Biofilm production by Listeria monocytogenes strains: detection with colorimetric analysis

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.

Coatings Based on Two-Dimensionally Ordered Linear Chain Carbon for Protection of Titanium Implants from Microbial Colonization

Purpose of the study – to evaluate the antibacterial activity and biological compatibility of alloy coatings based on two-dimensionally ordered linear chain carbon (TDOLCC).Materials and Methods. Coatings based on TDOLCC were synthesized using alloying additions like nitrogen (TDOLCC+N) and silver (TDOLCC+Ag) on the surfaces of titanium plates and polystyrene plates by the ion-stimulated carbon condensation in a vacuum. The authors examined the superficial bactericidal activity of the coatings and its resistance to mechanical effects. Coated plates were evaluated in respect of rate of microbial biofilms formation by clinical isolates with multiple and extreme antibiotic resistance. Specimens were colored with crystal violet solution to visualize the biofilms. Cytotoxic effect of coatings was evaluated in respect of primary culture of fibroblasts and keratinocyte cell line HaCaT.Results. The authors observed pronounced superficial bactericidal effect of TDOLCC+Ag coating in respect of microorganisms of several taxonomic groups independently of their resistance to antibacterial drugs. TDOLCC+Ag coating proved capable to completely prevent microbial biofilm formation by antibiotic resistant clinical isolates of S. aureus and P. aeruginosa. Silvercontaining coating demonstrated mechanical resistance and preservation of close to baseline level of superficial bactericidal activity even after lengthy abrasion treatment. TDOLCC based coatings did not cause any cytotoxic effects. Structure of monolayers formed in cavities coated by TDOLCC+N and TDOLCC+Ag was indistinguishable from the monolayers in cavities of control plates.

MONITORING OF FORMATION OF BIOFILMS BY OPPORTUNISTIC AND PATHOGENIC BACTERIA

The aim of the work is monitoring the formation of biofilms by opportunistic and pathogenic microorganisms. Materials and methods. The cultures of the genera Salmonella, Escherichia, Yersinia, Proteus, Citrobacter, Enterobacter, Prtovidenzia, Morganella, Klebsiella, Cronobacter, Pseudomonas, Bacillus, Staphylococcus were used in the work. The studied microorganisms were cultured in polystyrene 96-well plates. For this purpose, a daily culture of microorganisms was introduced into the wells with meat-peptone broth, having previously established a concentration of 104 mc / ml, and incubated for 24...96 hours at temperature of 37 °C. Then the medium with plankton cells was removed from the wells. 200 μl of filtered 0,1% crystal violet solution was poured into the wells of the plate and the plates were kept for 10...15 min at room temperature. Then dye was removed from the wells. Unbound dye was thoroughly washed with saline or distilled water. The plates were turned over on filter paper and dried. The presence and density of biomatrix (biofilm) was determined visually by the intensity of staining the surfaces of plates. Then, for the extraction of paint from the film, 200 μl of 96% ethanol was added to the wells and the optical density was measured on KFK-3KM spectrophotometer at a wavelength of 590 nm. Results of research. The results of the experiments allowed us to assert that within 48 hours of cultivation microorganisms form a mature biofilm, which can serve as a model for studying the process of biofilm formation. Biofilm of microorganisms of different taxonomic groups differs in density. In addition, even bacteria belonging to the same genus, under the same conditions, can form a biofilm, the density of which differs by 30...60%.

Adsorption Properties of the Macro/Nano-Clay in Crystal Violet Solution

In this study, different appearances, structures and morphologies of macro-clay G105-Polymer grade montmorillonite (PMMT) and nanoclay Lithium magnesium silicate (LMSH) were compared and analyzed, which were used in different conditions of non-adsorption and adsorption of crystal violent (CV). Adsorption kinetics and properties of the macro/nanoclay in CV solution were studied, and adsorption time, dye concentration and environmental pH were the main factors on the inspection in this work. The adsorption mechanisms of the macro-/nanoclay on CV were investigated. The results showed that adsorption actions of PMMT and LMSH on CV were some similar, such as time-dependent, linear growth of adsorbed CV amount with increasing CV concentration, adsorption capacity of macro-/nanoclay declined firstly and then increased in 10mg/L CV solution along with increasing pH value. But the adsorption differences in two systems of PMMT-CV and LMSH-CV were obvious. Adsorption behavior of LMSH-CV system showed two adsorption phenomena. Under neutral conditions, adsorbed CV amount in 0.5g LMSH was 1.69 times that of PMMT. Adsorption amount of LMSH was more significant to be affected by dye concentration and pH conditions. The research achievements further showed that adsorption mechanism was the root cause of the following difference. The adsorption of PMMT-CV relied mainly on iron-exchange effect of layers replaceable cations and surface adsorbed ions with CV cations, while the significant adsorption of LMSH-CV was mainly due to the electrostatic interaction from layers negative charge distribution and CV cationic properties in the solution, partly surface ions exchanges also existed in the adsorption process of LMSH-CV.

Properties of PbO2 Electrode Modified by AEO-7 for Electrocatalytic Degradation of Crystal Violet

In order to improve the performance of PbO2electrode, A kind of surface active agent – AEO-7 was added into nitric acid bath and modified PbO2electrode was prepared on stainless steel substrate by anodic oxidation. The modified variations on surface morphology of the electrodes were examined by Scanning Electron Microscopy(SEM); The polarization curves, Tafel curves and Cyclic voltammograms of modified and unmodified PbO2electrodes were compared in Crystal violet solution; The electrocatalytic activities were inspected by electrocatalytic degradation of crystal violet. It was found that the modified electrode showed higher oxygen evolution potential, better corrosion resistance, and more excellent electrocatalytic activity than the unmodified electrode.