 min, followed by 99 °C for 10 min. After centrifugation (2 min; 11,000 rpm), the supernatant was used as template for the PCR.
Difco columbia blood agar base eh
Manufactured by BD
Sourced in Switzerland
Difco™ Columbia Blood Agar Base EH is a general-purpose medium used for the cultivation and identification of a wide variety of microorganisms, including fastidious bacteria. It provides the necessary growth factors and nutrients for microbial growth.
Automatically generated - may contain errors
Lab products found in correlation
Enterobacteriaceae enrichment broth, by BD (4 mentions)
Sheep blood sb055, by Thermo Fisher Scientific (3 mentions)
Lightcycler r 2.0 instrument, by Roche (2 mentions)
Quantifast multiplex pcr kit, by Qiagen (2 mentions)
Rapid e coli 2 agar, by Bio-Rad (1 mentions)
Rapid e coli agar, by Bio-Rad (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Fraser supplement, by Thermo Fisher Scientific (1 mentions)
10 protocols using difco columbia blood agar base eh
1
Enterobacteriaceae Enrichment and Detection
2
STEC Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specimens were cultured on MacConkey agar using the streak plate technique. From each plate, six individual colonies, if possible of different morphology, were picked und subcultured on sheep blood agar (Difco TM Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland). Isolates that were confirmed to possess stx (stx1 and/or stx2) by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA) (EURL, 2013a) were selected for further analysis. From plates yielding more than one stx positive colony, one isolate was randomly chosen for subsequent characterization. Proportions of STEC in stool samples were defined as the numbers of stx positive colonies among six E. coli colonies.
3
Enrichment and Detection of Shiga Toxin Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each fecal sample was enriched at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) for 24 h at 37 °C. One loopful of each of the enrichment cultures was cultured on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland) using the streak plate technique. The resulting colonies were washed off with 2 ml 0.85% NaCl and DNA was extracted by a standard lysis protocol. Screening for stx1 and stx2 genes was performed by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA) using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland) according to the guidelines of the European Union Reference Laboratory (EURL) [25 ].
4
Enrichment and Detection of Shiga Toxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each sample (10g) was enriched at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) for 24 h at 37 °C. One loopful of each of the enrichment cultures was cultured on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland), using the streak-plate method. The resulting colonies were washed off with 2 mL 0.85% NaCl. Samples were then screened by real-time PCR for stx1 and stx2, using the Assurance GDS® for Shiga Toxin Genes (Bio Control Systems, Bellevue, WA, USA).
5
Rapid Detection of Shiga Toxin Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of Shiga toxin genes (stx), 10 g of each sample were enriched first at a 1:10 ratio in modified tryptic soy broth (Oxoid) with 1 mg/L acriflavin (24 h, 37°C) and subsequently subcultured (24 h, 37°C) on sheep blood agar (Difco Columbia blood agar base EH, Becton Dickinson; 5% sheep blood SB055, Oxoid). After washing off the colonies (0.85% NaCl), samples were screened by the Assurance GDS assay for Shiga toxin genes (Bio Control Systems, Bellevue, WA, USA). For isolation of Shiga toxin-producing Escherichia coli (STEC), stx-positive enrichment cultures were streaked in triplicate on Rapid E. coli 2 agar (Bio-Rad; 24 h, 37°C). Three typical E. coli colonies were picked from each plate, subcultured (plate count agar; 24 h, 37°C) and tested for Shiga toxin genes (EU Reference Laboratory, 2013) by real-time PCR (LightCycler, Roche Diagtnostics, Rotkreuz, Switzerland) using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland).
Obtained STEC isolates were examined by PCR for stx1, stx2, and subtypes, eae (intimin) and the top-five serogroups O26, O103, O111, O145, and O157 (EU Reference Laboratory, 2013, 2014).
Obtained STEC isolates were examined by PCR for stx1, stx2, and subtypes, eae (intimin) and the top-five serogroups O26, O103, O111, O145, and O157 (EU Reference Laboratory, 2013, 2014).
6
Isolation and Characterization of Shiga Toxin-producing Escherichia coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In the event of a stx positive PCR result, one loopful each of the enrichment broth was streaked onto on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland) and onto RAPID'E. coli Agar (BioRad, Basel, Switzerland) to obtain single colonies of E. coli.From each plate, individual colonies were picked and suspended in 0.5 ml NaCl 0.85%. Isolates that were confirmed to possess stx (stx1and/or stx2) by real-time PCR were subcultured on sheep blood agar and the presence of stx was confirmed using the Assurance GDS® for Shiga Toxin Genes (Bio Control Systems, Bellevue, WA, USA). From plates yielding more than one stx positive colony, one isolate was randomly chosen for subsequent characterization.
7
Enrichment and Detection of Shiga Toxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each sample (10 g) was enriched at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) for 24 h at 37 °C. One loopful of each of the enrichment cultures was cultured on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland) using the streak-plate method. The resulting colonies were suspended in 2 ml 0.85% NaCl. Samples were then screened by real-time PCR for stx1 and stx2 using the Assurance GDS® for Shiga Toxin Genes (Bio Control Systems, Bellevue, WA, USA).
8
Yersinia Detection from Fecal Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A subset of each fecal sample (approx. 1 g) was enriched (24 h, 30 °C) at a 1:10 ratio in BPW (Oxoid). The enriched samples (one loopful: approx. 10 µl) were subcultured for 24 h at 30 °C on cefsulodinirgasan-novobiosin (CIN) agar (Oxoid). Presumptive Yersinia colonies (red bull’s-eye surrounded by a transparent border) were streaken (24 h, 37 °C) on sheep blood agar (Difco Columbia blood agar base EH, Becton–Dickinson, Allschwil, Switzerland; 5% sheep blood SB055, Oxoid) and then tested for urease activity. Urease-positive colonies were identified with MALDI-TOF mass spectrometry [6 (link)].
9
Screening Reindeer Fecal Samples for Shiga Toxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A subset of each fecal sample (about 1 g) was enriched (18–24 h, 37 °C) at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton–Dickinson). After incubation (24 h, 37 °C) of the enriched samples on sheep blood agar (Difco Columbia blood agar base EH, Becton–Dickinson; 5% sheep blood SB055, Oxoid), the colonies were washed off with 2 ml of 0.85% saline solution. To screen the samples by real-time polymerase chain reaction (PCR) for stx1 and stx2, the Assurance GDS® Assay MPX ID for Top STEC (Tq 71019-52; Bio Control Systems, Bellevue, WA, USA) was applied [7 (link)]. To compare the frequencies of stx1, stx2, and both stx1 and stx2 among fecal samples and the occurrence of Shiga toxin genes among fecal samples of reindeer from different areas of origin, contingency tables (Fisher’s exact test) were used.
10
Isolation and Identification of Listeria monocytogenes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Examination for L. monocytogenes was done in accordance with ISO 11290-1:2005-01. A subset of each fecal sample (approx. 1 g) was enriched (24 h, 30 °C) at a 1:10 ratio in Fraser broth with half Fraser supplement (Oxoid). From the first enrichment, 0.1 ml were incubated (24 h, 37 °C) in 10 ml of Fraser broth with Fraser supplement (Oxoid). The enriched samples (one loopful: approx. 10 µl) were subcultured for 48 h at 37 °C on chromogenic Ottaviani Agosti agar (bioMérieux, Marcy l’Etoile, France). Presumptive L. monocytogenes colonies (turquoise–blue colonies surrounded by an opaque halo on the chromogenic agar) were streaken (24 h, 37 °C) on sheep blood agar (Difco Columbia blood agar base EH, Becton–Dickinson, Allschwil, Switzerland; 5% sheep blood SB055, Oxoid) for evaluation of hemolysis. Isolated L. monocytogenes were serotyped using the commercial set of Listeria O-factor and H-factor antisera from Denka Seiken (Pharma Consulting, Burgdorf, Switzerland).
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!