Microscopic examination of attached pneumococci was done in 6-well plates added with 1 ml of medium and incubation in anaerobiosis. The use of 6-well plates allowed microscopic examination of cells at the bottom of wells using a normal light microscope (not inverted), since they permit insertion of the microscope objective within the wells. Microtiter biofilm methodology: model based on enriched stationary phase inoculum Cells grown to early stationary phase were inoculated 1:10 into TSB or BHI medium, either undiluted, diluted Selinexor ic50 1:2, 1:3 or 1:4, with or without supplementation with CSP (concentrations as above) [24]. In this model biofilms were grown in 96 well plates for quantification only or in 6-well plates for
microscopic examination. Plates were incubated at 37°C in a CO2-enriched atmosphere. To permit duration for more than 24 hours 50% of spent medium was exchanged twice daily with fresh prewarmed medium. AT the termination of the experiment wells were washed three times, and the biofilm was detected by crystal violet staining. Staining was done after desiccation at 50°C and staining with 1% crystal violet for 30 min followed by microscopic examination. For quantification stain was detached with 70% ethanol solution for 30 min and quantitative analysis was performed
after transfer of the ethanol to a new mictotiter plate by measuring crystal violet absorbance at 590 nm. Continuous flow biofilm model The continuous flow biofilm model system used in this work had been developed by CDC [17]. In the original work [17], the CDC bioreactor was connected to a FTIR laser spectrometer holding an attenuated total reflectance (ATR) flow cell. Selleckchem Dactolisib The current study was performed with the
CDC bioreactor system alone. The bioreactor contained eight removal rods, each of which holds three removable polycarbonate coupons. Each coupon has a diameter of 1.3 cm which provides the surface for biofilm growth. Following assembly of the bioreactor, 400 ml of BHI broth supplemented with casein [0.5%] and yeast extract [0.2%] was added to the bioreactor and sterilized in an autoclave. Then, the Anidulafungin (LY303366) bioreactor was placed in a Class II bioSafety Hood, and inoculated with 9 ml of a monoculture of the designated S. pneumoniae strain. Immediately, the inoculated bioreactor was placed in a water bath heater that maintained a temperature of approximately 35°C, and connected to a pre-sterilized carboy that contained 4 litre of 10% BHI plus supplements. During each experiment, the environment of the bioreactor was purged continuously with a filter-sterilized compressed gas mixture (5% oxygen, 10% Carbon dioxide, 85% nitrogen). Immediately after inoculation, the reactor was operated in batch mode (closed system) for 12 hours, during which growth was agitated by a magnetic stirrer (Barnstead, Inc., Dubuque, IA) at 60 rpm. Continuous flow (open system) was initiated by pumping 10% BHI broth with a Masterflex peristaltic pump (Cole Parmer, Niles, Ill) at a flow rate of 0.
Related posts:
- , 2007a) In all cases, mutations were created by conjugal transf
- In accordance for the Certificate of Examination, the written con
- Chemical compound library empty plasmids were transfected into MCF-7 and MDAMB 231 cells
- Human BMSCs have been purchased from Cambrex and initially grown within a Dulbec
- Dovitinib in numerous myeloma following stem cell transplant