Overnight cultures of B. pseudomallei wild-type, 6H4 mutant and the complemented strains were washed and subcultured at a concentration of 1×107 colony forming units (CFU)/mL in M9 medium supplemented with 0.1% casamino acids and 40 mM sodium nitrate (NaNO3). The latter was included as a major source of nitrogen. The bacterial culture was incubated statically at 37°C under aerobic or anaerobic atmospheres for comparison purposes. Anaerobic growth studies were performed within an anaerobic jar using an Oxoid AnaeroGen sachet (Thermo Scientific, MA, USA). B. pseudomallei growth at various time points was observed by the optical density at 600 nm (OD600). The measurement and detection of viable bacteria was observed by plating serial dilutions of bacterial samples on LB agar and colony counts.
Oxoid anaerogen sachet
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Oxoid AnaeroGen Sachet is a reagent used to create an anaerobic (oxygen-free) environment within an airtight container. It is designed to rapidly remove oxygen and maintain low oxygen levels, supporting the growth of anaerobic microorganisms.
Automatically generated - may contain errors
5 protocols using oxoid anaerogen sachet
1
Anaerobic Growth Analysis of Burkholderia pseudomallei
2
Metabolic Labeling of Bacterial Glycans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial cells were grown in rich liquid media supplemented with 0.5 mM Ac4GlcNAz, Ac4GalNAz, or the azide-free control Ac4GlcNAc for 2–4 days, depending on the doubling rate of the organism. H. pylori was metabolically labeled for 4 days under microaerophilic conditions (14% CO2, 37 °C), C. jejuni was metabolically labeled for 3 days under microaerophilic conditions, and B. fragilis was metabolically labeled for 2 days under anaerobic conditions (created by a Thermo Scientific Oxoid AnaeroGen Sachet in an airtight container; 37 °C) using 0.1 mM Ac4GalNAz according to previous literature.69 (link) Cells were then harvested, washed with phosphate buffered saline (PBS), and whole cells were analyzed by flow cytometry or lysed for subsequent western blot analysis, as described below.
3
Fecal Sample Collection and Calprotectin Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The entire bowel movement was collected within 4 h of defecation and was transferred to the laboratory on ice under anaerobic conditions (Oxoid AnaeroGen Sachet; ThermoFisher Scientific)[9 (link)]. Samples were homogenised and aliquots were stored in – 80 °C. Faecal calprotectin was measured using the CALP0170 kit (CalproLab, Lysaker, Norway) [17 (link)].
4
Preserving Anaerobic Fecal Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fecal samples were collected from two adult donors with IBD; both were female and aged 20-25 years. Immediately upon collecting the sample, an Oxoid™ AnaeroGen™ sachet (Thermo Fisher Scientific, MA, USA) was placed into the sample box which removed all oxygen. Next, anaerobic phosphate-buffered saline was added to the sample and a fecal slurry was prepared by homogenization in a stomacher. Large particles were removed by brief centrifugation. An equal volume of cryoprotectant solution was added to the fecal slurry in an anaerobic workstation and the slurry was snap-frozen in liquid nitrogen and stored at -80 °C until used in the study. Ethical approval of the University Hospital Ghent (reference no. B670201836585) was obtained for the collection of the fecal samples, and written informed consent was obtained prior to sample collection.
5
Transcriptome Analysis of A. pleuropneumoniae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiments were conducted with the A. pleuropneumoniae serotype 5 reference strain L20. The strain was grown in Brain Heart Infusion (BHI) supplemented with nicotinamide adenine dinucleotide (NAD—10 µg ml−1) at 37°C until early stationary phase (8 h) under aerobic (5% CO2) and anaerobic conditions (anaerobic jar with Oxoid Anaerogen Sachet, Thermo Scientific). Total RNA extraction was performed by cell disruption using the Lysing Matrix B tubes (MP Biomedicals), followed by the procedures of the miRNeasy Mini Kit (QIAGEN), according to the manufacturer's instructions. After extraction, the concentration and purity of the RNA was determined by Nanodrop and 2100 Bioanalyzer (Agilent Technologies). The resulting total RNA (purity 1.8–1.9, A260/A280 ratio) was treated with one unit of RQ1 DNAse (Promega) per µg of nucleic acid, and incubated for 60 min at 37°C.
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!