Computational protocol: Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates

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Protocol publication

[…] Pneumococcal biofilms were also visualized directly using CLSM and the BacLight Bacterial Viability Kit. Biofilms were grown for 1, 3, and 6 days and stained according to the manufacturer's directions, rinsed, immersed in Hank's balanced salt solution (HBSS) with Ca+2 and Mg+2 and immediately examined with a Leica DM RXE microscope attached to a TCS SP2 AOBS confocal system (Leica Microsystems, Exton, PA) using a 63× water immersion lens and the sequential scanning mode. Images were analyzed using the Leica LCS software, COMSTAT [] and Imaris Software (Bitplane; St. Paul, MN). [...] To combine the information from the 3 biofilm assays (crystal violet, 6 day CFU/cm2 count and COMSTAT data) to give an overall assessment of the degree of biofilm formation of each pneumococcal strain, we used a simple normalized ranking system based on a "biofilm forming index" (BFI) for each assay. The Shapiro-Wilk normality test (The R Project for Statistical Computing: R statistical software version 2.6.2 (2008 02 08) freely available at ) was used to determine whether the data sets needed to be transformed to achieve a normal distribution (P > 0.05). Of the data sets for the 6 pneumococcal strains, 4 of 6 were normal for the CV assay and viable cell count, and 1 of 6 was normal for COMSTAT. Log10 transformation of the data resulted in all data sets conforming to a normal distribution (P > 0.05). The BFI for each of the individual assays for each of the six strains was determined using: (1)BFIOD or BFICFU or BFICOMSTAT = (Log(10) Strain value - Log(10) lowest value)/(Log(10) highest value - Log(10) lowest value) Thus each strain was assigned a value ranging from "I", for the least biofilm, to "1" for the most biofilm.The data from the 3 assays was combined to find the overall combined (BFI) from: (2)BFI = (BFIOD + BFICFU + BFICOMSTAT)/3 […]

Pipeline specifications

Software tools Comstat, Imaris, The R Project for Statistical Computing
Application Laser scanning microscopy
Organisms Streptococcus pneumoniae, Homo sapiens, Mycoplasma pneumoniae
Diseases Otitis Media, Respiratory Tract Infections
Chemicals Azithromycin