In this method, the target bacteria were first incorporated into the agar medium with a final density of 103 CFU/mL. After solidification of the medium, cellulose acetate membranes (Sartorius Stedim Biotech GmbH, Göttingen, Germany) were applied onto the agar surface. Different inocula of the probiotics were pipetted onto the membranes; then, the plates were incubated anaerobically at 33 °C for up to 7 days. Tests with S. salivarius M18: The pathogens S. aureus and C. acnes were incorporated into BHI and WC agar, respectively. This probiotic was applied onto the membrane in suspension of 2 × 106 CFU/mL and tenfold concentrated. Tests with S. salivarius K12: 108 CFU/mL were applied onto the membrane. In all the tests, the liquid culture medium was used as control. The volume of each probiotic suspension or culture medium (control) applied onto the membrane spots was 20 µL.
Cellulose acetate membrane
Manufactured by Sartorius
Sourced in Germany
The Cellulose Acetate Membrane is a laboratory filter membrane made of cellulose acetate material. It is designed for filtration and separation applications in various research and analytical settings.
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Lab products found in correlation
Bioxtra, by Merck Group (2 mentions)
Vibra cell 400w, by Sonics (1 mentions)
Vivaflow 200, by Sartorius (1 mentions)
Tewameter tm300, by Courage + Khazaka (1 mentions)
Auriga crossbeam microscope, by Zeiss (1 mentions)
N n dimethyldodecylamine n oxide, by Merck Group (1 mentions)
Bioreagent, by Merck Group (1 mentions)
Azocasein, by Merck Group (1 mentions)
Mab1574, by Merck Group (1 mentions)
Oligomer specific a11 polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
Harmine, by Merck Group (1 mentions)
Fitc conjugated anti mouse antibody, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Simplicity uv apparatus, by Merck Group (1 mentions)
Ammonium acetate nh4oac, by Merck Group (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Pelargonidin, by Merck Group (1 mentions)
Cyanidin, by Merck Group (1 mentions)
25 protocols using cellulose acetate membrane
1
Evaluating Probiotic Inhibition of Pathogens
2
In Vitro Release Kinetics of EN and TA from MSNs-Loaded Scaffold
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The in vitro release of EN and TA from the MSNs-loaded scaffold was carried out by using the Franz diffusion cell method [45 (link)]. The drug-release process was performed in freshly prepared phosphate buffer at pH 7.4 and 37 °C. Cellulose acetate membranes (Sartorius®, Darmstadt, Germany, 0.45 μm average pore size) were soaked in distilled water for 30 min before loading the SC formulation and then fixed on top of the receptor chambers of the cells after adding the release media in the chamber. The donor compartment was tightly clamped on the membrane fixed on top of the receiver compartment. About 150 mg of the SC formulation was loaded in the donor chamber already containing 10 mL of the release medium at a stirring speed of 50 rpm. At predetermined time intervals, about 0.5 mL of the sample from the receptor chambers of the Franz diffusion cell were taken and 0.5 mL of freshly prepared media was added in that chamber. After the appropriate dilution, the amount of EN and TA in the obtained samples was determined using a previously determined HPLC technique.
3
Antimicrobial Liposomal Biofilm Inhibition
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The liposomal formulations were next tested for their inhibitory effects on biofilm viability, as previously described (Graziano et al., 2015 (link)) (39). Cellulose acetate membranes (25 mm diameter, 0.2 µM pores) (Sartorius Stedim GmbH, Guxhagen, Hessen, Germany) were used as substrates for S. aureus biofilm formation. The membranes were placed in 6-well plates containing BHI medium supplemented with 1% glucose and bacterial suspension (approximately 1 × 106 CFU/ml in each well). The plates were incubated at 37°C for 24 h. Then the membranes were transferred to new plates containing fresh BHI plus 1% glucose, and biofilms were treated with the formulations at 1 × MIC, 10 × MIC, and 50 × MIC for 24 h. Treated biofilm-coated membranes were gently washed (three times) through immersion into 5 ml of 0.9% NaCl. Then, the membranes were transferred to other tubes containing freshly 5 ml of 0.9% NaCl and then sonicated with six pulses of 9.9 s, 5 s time-interval, 5% amplitude (VibraCell 400W, Sonics & Materials Inc., Newtown, CT, USA) and vortexed at 3,800 rpm for 30 s. Ten-microliter aliquots were collected from each tube, serially diluted, and plated for CFUs onto TSA. The plates were incubated at 37°C for 24 h.
4
In Vitro Release of Loteprednol Etabonate Ointments
5
Panomycocin Production and Antifungal Evaluation
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Panomycocin-producing strain W. anomalus NCYC 434 was cultured in 5 L of YEPD at 20°C until the stationary phase in a 7 L bioreactor (BioBundle; Applikon, Delft, the Netherlands) at an impeller speed of 450 rpm. The pH of the culture medium was maintained at 4.5 by automatic addition of 2 M KOH (ADI 1030 Bio-Controller; Applikon). The culture chamber was fed with sterilized air by a mass-flow control unit with a flow rate of 2 L/min to ensure dissolved oxygen concentration above 30% measured by Applikon oxygen probe. The culture medium was clarified by centrifugation (5,000 rpm for 10 min at 4°C) and filtered through 0.45 and 0.2 µm cellulose acetate membranes (Sartorius AG, Göttingen, Germany). Panomycocin in the cell-free medium was concentrated by ultrafiltration using 30 and 5 kDa cut-off ultrafilters (Vivaflow200; Sartorius AG).
The crude panomycocin was purified and analyzed as described previously.12 (link) The biological activity of the pure panomycocin was tested on vaginal C. albicans and C. glabrata isolates using the microdilution broth method according to the Clinical and Laboratory Standards Institute reference document M27-A325 with modifications (assay medium pH was adjusted to 4.5 and the assay was performed at 30°C).
The crude panomycocin was purified and analyzed as described previously.12 (link) The biological activity of the pure panomycocin was tested on vaginal C. albicans and C. glabrata isolates using the microdilution broth method according to the Clinical and Laboratory Standards Institute reference document M27-A325 with modifications (assay medium pH was adjusted to 4.5 and the assay was performed at 30°C).
6
Evaluating Water Evaporation of SMEDDS Films
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The ability to retain water evaporation in vitro was evaluated using the Tewameter®TM 300 (Courage&Khazaka GmbH, Cologne, Germany). Franz diffusion cells were filled with purified water and covered with cellulose acetate membranes (pore size 0.45 µm, Sartorius Stedim Biotech GmbH, Göttingen, Germany) previously soaked in purified water for 24 h, which provided physical support for the tested films. First, the basal water evaporation through the artificial membrane was measured. Then, the dried film (2 cm × 2 cm) was placed on the membrane and the water evaporation was again determined. After the film was moistened, the measured values began to increase. The measurement was stopped when the reading reached the basal value. The measurements were performed for films prepared from SMEDDS hydrogels with or without embedded BDP. The experiment was performed in triplicate. Data are expressed as averages.
7
In Vitro Release of Loteprednol Etabonate Ointments
8
Characterization of Polystyrene Nanoparticles
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Sodium dodecyl sulfate (SDS, BioXtra ≥99.0%, Sigma-Aldrich 151213), sodium chloride (NaCl, AnalaR ≥99.5%, VWR Chemicals 7647145), dodecyldimethylamine oxide (DDAO, 30 wt% in water, Sigma-Aldrich 1643205), 80 nm (nominal) polystyrene latex particles (PS NP, 3.55 × 1012 mL -1 , Agar Scientific AGS130), 100 nm (nominal) PS NP (10 wt%, Sigma-Aldrich 43 302), 200 nm (nominal) PS NP (10 wt%, Sigma-Aldrich 69057), and LUDOX AS-40 colloidal silica particles (40 wt%, Sigma-Aldrich 7631869) were used as received. Aqueous solutions were prepared with ultra-pure 18.2 kΩ cm water, and filtered through a 0.2 μm cellulose acetate membrane (Sartorius), except 200 nm PS NP solutions, where a 0.5 μm pore size was used. Polystyrene (MW = 100 kg mol -1 , BDH Chemicals 29789) solutions were prepared in toluene (AnalaR ≥99.5%, VWR Chemicals 108883) and filtered (0.2 μm PTFE, Pall Corporation). All solutions were homogenised on a roller mixer and allowed to rest for 24 h.
The average size and size distribution of the PS NP (with nominal diameters of 80, 100, and 200 nm) were measured by scanning electron microscopy (SEM). The suspensions were deposited on stubs and left to evaporate at 5 °C for 5 days before being coated in ∼10 nm of gold. These were imaged at 5 kV to prevent damage to the sample with a typical working distance of 3.5-5 mm using a Zeiss Auriga Crossbeam microscope.
The average size and size distribution of the PS NP (with nominal diameters of 80, 100, and 200 nm) were measured by scanning electron microscopy (SEM). The suspensions were deposited on stubs and left to evaporate at 5 °C for 5 days before being coated in ∼10 nm of gold. These were imaged at 5 kV to prevent damage to the sample with a typical working distance of 3.5-5 mm using a Zeiss Auriga Crossbeam microscope.
9
Aggregated Protein Examination via Dot Blot
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Cells were lysed in 2% SDS solution and diluted to a final volume of 200 μL. Protein samples were then loaded to a 48-well Bio-Dot microfiltration apparatus and filtered with cellulose acetate membrane (pore size = 0.2 μm; Sartorius Stedim Biotech, Goettingen, Germany). The membrane was then blocked with 5% non-fat milk prior to primary antibody incubation (anti-myc, same condition used for western blotting) for aggregated protein examination.
10
In Vitro Drug Release Profiling
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Franz diffusion cells (LGA, Berkeley, CA) with an exposed surface area of 0.75 cm2 and a receiver compartment volume of 4.5 mL were used to determine the release profile of pure CPS and CPS-LN. The receptor compartment was filled with a water–ethanol solution (50:50) to create the sink condition, since CPS has a limited solubility in buffer. The solution has been stirred at 500 rpm and thermostated at 32 ± 1 °C during all of the experiments [24 (link)]. The testing samples (350 µL) were placed in the donor cell maintaining a complete and intimate contact with the surface of a cellulose acetate membrane (0.2 µm pore size, 25 mm diameter, Sartorius; Göttingen, Germany). Each experiment was run in duplicate for 24 h. At predetermined intervals, samples (200 µL) of receiving solution were withdrawn and replaced with fresh solution. The samples were analyzed for drug content by High Performance Lipid Chromatography (HPLC), as described below.
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