Abstract：
:For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Walberg M,Gaustad P,Steen HBdoi
10.1016/s0167-7012(98)00118-3subject
Has Abstractpub_date
1999-03-01 00:00:00pages
167-76issue
2eissn
0167-7012issn
1872-8359pii
S0167701298001183journal_volume
35pub_type
杂志文章abstract：:The Carba NP test was developed to detect carbapenemase-producing Enterobacteriaceae, and uses imipenem as the reaction substrate. In Japan, IMP-6 metallo-β-lactamase (MBL) producers, which are usually resistant to meropenem but susceptible to imipenem, and IMP-1 MBL producers, which are usually resistant to both carb...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.12.015
更新日期：2017-02-01 00:00:00
abstract：:Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In th...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.12.012
更新日期：2019-02-01 00:00:00
abstract：:Preservation of strains is important to further studies and characterization; we have shown a technique of long-term preservation of Neisseria spp. and Haemophilus spp. by freezing cotton tip swabs impregnated with fastidious organisms at -80 °C without the addition of cryoprotectants. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105969
更新日期：2020-08-01 00:00:00
abstract：:The aim of the present investigation was to develop a novel approach for rapid identification and differentiation of Pseudomonas aeruginosa clones from Canadian cystic fibrosis patients by capillary electrophoresis-mass spectrometry. We screened P. aeruginosa isolates for lipopolysaccharide structure and presence/abse...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.10.008
更新日期：2009-02-01 00:00:00
abstract：:We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased s...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.04.009
更新日期：2016-06-01 00:00:00
abstract：:In this report, a passive microfluidic chip design was developed for fast and sensitive fluorometric determination of Escherichia coli (E. coli) based on sandwich immunoassay. Initially, magnetic nanoparticles (MNPs) and chitosan modified mercaptopropionic acid capped cadmium telluride (CdTe) quantum dots (QDs) were f...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.105680
更新日期：2019-09-01 00:00:00
abstract：:A fused protein composed of a carbohydrate-binding module and green fluorescence protein (GFP) was developed to measure the exopolysaccharides (EPShs) present in Escherichia coli microcolonies. The cleavage of the GFP part of this protein using a site-specific protease allowed for the non-invasive and quantitative eva...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.05.013
更新日期：2015-07-01 00:00:00
abstract：:Coagulase negative staphylococci (CoNS) are a leading cause of infections in preterm infants, mostly involved in late-onset infection in low birth weight neonates. The epidemiology and pathophysiology of these infections remain unclear, notably because the causing agents are gathered in the artificial CoNS group. The ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.12.022
更新日期：2014-03-01 00:00:00
abstract：:Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.09.016
更新日期：2010-12-01 00:00:00
abstract：:The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The p...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2006.03.022
更新日期：2006-11-01 00:00:00
abstract：:A 384-well-based antibacterial assay amenable for high-throughput screening and combination testing is described. The assay uses 100-500nL of test compounds and tolerates up to 2.5% dimethyl sulfoxide concentrations. It can be used for screening compound libraries and testing combinatory/synergistic/antagonistic effec...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.09.019
更新日期：2015-11-01 00:00:00
abstract：:We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was the definitive standar...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(99)00080-9
更新日期：1999-10-01 00:00:00
abstract：:Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil: the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean. Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid c...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2003.08.002
更新日期：2003-12-01 00:00:00
abstract：:The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian childr...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(02)00158-6
更新日期：2003-02-01 00:00:00
abstract：:In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as contro...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.03.010
更新日期：2016-06-01 00:00:00
abstract：:Much of our knowledge concerned with microbial cells is based on population-based analysis of cultures, which give useful insights into average responses but neither on individual cells nor subpopulations. In this work we demonstrate how to access and utilise large amounts of valuable information concerned with cell p...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.01.011
更新日期：2009-04-01 00:00:00
abstract：:The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/F...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2005.06.014
更新日期：2006-04-01 00:00:00
abstract：:Sequences in public databases may contain a number of sequencing errors. A double binomial model describing the distribution of indel-excluded similarity coefficients (S) among repeatedly sequenced 16S rRNA was previously developed and it produced a confidence interval of S useful for testing sequence identity among s...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2007.11.013
更新日期：2008-02-01 00:00:00
abstract：:A novel in vitro onychomycosis model was developed to easily predict the topical activity potential of novel antifungal drugs. The model encompasses drug activity and diffusion through bovine hoof slices in a single experimental set-up. Results correspond well with the antifungal susceptibility assay and Franz cell di...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.03.009
更新日期：2015-05-01 00:00:00
abstract：:Specific molecular determination and classification of ammonia-oxidizing bacteria have relied on the use of conventional markers such as 16S rDNA. However, this gene does not satisfactorily provide a wide vision of all phylogenetic lineages. Despite the initial expectations, the use of functional genes as for example ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2003.11.019
更新日期：2004-04-01 00:00:00
abstract：:In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR techno...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.10.004
更新日期：2011-01-01 00:00:00
abstract：:In H. polymorpha, there is still a lack of a highly efficient gene disruption method. To help address this issue, we presented a simple and efficient method for both single and multiple gene disruptions in H. polymorpha. The knockout system combined a variation of sticky-end polymerase chain reaction method (SEP), spl...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.09.004
更新日期：2009-12-01 00:00:00
abstract：:This study proposes a high throughput method based on Confocal Laser Scanning Microscopy (CLSM) combined with the use of 96-wells microtiter plates compatible with high resolution imaging for the study of biofilm formation and structure. As an illustration, the three-dimensional structures of biofilms formed by 60 opp...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.04.006
更新日期：2010-07-01 00:00:00
abstract：:Rapid enumeration of Escherichia coli strains by quantitative real-time PCR targeting the uidA gene was developed and confirmed for minced beef, tuna and raw oyster. Higher sensitivity (1 CFU/g of E. coli in all three food samples) was obtained by incubating for 7 h in TSB. Colony-directed E. coli specific TaqMan PCR ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.08.002
更新日期：2009-10-01 00:00:00
abstract：:Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters....
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.08.010
更新日期：2015-11-01 00:00:00
abstract：:Efficient selection of new silage inoculant strains from a collection of over 10,000 isolates of lactic acid bacteria (LAB) requires excellent strain discrimination. Toward that end, we constructed a GelCompar II database of DNA fingerprint patterns of ethidium bromide-stained EcoRI fragments of total LAB DNA separate...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(03)00186-6
更新日期：2003-12-01 00:00:00
abstract：:A discontinuous lactate dehydrogenase coupled assay is described for the evaluation of the pyruvate carboxylase activity (Pc, EC 6.4.1.1) in a glutamate overproducing strain of Corynebacterium glutamicum. After an initial permeabilisation period of the cells, the method consisted of the fluorometric determination of t...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(99)00104-9
更新日期：1999-12-01 00:00:00
abstract：:We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae. The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such a...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(00)00171-8
更新日期：2000-09-01 00:00:00
abstract：:Treatment with 70% ethanol has been proposed as a safe and effective way to inactive bacteria for transport between laboratories prior to identification by MALDI-TOF MS. Ethanol alone does not inactivate spore-forming bacteria and additional chemical or physical treatment is necessary to guarantee inactivation of bact...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105893
更新日期：2020-05-01 00:00:00
abstract：:The need for improved pathogen separation and concentration methods to reduce time-to-detection for foodborne pathogens is well recognized. Apolipoprotein H (ApoH) is an acute phase human plasma protein that has been previously shown to interact with viruses, lipopolysaccharides (LPS) and bacterial proteins. The purpo...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.07.014
更新日期：2016-09-01 00:00:00
