In rest of the wells, spent medium was replaced with fresh media and plate was reincubated at 37°C overnight. This procedure was repeated until 7th day of experiment. Bacteriophage treatment of biofilm grown in minimal media supplemented with cobalt (CoSO4) and iron (FeCl3) salts To selleck determine the efficacy of bacteriophage alone as well as in combination with the iron anatagonizing molecule in treating the biofilms

of K. pneumoniae B5055, 100 μl of bacterial culture Selleck CA4P was inoculated in different wells of microtiter plate containing 100 μl of minimal media supplemented with 10 μM FeCl3 and/or 500 μM of Cobalt sulphate (CoSO4) and incubated at 37°C overnight. Unadhered bacteria were removed from two set of wells supplemented with 10 μM FeCl3 and learn more 10 μM FeCl3+ 500 μM CoSO4 on different days. Thereafter, these biofilms were exposed to bacteriophage (KPO1K2/NDP)

at multiplicity of infection [m.o.i: ratio of infectious agent (e.g. phage or virus) to infection target (e.g. bacterial cell)] of 1 for 3 h followed by washing with 0.85% NaCl and enumeration of viable cells from 8 wells. A set of two wells containing biofilm grown in unsupplemented, iron supplemented minimal media alone and with the addition of CoSO4 served as controls and were also processed as mentioned previously on each day. In rest of the wells, spent medium was replaced with fresh media and plate was re-incubated at 37°C overnight. This procedure was repeated until 7th day of experiment. Development

of biofilm on glass coverslip To determine the effectivness of treatment with various combinations qualitatively, biofilms were grown on glass coverslips (18 mm × 18 mm; 0.08–0.12 mm; Corning Glass, USA) at air–liquid interface by the Tipbox batch culture method of Hughes et al. [7] as standardized in our laboratory by Verma et al. [18]. Tip-box mounted coverslips and minimal M9 media supplemented with 10 μM FeCl3 with or without 500 μM CoSO4 were sterilized separately. 100 μl bacterial culture (108 CFU/ ml) was added to the media which was then poured into the tip box. The whole very set-up was incubated at 37°C. Spent growth medium in the culture boxes was replaced every 24 h. On 3rd and 7th day 16 coverslips (4 corresponding to each group) were removed, rinsed thoroughly with sterile 0.85% NaCl and 8 were incubated with bacteriophage (MOI = 1) for 3 hours. After treatment, biofilm laden coverslip was washed with sterile sodium phosphate buffer (pH 7.2), stained for 15 min in dark with the components of LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen), washed with 0.85% NaCl and observed under oil immersion 100× objective, with a B2A filter set fitted in a fluorescent microscope (Nikon). The images were captured using an image acquisition system by Nikon. The untreated cover-slips were also processed in a similar way as treated ones.