MONITORING OF FORMATION OF BIOFILMS BY OPPORTUNISTIC AND PATHOGENIC BACTERIA

2019 ◽  
Vol 1 (2) ◽  
pp. 174-182
Author(s):  
A. B. Kononenko ◽  
◽  
D. A. Bannikova ◽  
S. V. Britova ◽  
I. B. Pavlova ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Filter Paper ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Room Temperature ◽  
Distilled Water ◽  
Pathogenic Microorganisms ◽  
Crystal Violet Solution ◽  
Taxonomic Groups ◽  
Peptone Broth

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%.

METHODOLOGICAL APPROACHES TO THE STUDY OF FORMATION OF BIOFILMS BY CONDITIONALLY PATHOGENIC AND PATHOGENIC ENTEROSBACTERIES

2018 ◽  
Vol 1 (1) ◽  
pp. 50-58
Author(s):  
A. B. Kononenko ◽  
◽  
I. B. Pavlova ◽  
D. A. Bannikova ◽  
S. V. Britova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nutrient Medium ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Osmic Acid ◽  
Liquid Nutrient Medium ◽  
Petri Dishes ◽  
Peptone Broth ◽  
Scanning Electron

To study the process of biofilm formation, microorganisms were cultured in 96-well plates, on meat-peptone broth, stained with a 0,1% solution of crystalline violet for 10...15 minutes, after which the unbound dye was washed off. The quantitative accounting of the bound dye was carried out by spectrophotometry at a wavelength of 490 nm. The technique for making bacterial preparations for light and scanning electron microscopy on dodged glasses immersed in Petri dishes with a liquid nutrient medium is proposed. A suspension of bacteria at a concentration of 105 m.k/ml in a volume of 5 ml was shaken on Vortex apparatus and introduced into Petri dishes with 20 ml of meat-peptone broth. Sterile non-greased cover glasses were placed on sterile object glasses and immersed in a liquid nutrient medium in Petri dishes. The material was incubated for 18...24 hours at 37 °C. Then the cover slips were removed with tweezers and some of them were stained with 1% aqueous solution of methylene blue (for light microscopy), and some were placed in Petri dishes with bottomed filters (for electron microscopy). The latter, in order to preserve natural architectonics, were fixed in vivo by pairs of 25% glutaraldehyde for 3...5 hours. Vapors of 2...4% osmic acid solution were used for 2...3-minutes to contrast the preparations. After treatment with vapors of osmic acid, biofilms with included bacteria acquired yellowish or brown color. The obtained preparations after dehydration with propylene oxide vapors and spraying with gold ions were examined in a scanning electron microscope (SEM). The technique allows us to study the phases of development of biofilms and obtain objective data on the morphology of populations of pathogenic and conditionally pathogenic bacteria without disturbing natural architectonics. It is shown that the intensity of biofilm formation by pathogenic microorganisms, such as salmonella, Yersinia, Staphylococcus aureus was slightly higher than that of non-pathogenic: Escherichia, Proteus, Citrobacter, Enterobacter.

scholarly journals Biofilm production by Listeria monocytogenes strains: detection with colorimetric analysis

2020 ◽  
Vol 30 (Supplement_5) ◽  
Author(s):  
P Centorame ◽  
L Iacone ◽  
R Salini ◽  
A Ciarulli ◽  
F Guidi ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Colorimetric Analysis ◽  
Solution Versus ◽  
Crystal Violet Solution ◽  
Biofilm Production ◽  
Staining Methods

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.

scholarly journals Effect of Lactobacillus salivarius supernatant against growth and biofilm formation of some pathogenic microorganisms

2016 ◽  
Vol 13 (2) ◽  
pp. 247-252
Author(s):  
Baghdad Science Journal
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Inhibition Zone ◽  
Inhibitory Effect ◽  
Pathogenic Microorganisms ◽  
Lactobacillus Salivarius ◽  
Inhibition Effect ◽  
Probiotic Lactobacillus ◽  
Diffusion Assay ◽  
Klebsiella Spp

The Inhbititory effect of cocentrated and non-cocentrated supernatant of the probiotic Lactobacillus salivarius against growth of some potential pathogenic microorganisms which included Pseudomonas eruginosa, Klebsiella spp, Escherichia coli and Candida albicans. The results were diffusion assay revealed that concentrated and non-concentrated supernatant had inhitory effect against pathogenic bacteria with inhibition zone renged between 13-17mm while inhibition effect of concentrated supernatant against C.albicans was inhibition zone 8mm. On the other hand, the effect of these suprnatant against biofilm formation of the tested microorganisms was studied. The result showed that the concentrated supernatant had inhibitory effect on biofilm formation for all tested microorganisms with percentage (28-29)% against tested bacteria and (23)% against C.albicans.

scholarly journals Features of formation of Yersinia enterocolitica biofilms

2019 ◽  
Vol 12 (1) ◽  
pp. 136-140 ◽  
Author(s):  
E. Lenchenko ◽  
D. Lozovoy ◽  
A. Strizhakov ◽  
Yu Vatnikov ◽  
V. Byakhova ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Optical Density ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Osmic Acid ◽  
Persistent Infections ◽  
Sample Density ◽  
Dark Color ◽  
Livestock Products ◽  
Peptone Broth

Aim: The work aimed to study the morphology of colonies and their comparison by features of the formation of Yersinia enterocolitica biofilms. Materials and Methods: Bacteria were cultured on a Yersinia Selective Agar medium ("CIN-agar") at 28°C for 24 h. The microorganisms were grown in meat-peptone broth with 1.0% glucose to measure the absolute values of the optical density of the culture. The optical density of the liquid was determined in a microplate photometric analyzer Immunochem-2100 (HTI, USA) at a wavelength of 490 nm. For the study of biofilms, the specimens were fixed for 3-5 h in pairs of 25.0% solution of glutaraldehyde (according to DV), and pairs of a 1.0% aqueous solution of osmic acid (OSO4) were used for contrasting for 2-3 min. The specimens were examined with stereoscopic microscopy "BIOMED MS-1 Stereo" (Russia) and scanning electron microscope "TM 3030 plus" (Holland). Results: With stereoscopic microscopy of the colonies of Y. enterocolitica, the S-forms had an elevated intensely colored center, radial striation along the periphery, smooth edges, d ≤ 1.0 mm. R-form colonies had a dark color and a dry surface, were tuberous and had a dense center with a peripheral ridge, rugged edges, d ≥ 1.5 mm. The optical density of the Y. enterocolitica S-form showed that this type of microorganism belongs to the moderate producers of biofilms since the optical density of the sample (density of the sample - Ds) exceeded the optical density of control (density of the control - Dc) by 3 times. In Y. enterocolitica R-form (D ≤ 0.197) weakly produced biofilms, the optical density of the sample exceeded the optical density of the control by <2 times. Conclusion: The ability to form biofilms, the variability of phenotypic features, and the multiplicity of virulence factors of bacteria significantly reduce the effectiveness of diagnostic studies. The development of accelerated methods of detection and differentiation of the virulent properties of pathogenic bacteria will allow scientifically to substantiate and develop a set of measures aimed at preventing animal diseases and obtaining safe livestock products to prevent human diseases. Thus, we need to pay attention to which forms of colonies do Y. enterocolitica form on solid nutrient media: S- or R-forms. Through this study, we know that bacteria-forming S-shaped colonies are more capable of forming biofilms than R-forms. It means that they are more pathogenic and can cause persistent infections due to adhesion and biofilm formation.

Scanning Electron Microscopy-cuticular Lesions on the Roundworm Ascaris Suum

1970 ◽  
Vol 28 ◽  
pp. 114-115
Author(s):  
P. A. Madden ◽  
W. R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Freeze Drying ◽  
Room Temperature ◽  
Secondary Electron ◽  
Distilled Water ◽  
Freeze Dried ◽  
Silver Paint ◽  
To Come ◽  
Scanning Electron

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.

SEM Observations on the Germination of Euonymous Americanus

1985 ◽  
Vol 43 ◽  
pp. 508-509
Author(s):  
D.R. Hill ◽  
J.R. McCurry ◽  
L.P. Elliott ◽  
G. Howard
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Filter Paper ◽  
Room Temperature ◽  
Food Source ◽  
Environmental Chamber ◽  
Dormant Stage ◽  
Scanning Electron

Germination of Euonymous americanus in the laboratory has previously been unsuccessful. Ability to germinate Euonymous americanus. commonly known as the american strawberry bush, is important in that it represents a valuable food source for the white-tailed deer. Utilizing the knowledge that its seeds spend a period of time in the rumin fluid of deer during their dormant stage, we were successful in initiating germination. After a three month drying period, the seeds were placed in 25 ml of buffered rumin fluid, pH 8 at 40°C for 48 hrs anaerobically. They were then allowed to dry at room temperature for 24 hrs, placed on moistened filter paper and enclosed within an environmental chamber. Approximately four weeks later germination was detected and verified by scanning electron microscopy; light microscopy provided inadequate resolution. An important point to note in this procedure is that scarification, which was thought to be vital for germination, proved to be unnecessary for successful germination to occur. It is believed that germination was propagated by the secretion of enzymes or prescence of acids produced by microorganisms found in the rumin fluid since sterilized rumin failed to bring about germination.

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria

2020 ◽  
Vol 21 (4) ◽  
pp. 270-286 ◽  
Author(s):  
Fazlurrahman Khan ◽  
Dung T.N. Pham ◽  
Sandra F. Oloketuyi ◽  
Young-Mog Kim
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Extracellular Polymeric Substances ◽  
Quorum Quenching ◽  
Resistance Mechanisms ◽  
Bacterial Biofilm ◽  
Bacterial Cells ◽  
High Concentration ◽  
Biofilm Inhibition ◽  
Abiotic Surfaces

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics. Therefore, the resistance of bacterial biofilm-forming cells could be increased up to 1000 times than the planktonic cells, hence requiring a significantly high concentration of antibiotics for treatment. Methods: Up to the present, several methodologies employing antibiotics as an anti-biofilm, antivirulence or quorum quenching agent have been developed for biofilm inhibition and eradication of a pre-formed mature biofilm. Results: Among the anti-biofilm strategies being tested, the sub-minimal inhibitory concentration of several antibiotics either alone or in combination has been shown to inhibit biofilm formation and down-regulate the production of virulence factors. The combinatorial strategies include (1) combination of multiple antibiotics, (2) combination of antibiotics with non-antibiotic agents and (3) loading of antibiotics onto a carrier. Conclusion: The present review paper describes the role of several antibiotics as biofilm inhibitors and also the alternative strategies adopted for applications in eradicating and inhibiting the formation of biofilm by pathogenic bacteria.

Cholesterol in Serum and Lipoprotein Fractions

1956 ◽  
Vol 2 (3) ◽  
pp. 145-159 ◽  
Author(s):  
Joseph T Anderson ◽  
Ancel Keys
Keyword(s):  
Human Serum ◽  
Total Cholesterol ◽  
Filter Paper ◽  
Room Temperature ◽  
Paper Electrophoresis ◽  
Cholesterol Content ◽  
Intraindividual Variability ◽  
Detailed Data ◽  
The Stability ◽  
Source Of Error

Abstract 1. Methods are described for the separation, by paper electrophoresis and by cold ethanol, of α- and β-lipoproteins in 0.1 ml. of serum, with subsequent analysis of cholesterol in the separated portions. 2. It is shown that both methods of separation yield separated fractions containing substantially the same amounts of cholesterol. 3. Detailed data are given on the errors of measurement for total cholesterol and for cholesterol in the separated lipoprotein fractions. 4. Studies are reported on the stability of cholesterol in stored serum and on paper electrophoresis strips. It is shown that simple drying on filter paper causes no change in cholesterol content and yields a product that is stable for many weeks at ordinary room temperature. 5. The sources of variability in human serum cholesterol values are examined and it is shown that spontaneous intraindividual variability is a much greater source of error than the errors of measurement with these methods.

scholarly journals Extract from phyllosphere bacteria with antibiofilm and quorum quenching activity to control several fish pathogenic bacteria

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Olivia Nathalia ◽  
Diana Elizabeth Waturangi
Keyword(s):  
Streptococcus Agalactiae ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Vibrio Harveyi ◽  
Quorum Quenching ◽  
Indicator Bacteria ◽  
Antibiofilm Activity ◽  
Fish Pathogen ◽  
Phyllosphere Bacteria

Abstract Objective The objective of this research were to screen quorum quenching activity compound from phyllosphere bacteria as well as antibiofilm activity against several fish pathogen bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. Results We found eight phyllosphere bacteria isolates with potential quorum quenching activity to inhibit Chromobacterium violaceum as indicator bacteria. Crude extracts (20 mg/mL) showed various antibiofilm activity against fish pathogenic bacteria used in this study. Isolate JB 17B showed the highest activity to inhibit biofilm formation of A. hydrophila and V. harveyi, meanwhile isolate JB 3B showed the highest activity to inhibit biofilm of S. agalactiae. From destruction assay, isolate JB 8F showed the highest activity to disrupt biofilm of A. hydrophila isolate JB 20B showed the highest activity to disrupt biofilm of V. harveyi, isolate JB 17B also showed the highest activity to disrupt biofilm of S. agalactiae.

scholarly journals Green Biosynthesis of Silver Nanoparticles Using Leaf Extract of Carissa carandas L. and Their Antioxidant and Antimicrobial Activity against Human Pathogenic Bacteria

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 299
Author(s):  
Reetika Singh ◽  
Christophe Hano ◽  
Gopal Nath ◽  
Bechan Sharma
Keyword(s):  
Silver Nanoparticles ◽  
Pathogenic Bacteria ◽  
Leaf Extract ◽  
Room Temperature ◽  
Xrd Analysis ◽  
Antibacterial Activities ◽  
Effect Of Temperature ◽  
Human Pathogenic Bacteria ◽  
Carissa Carandas ◽  
Antioxidant And Antimicrobial Activity

Carissa carandas L. is traditionally used as antibacterial medicine and accumulates many antioxidant phytochemicals. Here, we expand this traditional usage with the green biosynthesis of silver nanoparticles (AgNPs) achieved using a Carissa carandas L. leaf extract as a reducing and capping agent. The green synthesis of AgNPs reaction was carried out using 1mM silver nitrate and leaf extract. The effect of temperature on the synthesis of AgNPs was examined using room temperature (25 °C) and 60 °C. The silver nanoparticles were formed in one hour by stirring at room temperature. In this case, a yellowish brown colour was developed. The successful formation of silver nanoparticles was confirmed by UV–Vis, Fourier transform infrared (FT-IR) and X-ray diffraction (XRD) analysis. The characteristic peaks of the UV-vis spectrum and XRD confirmed the synthesis of AgNPs. The biosynthesised AgNPs showed potential antioxidant activity through DPPH assay. These AgNPs also exhibited potential antibacterial activity against human pathogenic bacteria. The results were compared with the antioxidant and antibacterial activities of the plant extract, and clearly suggest that the green biosynthesized AgNPs can constitute an effective antioxidant and antibacterial agent.

Sign in / Sign up

Export Citation Format

Share Document