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1) Product Images from "Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism"
Article Title: Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism
Journal: PLoS ONE
Figure Legend Snippet: PittGG biofilm formation is stimulated by ampicillin in a narrow concentration range. Crystal violet assay of 1-day PittGG biofilms, formed in the presence of increasing amounts of ampicillin up 300 ng/mL, showed a stimulation of biofilm formation at 170 ng/mL concentration.
Techniques Used: Concentration Assay, Crystal Violet Assay
Figure Legend Snippet: Sub-inhibitory concentrations of ampicillin result in an increase in dead NTHi bacteria in newly formed biofilms. Confocal laser scanning microscopy (cLSM) of biofilms formed by NTHi strains 2019 and PittGG in the absence (− amp) and presence (+ amp) of sub-inhibitory concentrations of ampicillin were stained using the LIVE/DEAD viability assay. 2019 were exposed to 90 ng/mL ampicillin and PittGG to 170 ng/mL ampicillin. Live bacteria were colored green and dead bacteria red. From above, biofilms formed in the absence of ampicillin were mostly green (A C), indicating living (or intact) NTHi bacteria. Clumps of bacteria in both biofilms stained red (A C) indicating the presence of dead, or structurally compromised bacteria. Biofilms formed by 2019 (A) contained fewer aggregates of red bacteria than did PittGG (C) contained more. In the presence of ampicillin (B D) the bacteria in the biofilms were mostly red, indicating a large number of dead bacteria. Green-stained bacteria were still present in both biofilms but appeared more aggregated in the presence of antibiotic (B D). Large amounts of aggregated red bacteria were present in both biofilms, with the aggregates being larger in the PittGG biofilm (Fig. D). Z-stack projections of the biofilms (E–H) showed that all the biofilms were denser at the base of the biofilm, whether exposed to ampicillin or not. In the absence of ampicillin, 2019 (E) and PittGG (G) biofilms comprised green, or intact bacteria. In the presence of ampicillin the biofilm contained mostly structurally compromised bacterial cells, which were colored red or yellow. The 2019 (F) and PittGG (H) biofilms formed in the presence of ampicillin were higher than the comparable biofilms formed without exposure to ampicillin (E G). All images; scale bar (G) = 20 µm.
Techniques Used: Confocal Laser Scanning Microscopy, Staining, Viability Assay
Figure Legend Snippet: A sub-inhibitory concentration of ampicillin changes the composition of forming NTHi biofilms. Two strains of NTHi, 2019 and PittGG, exposed to sub-inhibitory concentrations (90 ng/mL: 2019; 170 ng/mL: PittGG) of ampicillin for 24 hr, showed increases in biofilm biomass as measured by dry weight (A. Biomass), and percent protein content (B. Protein Content). In contrast, the presence of the antibiotic during biofilm formation resulted in a decrease in viable (or culturable) bacteria (C. Viable Bacteria). Strain PittGG showed the most noticeable changes in dry weight, protein content and numbers of viable bacteria resulting from exposure to 170 ng/mL ampicillin (t-test p =
Techniques Used: Concentration Assay, T-Test
Figure Legend Snippet: Biofilm formation is stimulated by beta-lactam antibiotics. Crystal violet assays of 1-day NTHi biofilms formed in the presence of amoxicillin or amoxicillin. Strains 2019, 9274 and PittEE reacted to inhibitory concentrations of antibiotic (grey vertical bars, Biomass OD 600 ) by producing more crystal violet stainable biofilm (line graph). This stimulatory effect was different for each bacterial strain. PittGG did not react with amoxicillin and produced an ambiguous reaction to ampicillin. PittAA and PittII were not affected by amoxicillin or ampicillin in the concentration ranges studied. Cefuroxime (0–950 ng/mL range) showed a biofilm-stimulatory effect on all the NTHi strains under study. Each strain exhibited biofilm stimulation in different concentrations of antibiotic. Strain 2019 was maximally stimulated at 100 ng/mL cefuroxime, PittGG and PittII at 220 ng/mL, 9274, PittAA at 300 ng/mL and PittEE at 525 ng/mL.
Techniques Used: Produced, Concentration Assay
Figure Legend Snippet: Sub-inhibitory concentrations of ampicillin change the ultrastructure of newly formed NTHi biofilms. Scanning electron microscopy (SEM) images comparing NTHi biofilms formed in the absence and presence of ampicillin on Thermanox coverslips. A–C: SEM of 2019 biofilms. A) At low power the 2019 biofilm is seen as a mat covering the Thermanox substrate. Bar = 500 µm. B) higher magnification shows the biofilm to be composed of partitions forming empty spaces, or cells, covered with a film of amorphous material (arrow). Bar = 10 µm. C) the partitions within the biofilm are composed primarily of bacterial cells aggregated into flat sheets. Bar = 5 µm. D–F: SEM of 2019 biofilms formed in 90 ng/mL ampicillin. D) The 2019 biofilm formed with ampicillin covers the substrate. Bar = 500 µm. E) The biofilm is composed of partitions around empty spaces with a flat sheet of amorphous material (arrow) over the top. Bar = 50 µm F) The biofilm partitions are composed of aggregated bacteria embedded in sheets of amorphous material. Bar = 5 µm. G–I: SEM of PittGG biofilms. G) The PittGG bacteria form a biofilm over the Thermanox surface. Bar = 500 µm. H) The biofilm is composed of bacterial cells aggregated into poorly defined partitions and covered with a layer of amorphous material (arrow). Bar = 10 µm. I) The biofilm is composed of bacterial cells aggregated into widely spaced strands, and empty space. Bar = 5 µm. J–L: SEM of PittGG biofilms formed in 170 ng/mL ampicillin. J) In the presence of ampicillin the PittGG bacteria form a biofilm comprised of a thick mat, which appears to be strongly attached to itself but less well attached to the substrate. Bar = 500 µm. K) The biofilm mats appear to be composed mostly of amorphous material, a layer of which covers the biofilm (arrow), and arranged into tightly-packed thin partitions. Bar = 50 µm. L) the biofilm is composed of amorphous material formed into thin partitions. The few bacteria detected were embedded in the thin partitions. Bar = 5 µm.
Techniques Used: Electron Microscopy
Figure Legend Snippet: NTHi biofilms protect against a lethal dose of cefuroxime. Biofilms of NTHi strains 2019 and PittGG were formed overnight in the absence of antibiotic (Untreated), or in the presence of 170 ng/mL ampicillin (AMP), 230 ng/mL amoxicillin (AMOX) or 170 ng/mL cefuroxime (CEF). The amounts of antibiotic were chosen for their ability to stimulate biofilm formation in these two NTHi strains. After washing, the biofilms were exposed to 10 µg/mL of cefuroxime for a further 24 hr. The percentages of viable bacteria present in each biofilm are tabulated here. Not shown: planktonic NTHi bacteria are killed in the presence of 10 µg/mL cefuroxime. Although all of the formed biofilms (in the presence or absence of antibiotic) protected against cefuroxime, the amoxicillin-stimulated biofilm was able to protect bacteria from the lethal effects of the cefuroxime. p-values are documented in Table S3 .
Figure Legend Snippet: mRNA analysis of PittGG biofilms. A. Principal component analysis of microarray gene expression of the PittGG biofilm bacteria (NTHi) exposed to 170 ng/mL ampicillin. The major variations (PC1, PC2, and PC3) are visualized in 3-dimensions. The three PCs together accounted for about 85% of the variations present in the entire data set. A distinguishable grouping difference can be seen between ampicillin treated (in blue) and non-treated (in red) samples. B. Hierarchical cluster of significantly differentially expressed genes in the PittGG biofilm bacteria (NTHi) treated with 170 ng/mL ampicillin. Heatmap visualization of 59 significantly regulated genes by ampicillin treatment. Results from the 3 replicate experiments (R1, R2 and R3) of ampicillin-treated and untreated biofilms are presented. A hierarchical clustering was analysed on the probe sets representing the 59 genes including 8 up and 51 down regulated gene transcripts with filter criteria of at least 1.5 folds change plus P≤0.05 and FDR less than 0.05. Rows: samples; columns: genes. The up-regulated genes relating to carbohydrate metabolism are indicated.
Techniques Used: Microarray, Expressing
Figure Legend Snippet: Ultrastructural visualization of glycogen in PittGG biofilms. Resin-embedded thin sections of PittGG biofilms formed in the presence or absence of ampicillin (+/− Am) and treated with a glycogen stain or a no stain control (+/− gly). A–D Biofilms with ampicillin, sections stained for glycogen (periodic acid and sodium chlorite). The glycogen stain reacted with an extracellular granular substance (arrows) that was associated with biofilm bacteria. Exposure to antibiotic resulted in a bacterial size increase with some bacteria (A–C). Extracellular areas that were not in close proximity to bacteria cells (asterisk) did not contain the extracellular granular substance (B–D). E. Biofilm with antibiotic but only treated with sodium chlorite did not reveal extracellular granular substance. Large bacterial cells were present. F. Biofilm with no antibiotic, sections stained for glycogen. No extracellular granular substance was detected and bacterial cells had a normal diameter (approx. 500 nm). Some lysed cells were detected. G. Biofilm with no antibiotic, treated with sodium chlorite (no periodic acid). Bacterial cells appear normal with a few dying cells present (arrowhead). Scale bars = 500 nm.
Techniques Used: Staining
Figure Legend Snippet: Biofilm formation is variable between NTHi strains. Biofilm quantification using crystal violet assay showed the variability in biofilm formation between the NTHi strains in this study. Relative Biofilm Quantity was measured as OD 600 of crystal violet in DMSO; higher absorbance indicated more biofilm production. The PittAA and PittEE strains showed the least amount of biofilm attachment to the growth surface while PittII showed the most. The other three strains (2019, 9274 and PittGG) formed intermediate amounts of biofilm material (t-test analysis in Table S2 ).
Techniques Used: Crystal Violet Assay, T-Test