Abstract
Background
In literature, there are no standardized laboratory methods to detect formed biomass by colorimetric analysis. The purpose of this study was to compare three staining methods and two different wavelengths for determination of biofilm formation of Listeria monocytogenes (Lm) strains.
Methods
Three strains of Lm isolated from different origin were tested using 96 well polistirene plates at 12 °C and 30 °C, after incubation the wells were subjected to washing, detaching and staining with crystal violet (CV) at 0.2% and 2% (Panreac EU) in 95% ethanol and with Gram's crystal violet solution (Merck KGaA, Germany). The absorbance at 492nm and 540nm wavelengths was read using a spectrophotometer (SIRIO S, Seac, Firenze, Italia).
Results
The strains incubated at 12 °C displayed production of biofilm when stained with CV 2% and with Gram's crystal violet solution, both at 492 and 540 nm (with better evidence at 540 nm). If CV 0.2% was used to stain and reading at both optical densities there was evidence of weak or no biofilm production.
At 30 °C, the biofilm production was displayed at both temperature and with all the stains. For all the strains and for all the conditions tested, the absorbance was greater but not proportional using the Gram's crystal violet solution, versus the CV 0,2% and CV 2%, and absorbance was higher at 540nm versus at 492nm.
Conclusions
Results confirmed the lack of reproducibility of each of the method used to detect and quantify the biomass produced during a biofilm formation test in vitro and the absence of ratio between the different results obtained using different CV concentration and wavelengths for reading.
Key messages
Biofilm production at 12 °C could not be adequately detected staining the wells with CV 0,2%. Absorbance could be influenced by the solvent in the stain used (ethanol, methanol or phenol or mixtures). To obtain data for assessment of biomass formation, being the method characterized by poor reproducibility, the laboratory should use at least the same stain and wavelength.
Determination of Electric Charge and rH by Means of the Staining Methods in Prothallia, Especially in Spermatozoids of Ferns
