Novel Method for Quantitative Estimation of Biofilms

Abstract

Biofilm protects bacteria from stress and hostile environment. Crystal violet (CV) assay is the most popular method for biofilm determination adopted by different laboratories so far. However, biofilm layer formed at the liquid-air interphase known as pellicle is extremely sensitive to its washing and staining steps. Early phase biofilms are also prone to damage by the latter steps. In bacteria like mycobacteria, biofilm formation occurs largely at the liquid-air interphase which is susceptible to loss. In the proposed protocol, loss of such biofilm layer was prevented. In place of inverting and discarding the media which can lead to the loss of the aerobic biofilm layer in CV assay, media was removed from the formed biofilm with the help of a syringe and biofilm layer was allowed to dry. The staining and washing steps were avoided, and an organic solvent-tetrahydrofuran (THF) was deployed to dissolve the biofilm, and the absorbance was recorded at 595 nm. The protocol was tested for biofilm estimation of E. coli, B. subtilis and M. smegmatis, and compared with the traditional CV assays. Isoniazid drug molecule, a known inhibitor of M. smegmatis biofilm, was tested and its inhibitory effects were quantified by the proposed protocol. For ease in referring, this method has been described as the Syal method for biofilm quantification. This new method was found to be useful for the estimation of early phase biofilm and aerobic biofilm layer formed at the liquid-air interphase. The biofilms formed by all three tested bacteria-B. subtilis, E. coli and M. smegmatis, were precisely quantified.

Genetic Basis of High-Pressure Tolerance of a Vibrio parahaemolyticus Mutant and Its Pathogenicity

“…To test the virulence of the C4 and N11 strains in vitro , cells were spread onto the surface of Wagatsuma agar containing 5% defibrillated sheep whole blood and 7% NaCl and incubated at 37°C for 14 h. Biofilm formation was measured using the method of Syal (2017) . The two strains were cultured in APW medium supplemented with 3% NaCl at 37°C, and their biofilms were stained with 0.1% (w/v) crystal violet.…”

Anti-Biofilm Potential of Nanonized Eugenol againstPseudomonas aeruginosa

“…In principle, the intensity of blue color of film is directly proportional to biofilm biomass. In this study, biofilms were first washed with PBS to remove planktonic bacteria from well-plate and were then stained with 0.1% (w/v) aqueous solution of CV [SRL] for 30 min [(25)-(26)]. The stained biofilms were further washed with PBS to remove excess stain and were subsequently air dried for microscopic [Invitrogen, Evos FL auto] study.…”

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M. smegmatis

“…Biofilm inhibition assay : M. smegmatis mc 2 155 was grown in Sauton's medium supplemented with glucose (2 %) and Tween 80 (0.05 %) for biofilm production. The biofilm assay and quantification were performed as described previously . The method of biofilm formation of the mycobacterium was at the interface between the solid surface and solution .…”