U. urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3) were acquired from the American Tissue Culture Collection (ATCC; Uu8: ATCC 27618, Up3: ATCC 27815). As described previously [31 (link)], ureaplasma isolates were cultured in a liquid in-house medium (“broth”) containing 82% autoclaved pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA), 10% heat-inactivated horse serum, 1% urea, and 0.002% phenol red (all: Sigma-Aldrich, St. Louis, CA, USA). The medium was adjusted to pH 6.5 after passage through a 0.2-μm filter membrane (Sartorius, Goettingen, Germany). Using the ToxinSensor™ Endotoxin Detection System (GenScript, Piscataway, NJ, USA), we could verify an endotoxin level < 0.06 EU/ml broth. Serial 10-fold dilutions of the ureaplasma cultures were incubated for 18–20 h to obtain titers of 1 × 109–1 × 1010 color-changing units (CCU)/ml of viable organisms.
0.2 μm membrane filter
Manufactured by Sartorius
Sourced in Germany
The 0.2 μm membrane filter is a lab equipment product designed for filtration applications. It has a pore size of 0.2 micrometers, which allows it to remove small particles and microorganisms from liquids.
Automatically generated - may contain errors
Lab products found in correlation
Horse serum, by Merck Group (2 mentions)
Phenol red, by Merck Group (2 mentions)
Autoclaved pleuropneumonia like organism medium, by BD (2 mentions)
Urea, by Merck Group (2 mentions)
Toxinsensor endotoxin detection system, by GenScript (2 mentions)
Uv 1800, by Shimadzu (1 mentions)
Wr g7115a, by Agilent Technologies (1 mentions)
Copley cis 8000, by Copley Scientific (1 mentions)
6 protocols using 0.2 μm membrane filter
1
Culturing Ureaplasma Serovars for Analysis
2
Culturing and Titrating Ureaplasma Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U. urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3) were attained from the American Tissue Culture Collection (ATCC; Uu8 ATCC 27618, Up3 ATCC 27815). ureaplasma isolates were cultured in a liquid in-house medium (referred to as “broth”) containing 82% autoclaved pleuropneumonia-like organism medium (Becton, Dickinson & Company, Franklin Lakes, NJ, USA), 10% heat-inactivated horse serum (v/v), 1% urea (w/v), and 0.002% phenol red (w/v) (all from Sigma-Aldrich, St. Louis, CA, USA). After passage through a 0.2-μm filter membrane (Sartorius, Goettingen, Germany), the medium was adjusted to pH 6.5. The ToxinSensor™ Endotoxin Detection System (GenScript, Piscataway, NJ, USA) verified an endotoxin level < 0.06 EU/ml broth. As described previously [17 (link)], serial tenfold dilutions of the ureaplasma cultures were incubated for 18–20 h to obtain titers of 1 × 109–1 × 1010 color-changing units (CCU)/ml of viable bacteria. Corresponding amounts of ureaplasma DNA were verified and amounted to 5 × 107–6 × 108 copy numbers/ml (Institute of Medical Microbiology and Hospital Hygiene, Duesseldorf, Germany). Simultaneous culture on selective agar plates (medco Diagnostika GmbH, Ottobrunn, Germany) confirmed bacterial viability.
3
Etching and Staining Protocol for Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, the sections and slices were etched with 10% acetic acid for 5 min. The sample was then washed with distilled water and dried. In the next step, a drop of toluidine staining solution (0.5 mg/mL) was applied to the section with a syringe (B. Braun AG, Melsungen, Germany) for 15 min. Sterilization filtration with a 0.2 μm membrane filter (Sartorius, Göttingen, Germany) was used to ensure that no contamination or small dye particles were present in the staining solution. The staining solution was then briefly washed with water. DePeX was used to cover the slides.
4
Evaluating Antibacterial Potential of Probiotic CFS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibacterial activity of CFS on the growth of S. Typhimurium, L. monocytogenes, E. coli, and S. aureus was evaluated by following a previously described method [19 ] with slight modifications. The potential probiotic strains were centrifuged (10,000 rpm for 10 min 4°C), and CFS was collected and sterilized through 0.2 μm membrane filter (Sartorius, Minisart, Germany). The CFS (10 mL) of each LAB strain was mixed with 100 mL of cell culture of each pathogenic strain and incubated at 37°C for 24 h. The optical density (OD600 nm) was measured every 2 h through a Uv-Vis spectrophotometer (UV-1800, Shimadzu, Japan). The cell cultures of S. Typhimurium, L. monocytogenes, E. coli, and S. aureus without the addition of CFS were used as control.
5
Biogenic Amines Quantification in Cheese
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The eight biogenic amines (TRP, PHE, PUT, CAD, HIS, TYR, SPD, and SPM) were detected and quantified using high-performance liquid chromatography equipped with a diode array detector (WR G7115 A, Agilent Technologies, Santa Clara, CA, USA) at 254 nm, as described in Eerola et al. [38 (link)]. The extraction of BA’s was carried by homogenizing each cheese sample (5 g) with 20 mL of 0.4 M perchloric acid followed by centrifugation (3000 rpm, 10 min, 4 °C). A second extraction was performed again with 20 mL 0.4 M perchloric acid and both extracts were pooled together and the total volume was made up to 50 mL with 0.4 M perchloric acid. The extract was further filtered through Whatman paper No.1 (0.2 μm).
Before derivatization, the extract/standard solutions (1 mL) were initially alkalinized by adding 200 μL 2M NaOH and 300 μL saturated sodium bicarbonate. To this, 2 mL of dansyl chloride (1% w/v in acetone) was added and allowed to react in a dark room at 40 °C, 45 min with intermittent stirring. For removal of the residual dansyl chloride, 100 μL of 25% ammonium hydroxide was added to the mixture and kept in room temperature for 30 min. The sample volume was made up to 5 mL with acetonitrile and centrifuged at 3000 rpm for 10 min at 4 °C. The final supernatant was filtered using 0.2 μm membrane filter (Sartorius, Goettingen, Germany) and stored at −25 °C until HPLC analysis.
Before derivatization, the extract/standard solutions (1 mL) were initially alkalinized by adding 200 μL 2M NaOH and 300 μL saturated sodium bicarbonate. To this, 2 mL of dansyl chloride (1% w/v in acetone) was added and allowed to react in a dark room at 40 °C, 45 min with intermittent stirring. For removal of the residual dansyl chloride, 100 μL of 25% ammonium hydroxide was added to the mixture and kept in room temperature for 30 min. The sample volume was made up to 5 mL with acetonitrile and centrifuged at 3000 rpm for 10 min at 4 °C. The final supernatant was filtered using 0.2 μm membrane filter (Sartorius, Goettingen, Germany) and stored at −25 °C until HPLC analysis.
6
Dissolution Study of Paracetamol in Simulated GI Fluids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dissolution studies were carried out on a Copley CIS 8000 dissolution bath (Copley Scientific, UK) with a rotation speed of 50 rpm according to the BP Apparatus I basket method. The tests were performed in 900 mL of freshly prepared simulated gastric fluid (SGF, 2 g L -1 NaCl, 3.2 g L -1 pepsin, 80 mL L -1 1M HCl (BP 2011)) pH 1.2 for 2 hours at 37 ± 0.5 °C, followed by 6 hours in freshly prepared simulated intestinal fluid (SIF, 6.8 g L -1 KH2HPO4, 10 g L -1 pancreatin, 77 mL L -1 0.2 M NaOH (BP 2011)) pH 6.8. Samples of 10 mL were withdrawn at predetermined times, filtered through a 0.2 μm membrane filter (Sartorius Stedim Biotech GmbH, Germany) and replaced with an equivalent volume of fresh medium. The paracetamol concentration was determined spectrophotometrically (Lambda XLS UV/VIS, Perkin-Elmer, USA) at a wavelength of the pancreatic digestive enzymes were centrifuged at 13,000 rpm for 30 minutes prior to filtration and UV measurements. The dissolution studies were carried out in triplicate and the average drug release ± SD was calculated.
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!