Dear Kulandaivelu,

For Rhodamine B:

Fluorescent staining and confocal laser scanning microscopic observation

Following the secondary streptococcal adhesion phase, specimens were stained with calcein-AM (Molecular Probes/Invitrogen, Eugene, OR) for live cells and rhodamine-B (Wako Pure Chemical Industries Ltd., Osaka, Japan) for the whole structure. Calcein-AM diffuses passively into the cytoplasm, where it is converted into green-fluorescent calcein via native esterases. Calcein fluorescence is retained in live cells until plasma membrane permeability is compromised [14, 16, 17]. Rhodamine-B is a counterstaining dye, revealing the extent of biomass, independent of its activity [37]. Biofilm specimens were stained with 10 μg ml−1 calcein-AM in phosphate buffer for 2 h at 37°C, followed by staining with 5 μg ml−1 rhodamine-B for 5 min. A biofilm structure without the secondary adhesion phase served as a control.

Biofilms were imaged on a confocal laser scanning microscope (CLSM; FV-300, Olympus, Tokyo, Japan) using Ar 488-nm and He-Ne 543-nm lasers. Filters were set to 510–530 nm for the detection of calcein-AM and above 610 nm for rhodamine-B. A ×60 water-immersion objective lens was used for direct observation. Stacks of fluorescent images were collected in the z-dimension, and three-dimensional reconstruction was carried out using Imaris software (Bitplane AG, Zurich, Switzerland).

Concentration:  5 μg ml−1

 Solvent: phosphate buffer for 2 h at 37°C

To view the full paper, please see attached file.

For Alcian Blue:

Biofilm and capsule determinations were done post-growth by staining

washed (3×, PBS, pH 7.0) then dried honeycomb

plates with crystal violet (biofilm; Abs595 nm) or Alcian

blue (capsule; 1% w/v DDI), respectively [10].

To view the full paper, please see attached file.

Hoping this will be helpful,

Rafik

Thanks for your information and paper. Really useful information