Abstract：
:Recent developments in host-microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0-4.0log/mL, whereas a 5.0-5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Grootaert C,Boon N,Zeka F,Vanhoecke B,Bracke M,Verstraete W,Van de Wiele Tdoi
10.1016/j.mimet.2011.03.011subject
Has Abstractpub_date
2011-07-01 00:00:00pages
33-41issue
1eissn
0167-7012issn
1872-8359pii
S0167-7012(11)00110-2journal_volume
86pub_type
杂志文章abstract：:A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplifi...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.11.006
更新日期：2015-01-01 00:00:00
abstract：:Whole-cell bacterial biosensors can be applied for the screening of antibacterial properties of extracts. We constructed a biosensor panel consisting of four different bacterial biosensor strains: Escherichia, Staphylococcus, Acinetobacter and Pseudomonas for expanded screening potential. The functionality of the pane...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.106083
更新日期：2020-10-14 00:00:00
abstract：:A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(03)00091-5
更新日期：2003-10-01 00:00:00
abstract：:Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This stu...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.07.010
更新日期：2013-09-01 00:00:00
abstract：:Serology is commonly used for the diagnosis of acute Chlamydia pneumoniae infections and also for the diagnosis of complicated Chlamydia trachomatis infections. Furthermore, recent sero-epidemiological studies have linked C. pneumoniae infection with several diseases traditionally considered non-infectious. The object...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/s0167-7012(00)00209-8
更新日期：2000-11-01 00:00:00
abstract：:A simple generic toxicity method (test) is proposed using baker's yeast to mediate the reduction of the colourless triphenyltetrazolium chloride (TTC) to red, 1,3,5-triphenyl formazan, which can be extracted by dimethyl sulfoxide (DMSO), enabling the identification of reducible toxic compounds (e.g. cadmium, fipronil)...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.04.008
更新日期：2019-06-01 00:00:00
abstract：:Despite its long-standing history as a model organism, Neurospora crassa has limited tools for repetitive gene deletions utilizing recyclable self-excising marker systems. Here we describe, for the first time, the functionality of a bacterial recombination system employing β-recombinase acting on six recognition seque...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.12.004
更新日期：2013-03-01 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：:Lactic acid bacteria (LAB) play important roles in the food industry, animal husbandry and medicine which are closely related to human life. Modern gene engineering technology is the major means to reveal gene functions, study metabolic pathways and metabolomes of strains and improve the properties of strains. However...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/j.mimet.2020.105944
更新日期：2020-07-01 00:00:00
abstract：:Microbiological work requires a reliable source of cultures that are not only well defined and taxonomically determined, but are also adequately preserved without changes in their morphological, physiological and genetic traits. Here we describe an easy, cost effective and rapid method for reliably preserving filament...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.02.009
更新日期：2014-04-01 00:00:00
abstract：:An etiological relationship between periodontitis, a significant oral health problem, and the anaerobe Porphyromonas gingivalis may be related to the expression of a variety of putative virulence factors. The objective of the experiments described here was to develop a quantitative reverse transcription polymerase cha...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(01)00362-1
更新日期：2002-04-01 00:00:00
abstract：:A method to detect and enumerate bacterial colonies grown on membrane filters (MF) was described. The colonies were stained with an ethanolic solution of 0.1% Amido black 10B. The procedure yielded the rapid detection of colonies as compared to a conventional plate counting method. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(03)00170-2
更新日期：2003-11-01 00:00:00
abstract：:To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.02.011
更新日期：2016-04-01 00:00:00
abstract：:A new protocol was developed to detach bacteria from seagrass tissue and subsequently enumerate cells using flow cytometry (FCM). A method involving addition of the surfactant Tween 80 and vortexing resulted in maximum detachment efficiency of seagrass attached bacteria, providing a robust protocol for precisely enume...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.04.008
更新日期：2014-07-01 00:00:00
abstract：:This study compared an automated Escherichia coli and coliform detection system with the membrane filtration direct count technique for water testing. The automated instrument performed equal to or better than the membrane filtration test in analyzing E. coli-spiked samples and blind samples with interference from Pro...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.08.009
更新日期：2009-10-01 00:00:00
abstract：:MALDI-ToF MS can be used to identify microorganisms directly from blood cultures. This study compared two methods of sample preparation. Similar levels of genus- (91% vs 90%) and species-level identifications (79% vs 74%) were obtained with differential centrifugation and SDS methods. The SDS method is faster and requ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.05.026
更新日期：2016-08-01 00:00:00
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. c...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(98)00118-3
更新日期：1999-03-01 00:00:00
abstract：:The direct counts of bacteria inhabiting coral mucus were performed by flow cytometry testing four fluorescent dyes (SYBR®Green I, HCS, TOPRO®3, SYTO®62) with three different scleractinian species. Results obtained with SYTO62 were the most reliable based on the comparison with standardized epifluorescence counts and ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.05.032
更新日期：2016-09-01 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：: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：:Human viral contamination in drinking and recreational water may persist for extensive periods of time and cause a significant health risk concern. The aim of this study is to evaluate a viral recovery method using a new electropositive charged nanoalumina filter and to compare results with the widely used negatively ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.01.018
更新日期：2010-04-01 00:00:00
abstract：:Herein, an open-source method to generate quantitative bacterial growth data from high-throughput microplate assays is described. The bacterial lag time, maximum specific growth rate, doubling time and delta OD are reported. Our method was validated by carbohydrate utilization of lactobacilli, and visual inspection re...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.11.015
更新日期：2017-01-01 00:00:00
abstract：:Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/s0167-7012(03)00023-x
更新日期：2003-05-01 00:00:00
abstract：:Chemotactic behaviour is an important part of the lifestyle of motile bacteria and enables cells to respond to various environmental stimuli. The Hard Agar Plug (HAP) method is used to study the chemotactic behaviour of bacteria, including the fastidious microaerophile Campylobacter jejuni, an intestinal pathogen of h...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.02.008
更新日期：2018-03-01 00:00:00
abstract：:Nosocomial infections caused by Stenotrophomonas maltophilia resistant to SXT are increasingly reported worldwide. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of the ssrA and sul1, sul2 and sul3 genes was first established. The assays were performed on a Roch...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.12.002
更新日期：2019-01-01 00:00:00
abstract：:Streptococcus agalactiae, group B streptococci (GBS) is the leading cause of severe bacterial infections in newborns. GBS expression studies allowed the identification and characterization of virulence factors and a better understanding of the host-pathogen-environment interactions. The measurement of transcript level...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.05.011
更新日期：2012-09-01 00:00:00
abstract：:BD Phoenix™ is an automated bacterial identification and susceptibility testing system. Here, its performance in screening IMP-producing Enterobacteriaceae was evaluated. The system identified 97.8% of IMP producers as being nonsusceptible to imipenem or meropenem, which was higher than that identified by the broth mi...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2017.12.006
更新日期：2018-02-01 00:00:00
abstract：:Laser desorption-time of flight (LD-TOF) mass spectrometry-based detection of hemozoin was assessed for its performance characteristics as a rapid screening test for malaria. In spite of good specificity of >95%, poor sensitivity of 80.2% for microscopically positive samples makes the easy-to-apply and rapid approach ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.04.002
更新日期：2019-05-01 00:00:00
abstract：:Confocal microscopy-based monitoring of pH in biofilms is gaining increasing interest, as it allows for a quick assessment of horizontal pH gradients without mechanically perturbing the biofilm. Ratiometric monitoring of pH with the fluorescent dye C-SNARF-4 has been used to reliably map extracellular pH in the basal ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.08.007
更新日期：2018-09-01 00:00:00
abstract：:Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealin...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.08.003
更新日期：2014-11-01 00:00:00
