Concept: Growth medium
To compare the blood agar (BA), sabouraud dextrose agar (SDA) and chocolate agar (CA) for the isolation of fungi in patients with mycotic keratitis. Corneal Scrapings of 229 patients with clinically diagnosed microbial keratitis were inoculated on BA, SDA, CA. The culture media were evaluated for the rate and time taken for the fungal growth. Seventy six of 229 patients had fungal keratitis. Fungus grew on BA in 60/76(78.9 %), on SDA in 76/76 (100 %), on CA in 40/76(52.6 %) patients. The fungi which grew on BA (60/76) also grown on SDA at the same time. The colony morphologies of different fungi were better on SDA than BA/CA. Among the different culture media, SDA is essential for the isolation fungi in patients with mycotic keratitis.
The uptake of calcium phosphate nanoparticles (diameter 120 nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: Wortmannin, LY294002, nocodazole, chlorpromazine, and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localisation of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localised in lysosomes after 3 h of incubation.
To investigate the dynamic dissolution of quantum dots (QDs) in cell culture media, in this study we constructed an online automatic analytical system comprising a sequential in-tube solid phase extraction (SPE) device and an inductively coupled plasma (ICP) mass spectrometer. By means of selectively extracting QDs and cadmium ions (Cd(2+)) onto the interior surface of the polytetrafluoroethylene (PTFE) tube, this novel SPE device could be used to determine the degree of QD dissolution through a simple adjustment of sample acidity. To the best of our knowledge, this study is the first to exploit PTFE tubing as a selective SPE adsorbent for the online chemical differentiation of QDs and Cd(2+) ions with the goal of monitoring the phenomenon of QD dissolution in complicated biological matrices. We confirmed the analytical reliability of this system through comparison of the measured Cd-to-QD ratios to the expected values. When analyzing QDs and Cd(2+) ions at picomolar levels, a temporal resolution of 8 min was required to load sufficient amounts of the analytes to meet the sensitivity requirements of the ICP mass spectrometer. To demonstrate the practicability of this developed method, we measured the dynamic variations in the Cd-to-QD705 ratio in the presence of ascorbate as a physiological stimulant to generate reactive oxygen species in cell culture media and trigger the dissolution of QDs; our results suggest that the ascorbate-induced QD dissolution was dependent on the time, treatment concentration, and nature of the biomolecule.
Oil-in-water (o/w) emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The PHMB concentration in the aqueous phase was estimated by filtration centrifugation experiments. In parallel, PHMB concentration was assessed utilizing cytotoxicity assays (neutral red) on cultured murine fibroblasts (L929 cells) and tests of bactericidal efficacy on either Pseudomonas aeruginosa or Staphylococcus aureus. Biological tests were performed in cell culture medium. Filtration centrifugation experiments demonstrated much higher aqueous PHMB concentrations than did the assays for biologically effective PHMB. Therefore, biological test systems should preferably be used to verify effective PHMB concentrations. Tests of microbicidal efficacy in which the same 0.05% PHMB o/w emulsion was re-used 8 times revealed a drug delivery system activated by the presence of test bacteria.
Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities.
Like most cell types, hepatocytes constantly produce extracellular vesicles (EVs) such as exosomes and microvesicles that are released into the circulation to transport signaling molecules and cellular waste. Circulating EVs are being vigorously explored as biomarkers of diseases and toxicities, including drug induced liver injury (DILI). Emerging data suggest that a) blood-borne EVs contain liver-specific mRNAs and microRNAs (miRNAs), b) the levels can be remarkably elevated in response to DILI, and c) the increases correlate well with classical measures of liver damage. The expression profile of mRNAs in EVs and the compartmentalization of miRNAs within EVs or other blood fractions were found to be indicative of the offending drug involved in DILI, thus providing more informative assessment of liver injury than using alanine aminotransferase (ALT) alone. EVs in the urine and cell culture medium were also found to contain proteins or mRNAs that were indicative of DILI. However, major improvements in EV isolation methods are needed for the discovery, evaluation and quantification of possible DILI biomarkers in circulating EVs.
ABSTRACT We investigated the application capabilities of a laser optical sensor, BARDOT (bacterial rapid detection using optical scatter technology) to generate differentiating scatter patterns for the 20 most frequently reported serovars of Salmonella enterica. Initially, the study tested the classification ability of BARDOT by using six Salmonella serovars grown on brain heart infusion, brilliant green, xylose lysine deoxycholate, and xylose lysine tergitol 4 (XLT4) agar plates. Highly accurate discrimination (95.9%) was obtained by using scatter signatures collected from colonies grown on XLT4. Further verification used a total of 36 serovars (the top 20 plus 16) comprising 123 strains with classification precision levels of 88 to 100%. The similarities between the optical phenotypes of strains analyzed by BARDOT were in general agreement with the genotypes analyzed by pulsed-field gel electrophoresis (PFGE). BARDOT was evaluated for the real-time detection and identification of Salmonella colonies grown from inoculated (1.2 × 10(2) CFU/30 g) peanut butter, chicken breast, and spinach or from naturally contaminated meat. After a sequential enrichment in buffered peptone water and modified Rappaport Vassiliadis broth for 4 h each, followed by growth on XLT4 (~16 h), BARDOT detected S. Typhimurium with 84% accuracy in 24 h, returning results comparable to those of the USDA Food Safety and Inspection Service method, which requires ~72 h. BARDOT also detected Salmonella (90 to 100% accuracy) in the presence of background microbiota from naturally contaminated meat, verified by 16S rRNA sequencing and PFGE. Prolonged residence (28 days) of Salmonella in peanut butter did not affect the bacterial ability to form colonies with consistent optical phenotypes. This study shows BARDOT’s potential for nondestructive and high-throughput detection of Salmonella in food samples. IMPORTANCE High-throughput screening of food products for pathogens would have a significant impact on the reduction of food-borne hazards. A laser optical sensor was developed to screen pathogen colonies on an agar plate instantly without damaging the colonies; this method aids in early pathogen detection by the classical microbiological culture-based method. Here we demonstrate that this sensor was able to detect the 36 Salmonella serovars tested, including the top 20 serovars, and to identify isolates of the top 8 Salmonella serovars. Furthermore, it can detect Salmonella in food samples in the presence of background microbiota in 24 h, whereas the standard USDA Food Safety and Inspection Service method requires about 72 h.
Fifty-six foods and food ingredients were analyzed for populations of naturally occurring yeasts and molds using Petrifilm rapid yeast and mold (RYM) count plates, Petrifilm yeast and mold (YM) count plates, dichloran rose bengal chloramphenicol (DRBC) agar plates, acidified potato dextrose agar (APDA) plates, and dichloran 18% glycerol (DG18) agar plates. Colonies were counted after incubating plates for 48, 72, and 120 h at 25°C. Of 56 foods in which either yeasts or molds were detected on at least one medium incubated for 120 h, neither yeasts nor molds were detected in 55.4, 73.2, 21.4, 19.6, and 71.4% of foods plated on the five respective media and incubated for 48 h; 10.7, 14.3, 3.6, 1.8, and 19.6% of foods were negative after 72 h, and 3.6, 1.8, 0, 0, and 0% of foods were negative after 120 h. Considering all enumeration media, correlation coefficients were 0.03 to 0.97 at 48 h of incubation; these values increased to 0.75 to 0.99 at 120 h. Coefficients of variation for total yeasts and molds were as high as 30.0, 30.8, and 27.2% at 48, 72, and 120 h, respectively. The general order of performance was DRBC = APDA > RYM Petrifilm > YM Petrifilm ≥ DG18 when plates were incubated for 48 h, DRBC > APDA > RYM Petrifilm > YM Petrifilm ≥ DG18 when plates were incubated for 72 h, and DRBC > APDA > RYM Petrifilm > YM Petrifilm > DG18 when plates were incubated for 120 h. Differences in performance among media are attributed to the diversity of yeasts and molds likely to be present in test foods and differences in nutrient, pH, and water activity requirements for resuscitation of stressed cells and colony development.
Glioma patients often suffer from epileptic seizures because of the tumor’s impact on the brain physiology. Using the rat glioma cell line C6 as a model system, we performed long-term live recordings of the electrical activity of glioma populations in an ultrasensitive detection method. The transducer exploits large-area electrodes that maximize double-layer capacitance, thus increasing the sensitivity. This strategy allowed us to record glioma electrical activity. We show that although glioma cells are nonelectrogenic, they display a remarkable electrical burst activity in time. The low-frequency current noise after cell adhesion is dominated by the flow of Na(+) ions through voltage-gated ion channels. However, after an incubation period of many hours, the current noise markedly increased. This electric bursting phenomenon was not associated with apoptosis because the cells were viable and proliferative during the period of increased electric activity. We detected a rapid cell culture medium acidification accompanying this event. By using specific inhibitors, we showed that the electrical bursting activity was prompted by extracellular pH changes, which enhanced Na(+) ion flux through the psalmotoxin 1-sensitive acid-sensing ion channels. Our model of pH-triggered bursting was unambiguously supported by deliberate, external acidification of the cell culture medium. This unexpected, acidosis-driven electrical activity is likely to directly perturb, in vivo, the functionality of the healthy neuronal network in the vicinity of the tumor bulk and may contribute to seizures in glioma patients.
Bacteriophages are viruses that infect bacteria, and they are found everywhere their bacterial hosts are present, including the human body. To explore the presence of phages in clinical samples, we assessed 65 clinical samples (blood, ascitic fluid, urine, cerebrospinal fluid, and serum). Infectious tailed phages were detected in >45% of ascitic fluid and urine samples. Three examples of phage interference with bacterial isolation were observed. Phages prevented the confluent bacterial growth required for an antibiogram assay when the inoculum was taken from an agar plate containing lysis plaques, but not when taken from a single colony in a phage-free area. In addition, bacteria were isolated directly from ascitic fluid, but not after liquid enrichment culture of the same samples, since phage propagation lysed the bacteria. Lastly, Gram-negative bacilli observed in a urine sample did not grow on agar plates due to the high densities of infectious phages in the sample.