A higher surface area i.e. 324 mm2 of the cover slip allowed enhanced biofilm formation by approximately ALK inhibitor ~1 log on the cover slip in comparison to the microtiter plate (surface area = 32 mm2). Estimation of bacterial numbers in untreated biofilms at the air–liquid interface showed an increase, with a peak on 5th day (9.09 ± 0.15 Log10 CFU/ml) of

incubation, after which the biofilm bacterial counts decreased progressively (Figure 4). In biofilm treated with both phage and cobalt salt a mean log reduction of ~5 and ~ 2 logs was observed in comparison to the groups treated with phage or iron antagonizing molecule alone. The growth and treatment efficacy of biofilm formed at the air–liquid interface was ~1-2 logs better in comparison to biofilms grown

in microtiter plates therefore for further experiments biofilm were grown on glass coverslips at the air–liquid interface. On 3rd and 7th day, the bacterial viability in the treated/untreated biofilms was assessed by fluorescent microscopy. Figure 4 Kinetics of biofilm formation (on cover slips) by K. pneumoniae B5055 grown in minimal media (M9) supplemented with 10  μM FeCl 3 and treated with 500  μM cobalt salt (CoSO 4 ) and bacteriophage (KPO1K2) alone as well as in combination. **p < 0.005 [(10 μM FeCl3 +500 μM CoSO4 + Ø(KPO1K2) vs selleck chemical 10 μM FeCl3/10 μM FeCl3+ 500 μM CoSO4/10 μM FeCl3+ Ø(KPO1K2)], *p < 0.05 [(10 μM FeCl3 +500 μM CoSO4 + Ø(KPO1K2) vs 10 μM FeCl3+ 500 μM CoSO4], #p < 0.005 [(10 μM FeCl3 +500 μM CoSO4 + Ø(KPO1K2) vs 10 μM FeCl3/10 μM FeCl3+ Ø(KPO1K2)]. Assessment of fluorescent

stained biofilms on coverslip The LIVE/DEAD BacLight Bacterial Viability Kit has a mixture of SYTO® 9 green-flourescence nucleic acid stain (for intact live bacteria) and propidium iodide red flourescence nucleic acid stain (for membrane damaged or killed bacteria). Two types of cells were seen, green cells represented the intact or viable cells, red stained cells represented damaged or killed bacterial cells after treatment while yellow regions below showed the presence of both red and green coloured cells. As shown in [Figure 5(a)] a 3rd day biofilm consisting of sparsely populated green coloured rods formed in the iron supplemented media in comparison to 7th day old thicker and densely populated green coloured biofilm [Figure 5(a´)]. On the other hand, biofilm grown in additional cobalt supplemented media showed a lesser confluent growth of green colored cells along with some yellow and red cells on 3rd day [Figure 5(b)] as well as on 7th day [Figure 5(b´)] in comparison to biofilms grown in iron supplemented media.