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ELife Jun 2020Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and...
Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.
Topics: Bacteria; Bacteriological Techniques; Drug Resistance, Bacterial; Gastrointestinal Microbiome; High-Throughput Screening Assays
PubMed: 32553109
DOI: 10.7554/eLife.56998 -
International Journal of Clinical... 2022The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files Autoclave, Glass-bead sterilizer,...
AIM AND OBJECTIVE
The aim and objective of this study is to compare the efficacy of four various approaches of sterilizing endodontic hand files Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser.
MATERIALS AND METHODS
Fifty-two k-files of size #25 and length 21 mm were taken for the study. All the 52 files were presterilized in an endodontic instrument box autoclave. spore suspension was prepared and all the presterilized files were contaminated with stearothermophilus spore suspension in a sterile Petri dish under vacuum hood safety. Later, the test files were randomly divided into four groups of 13 each and subjected to four different methods of sterilization- Autoclave, Glass-bead sterilizer, Glutaraldehyde solution, and Diode laser. Files were then be placed in thioglycollate media containing test tubes and incubated in an incubator at 55°C and checked for turbidity at 3 days and 21 days.
RESULT
The result revealed that there was no Turbidity present in test tubes on both the 3rd and 21st day for autoclave. In all the remaining sterilization procedures there was some amount of turbidity present. In terms of sterilization provided autoclave provides complete sterilization and glutaraldehyde solution is the least effective.The specificity of was then confirmed with a sugar test viz., starch hydrolysis test which gave a positive result confirming the presence of .
CONCLUSION
We can conclude that autoclave is the perfect process of sterilization providing 100% sterility and although Glass-bead didn't provide 100% sterility, it can be used as an alternative if autoclave is not available.
HOW TO CITE THIS ARTICLE
Ameer B, Khatib MS, Peerzade SM, Comparing Sterilization of Endodontic Hand Files by Four Different Methods: An Study. Int J Clin Pediatr Dent 2022;15(2):149-152.
PubMed: 37457218
DOI: 10.5005/jp-journals-10005-2346 -
Proceedings of the National Academy of... Jun 2018Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often...
Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.
Topics: Cell Biology; Cytological Techniques; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques
PubMed: 29895687
DOI: 10.1073/pnas.1805449115 -
European Review For Medical and... Dec 2022Dexpanthenol is an ingredient in multiple topical pharmaceutical preparations thanks to its high penetration and localized concentration. It is included in many...
OBJECTIVE
Dexpanthenol is an ingredient in multiple topical pharmaceutical preparations thanks to its high penetration and localized concentration. It is included in many ointments or lotions for dermatological use, assisting in healing and reducing pruritus. Vaseline is a synthetic product obtained by distilling crude oil. It is commercially available in several grades. The study presented here examined how topically applied agents (dexpanthenol or vaseline) affect nasal epithelial cells in culture. In particular, the study aimed to identify any alterations to epithelial cells which might indicate toxicity.
MATERIALS AND METHODS
The nasal epithelial cells used were sourced from mucosal tissue fragments left over the following septorhinoplasty on five patients not suffering from rhinosinusitis. The first step was to dissect the mucosal fragments into smaller pieces on a sterilized Petri dish. These fragments were then placed into the DMEM-F12 cell culture medium, which had been freshly prepared. The dexpanthenol and vaseline were diluted in dimethylsulfoxide (DMSO) to a concentration of 5 mg/mL. The cells in the wells were exposed to varying concentrations of dexpanthenol or vaseline. The actual concentration of the test reagent to which the epithelial cells were exposed ranged from 0.15 mg/mL to 5 mg/mL. The exposure period was 24 hours. The cells were finally examined using a Leica SP5II confocal microscope. The features sought were DNA fragmentation, condensation of the nuclei, changes in the outer membrane, or cytoskeletal abnormality. These features, if present, indicate cytotoxicity.
RESULTS
The viability of the cultured nasal epithelial cells was unaltered by a 24-hour exposure to dexpanthenol, nor was the cellular proliferation rate affected at the level of statistical significance. There was evidence of a cytotoxic effect from exposing nasal epithelial cells to vaseline in liquid form for 24 hours. There was a reduction in cellular viability in the plates where the highest dose of vaseline (5 mg/mL) was used. Cellular viability was not affected significantly at any of the doses below 5 mg/mL.
CONCLUSIONS
The absence of cytotoxic effects from the application of dexpanthenol to the nasal mucosa indicates that this agent may be safely used within the nose. The cytotoxic effects of liquid vaseline observed in this trial (condensed nuclear chromatin, loss of cellular volume) indicate that this agent may be harmful when used intranasally. For patients who require nasal packing due to nose bleeds or following endoscopic sinus surgical procedures, dexpanthenol should be preferred to vaseline from the point of view of maximizing healing of a nasal injury.
Topics: Humans; Excipients; Petrolatum; Sinusitis; Pantothenic Acid
PubMed: 36524920
DOI: 10.26355/eurrev_202212_30496 -
IMA Fungus 2019Traditionally, fungal growth dynamics were assessed manually, limiting the research to a few environmental conditions and/or fungal species. Fortunately, more automated...
Traditionally, fungal growth dynamics were assessed manually, limiting the research to a few environmental conditions and/or fungal species. Fortunately, more automated ways of measurement are gaining momentum due to the availability of cheap imaging and processing equipment and the development of dedicated image analysis algorithms. In this paper, we use image analysis to assess the impact of environmental conditions on the growth dynamics of two economically important fungal species, and . Sixteen environmental conditions combining four temperatures (15, 20, 25 and 30 °C) and four relative humidity (RH) conditions (65, 70, 75 and 80% RH) were tested. Fungal growth characteristics were extracted from images of the growing fungi, taken at regular points in time. Advanced time series analysis was applied to quantitatively compare the effect of the environmental conditions on these growth characteristics. The evolution of the mycelial area and the number of tips over time resulted in typical sigmoidal growth curves. Other growth characteristics such as the mean hyphal segment length did not vary significantly over time. Temperature and RH usually had a combined effect on the growth dynamics of the mycelial area and the number of tips. When defining optimal growth conditions for a fungus, it is therefore of primordial importance that the effect of temperature and RH is assessed simultaneously. At the most extreme conditions we tested, the mycelium most probably experienced water stress when developing over the inert Petri dish surface. An RH of 65% (independent of temperature) for and a temperature of 30 °C (independent of RH) for both and therefore always resulted in limited fungal growth, while the optimal growing conditions were at 20 °C and 75% RH and at 25 °C and 80% RH for and at 20 °C and 75% RH for . The method applied in this study offers an updated and broader alternative to classical and narrowly focused studies on fungal growth dynamics, and is well suited to efficiently assess the effect of environmental conditions on fungal growth.
PubMed: 32647616
DOI: 10.1186/s43008-019-0009-3 -
PloS One 2020Spissistilus festinus (Say) (Hemiptera: Membracidae) was shown to transmit Grapevine red blotch virus (GRBV) in a greenhouse study. Grapevines infected with GRBV exhibit...
Spissistilus festinus (Say) (Hemiptera: Membracidae) was shown to transmit Grapevine red blotch virus (GRBV) in a greenhouse study. Grapevines infected with GRBV exhibit reduced sugar accumulation, altered secondary metabolite production and delayed berry maturation that negatively impacts wine quality and economics. Augmentative biocontrol may be a useful integrated pest management (IPM) tool for suppressing S. festinus populations in vineyards, but minimal research has been conducted on testing potential predators against the different life stages of S. festinus. The susceptibility of S. festinus adults and nymphs (1st through 5th instar) to predation by six commercially available biocontrol agents in petri dish and bell bean plant arenas was determined under greenhouse conditions. No significant mortality of S. festinus nymphs or adults occurred when exposed to Cryptolaemus montrouzieri adults, C. montrouzieri larvae and Sympherobius barberi adults in petri dish or bell bean plant arenas. Significant mortality of 1st and 2nd instar nymphs of S. festinus in the presence of Zelus renardii nymphs was observed in petri dish but not in bell bean arenas. Hippodamia convergens adults and Chrysoperla rufilabris larvae both consumed a significant number of S. festinus nymphs in petri dish and bell bean arenas. No significant predation of S. festinus adults was documented in this experiment. Results of this study aid in identifying predators that may be suitable candidates for additional field testing to determine their potential efficacy as biocontrol agents of S. festinus in a vineyard setting.
Topics: Animals; Coleoptera; Food Chain; Geminiviridae; Hemiptera; Models, Biological; Nymph; Predatory Behavior
PubMed: 33253247
DOI: 10.1371/journal.pone.0242775 -
Scientific Reports Jul 2023The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory...
The mechanisms governing chemotaxis in Chlamydomonas reinhardtii are largely unknown compared to those regulating phototaxis despite equal importance on the migratory response in the ciliated microalga. To study chemotaxis, we made a simple modification to a conventional Petri dish assay. Using the assay, a novel mechanism governing Chlamydomonas ammonium chemotaxis was revealed. First, we found that light exposure enhances the chemotactic response of wild-type Chlamydomonas strains, yet phototaxis-incompetent mutant strains, eye3-2 and ptx1, exhibit normal chemotaxis. This suggests that Chlamydomonas transduces the light signal pathway in chemotaxis differently from that in phototaxis. Second, we found that Chlamydomonas collectively migrate during chemotaxis but not phototaxis. Collective migration during chemotaxis is not clearly observed when the assay is conducted in the dark. Third, the Chlamydomonas strain CC-124 carrying agg1, the AGGREGATE1 gene (AGG1) null mutation, exhibited a more robust collective migratory response than strains carrying the wild-type AGG1 gene. The expression of a recombinant AGG1 protein in the CC-124 strain suppressed this collective migration during chemotaxis. Altogether, these findings suggest a unique mechanism; ammonium chemotaxis in Chlamydomonas is mainly driven by collective cell migration. Furthermore, it is proposed that collective migration is enhanced by light and suppressed by the AGG1 protein.
Topics: Chlamydomonas reinhardtii; Chemotaxis; Ammonium Compounds; Chlamydomonas; Cell Movement; Light
PubMed: 37402785
DOI: 10.1038/s41598-023-36818-6 -
Advanced Science (Weinheim,... Dec 2020There is an unmet demand for microfluidics in biomedicine. This paper describes contactless fabrication of microfluidic circuits on standard Petri dishes using just a...
There is an unmet demand for microfluidics in biomedicine. This paper describes contactless fabrication of microfluidic circuits on standard Petri dishes using just a dispensing needle, syringe pump, three-way traverse, cell-culture media, and an immiscible fluorocarbon (FC40). A submerged microjet of FC40 is projected through FC40 and media onto the bottom of a dish, where it washes media away to leave liquid fluorocarbon walls pinned to the substrate by interfacial forces. Such fluid walls can be built into almost any imaginable 2D circuit in minutes, which is exploited to clone cells in a way that beats the Poisson limit, subculture adherent cells, and feed arrays of cells continuously for a week. This general method should have wide application in biomedicine.
PubMed: 33304750
DOI: 10.1002/advs.202001854 -
Plants (Basel, Switzerland) May 2022Plant monoterpenes have received attention for their ecological functions and as potential surrogates for synthetic herbicides, but very little is known about the...
Plant monoterpenes have received attention for their ecological functions and as potential surrogates for synthetic herbicides, but very little is known about the processes that govern their behavior in the soil environment, and even less about the possible enantioselectivity in the functions and environmental behavior of chiral monoterpenes. We characterized the adsorption and dissipation of the two enantiomers of the chiral monoterpene pulegone in different soils, and their phytotoxicity to different plant species through Petri dish and soil bioassays. R- and S-pulegone displayed a low-to-moderate non-enantioselective adsorption on the soils that involved weak interaction mechanisms. Soil incubation experiments indicated that, once in the soil, R- and S-pulegone are expected to suffer rapid volatilization and scarcely enantioselective, biodegradation losses. In Petri dishes, the phytotoxicity of pulegone and its enantioselectivity to , , and was species-dependent. was the most sensitive species and showed higher susceptibility to S- than to R-pulegone. Biodegradation and volatilization losses greatly reduced the phytotoxic activity of S-pulegone applied to soil, but the addition of a highly-adsorptive organoclay stabilized the monoterpene and increased its phytotoxic effect. Stabilization by adsorption may represent an important mechanism by which the bioactivity of plant monoterpenes in soils can be increased.
PubMed: 35631720
DOI: 10.3390/plants11101296 -
Plant Disease Jun 2022Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of...
Grapevine is one of the most widely-planted fruit crops in the world, and is the most economically important fruit crop in the state of New York, USA. Symptoms of anthracnose on grapevine are similarly widely-reported on grapevine fruit and foliage, and such symptoms are commonly attributed to Elsinöe ampelina (Wilcox et al., 2015). However, similar symptoms, if not identical, to those associated with E. ampelina have been sporadically attributed to various species in the genus Colletotrichum. In September 2021, a survey was conducted in three research vineyards at Cornell AgriTech in Geneva, NY. Symptoms of anthracnose werebserved on four Vitis interspecific hybrid breeding lines in a 1 ha vineyard. Leaves, fruit, and petioles showing symptoms of anthracnose, i.e., sunken necrotic lesions with grayish centers and brownish margins, were collected. Symptomatic and healthy portions of surface-sterilized tissues were placed on PDA medium and incubated at 23oC for 7 days. Several petiole samples yielded colonies of white to greyish mycelium, with some red to orange pigmentation (Fig. 1A and 1B), similar to those described by Chowdappa et al. (2009) for Colletotrichum species isolated from grapevine in India. Cultures were allowed to sporulate. Slides from cultures were prepared and examined at 400X magnification. Conidia from cultures were cylindrical with rounded ends, 13.5-15.2 μm in length and 7.6-9.0 μm in width (Fig. 1C). Koch's postulates were fulfilled by inoculating detached healthy leaves of V. vinifera 'Chardonnay' that had been surface sterilized in 10% sodium hypochlorite and triple-rinsed in sterile distilled water. Drop inoculation was used from a suspension of 105 conidia/ml from the foregoing pure cultures as five 2 µL droplets per leaf. Inoculated detached leaves were maintained on water agar in a Petri dish at 23oC. Four days after inoculation, symptoms were observed and compared with the originally collected samples. Inoculated leaves displayed symptoms typically found on the collected tissues, and the original pathogen, as confirmed by colony morphology and conidial characteristics and dimensions, was reisolated from inoculated leaves, and not from non-inoculated controls. For molecular characterization, fungal DNA was isolated by using Qiagen DNeasy kit and amplified using the following primer pairs: ITS1/ITS4, TEF (Hyun et al., 2009), E. ampelina F/R (Santos et al. 2018), TUB2, ACT, HIS3, GAPDH and CHS1 (Damm et al., 2001). PCR products were purified using ExoSAP-IT, and samples were Sanger sequenced. Sequences were analyzed using Geneious Prime software, and the resulting sequences (NCBI accessions OL720215, OL720216, OL720217, OL720218, OL853836, OM982612, OM982613, OM982614, OM982615 and OM982616) had 94 to 100% identity to Colletotrichum fioriniae NCBI accessions MN944922.1, MK646015.1, MN944922.1, MN856415.1, KU847413.1, MN520490.1, MN544294.1, KY695259.1, MN535117.1 and MN544295.1. Symptoms of grapevine anthracnose caused by Colletotrichum species have been reported from India (Chowdappa et al., 2009) and Korea (Kim et al., 2021). To our knowledge this is the first report of grapevine anthracnose caused by C. fioriniae Anthracnose and ripe rot are diseases of increasing importance, particularly as new grapevine cultivars with resistance to powdery mildew or downy mildew are adopted. Taxonomy of the causal agents (E. ampelina and Colletotrichum spp.) has undergone considerable revision. Consequently, distribution and relative prevalence of the various taxa will require further study.
PubMed: 35771116
DOI: 10.1094/PDIS-03-22-0604-PDN