scholarly journals Double-Layer Plaque Assay for Quantification of Enteroviruses

2004 ◽  
Vol 70 (5) ◽  
pp. 2801-2805 ◽  
Author(s):  
Laura Mocé-Llivina ◽  
Francisco Lucena ◽  
Juan Jofre
Keyword(s):  
Double Layer ◽  
Carcinoma Cell ◽  
Plaque Assay ◽  
Most Probable Number ◽  
Suspended Cell ◽  
Probable Number ◽  
Membrane Filters ◽  
Naturally Occurring ◽  
Order Of Magnitude ◽  
Virus Quantification

ABSTRACT We describe here a double-layer plaque assay for the quantification of enteroviruses, combining a monolayer plaque assay and a suspended-cell plaque assay. The double-layer assay provides significantly greater counts than other methods of virus quantification of both suspensions of pure culture viruses and naturally occurring viruses. The counts obtained by this method are approximately one order of magnitude greater than those obtained with the more commonly used method, the monolayer plaque assay. We conclude that the methods available for quantifying viruses rank in efficiency as follows: double-layer plaque assay ≥ suspended-cell plaque assay > counting cytopathogenic virus adsorbed to cellulose nitrate membrane filters ≥ most probable number of cytopathogenic units > monolayer plaque assay. Moreover, the double-layer plaque assay allows the use of two different cell lines in the two layers. Using the human colonic carcinoma cell line CaCo2 facilitates the recovery of a greater number and diversity of naturally occurring enteroviruses in water than the monolayer agar method. In addition, the pretreatment of cells with 5-iodo-2′-deoxyuridine (IDU) prior to the quantification of enteroviruses by the double-layer plaque assay provides significantly higher recoveries than the use of IDU does with the other methods of quantification.

scholarly journals A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

2000 ◽  
Vol 66 (1) ◽  
pp. 194-198 ◽  
Author(s):  
Georgios T. Papageorgiou ◽  
Laura Mocé-Llivina ◽  
Christina G. Christodoulou ◽  
Francisco Lucena ◽  
Dina Akkelidou ◽  
...  
Keyword(s):  
Tap Water ◽  
Membrane Filter ◽  
Methodological Approach ◽  
Plaque Assay ◽  
Cellulose Nitrate ◽  
Assay Method ◽  
Most Probable Number ◽  
Cellulose Membrane ◽  
Probable Number ◽  
Membrane Filters

ABSTRACT We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

1985 ◽  
Vol 31 (8) ◽  
pp. 686-692 ◽  
Author(s):  
A. H. Havelaar ◽  
M. During ◽  
E. H. M. Delfgou-Van Asch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Filtration ◽  
Culture Media ◽  
Polluted Water ◽  
Most Probable Number ◽  
Cellulose Ester ◽  
Swimming Pools ◽  
Probable Number ◽  
Membrane Filters ◽  
Swimming Water

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 μg/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 μg/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide – nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them. In the most-probable-number technique, recovery of P. aeruginosa was shown to be excellent when using asparagine broth. Malachite green broth was strongly inhibitory to chlorine-injured P. aeruginosa.

Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples

1985 ◽  
Vol 31 (11) ◽  
pp. 977-980 ◽  
Author(s):  
Pierre Payment ◽  
Michel Trudel
Keyword(s):  
Cell Culture ◽  
Cytopathic Effect ◽  
Incubation Temperature ◽  
Plaque Assay ◽  
Virus Titer ◽  
Inoculum Size ◽  
Most Probable Number ◽  
Probable Number ◽  
Culture Density ◽  
Coxsackievirus B

The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 °C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.

Application of an Automated Hydrophobic Grid Membrane Filter Interpreter System at a Poultry Abattoir

1991 ◽  
Vol 54 (8) ◽  
pp. 619-622 ◽  
Author(s):  
W. BRUCE MCNAB ◽  
CHRISTINE M. FORSBERG ◽  
ROBERT C. CLARKE
Keyword(s):  
Membrane Filter ◽  
Geometric Mean ◽  
Aerobic Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
Membrane Filters ◽  
Government Inspection ◽  
Data Files ◽  
Good Repeatability ◽  
Set Up

The performance of a system to measure broiler carcass hygiene was investigated in the abattoir environment. The system involved: whole carcass rinses aided by a mechanical carcass shaker; filtration of rinse solutions though hydrophobic grid membrane filters (HGMF) (ISO-GRIDR, QA Laboratories Ltd.); and use of an automated HGMF interpreter. The interpreter recorded culture results in units of most probable number (MPN) of aerobic bacteria, in electronic data files (MI-100 HGMF Interpreter System, Richard Brancker Research Ltd.). Set-up and operation of the system by government inspection staff at an abattoir ran relatively smoothly with minimal interference to normal plant operation. The system demonstrated good repeatability in measuring log10 most probable number per gram of carcass (LgMPN/g), between repeat readings of the same filters (r=0.993 p<0.001), and good repeatability between repeat filters within the same carcass rinses (r=0.970 p<0.001). Overall, the LgMPN/g ranged from 0.258 to 3.955 with a mean of 2.276 and a variance of 0.324. These corresponded to MPN/g counts in the range of 2 to 9000 and a geometric mean of 188.8 MPN/g. A regression model was developed to investigate poultry supplier and abattoir effects on the variability of counts. A significant supplier effect was observed. The addition of two more carcass showers located just after the venting machine along the evisceration line was not associated with a change in carcass hygiene.

Detection of Enteroviruses: An Assessment of Ten Cell Lines

1985 ◽  
Vol 17 (10) ◽  
pp. 81-88 ◽  
Author(s):  
R. Morris
Keyword(s):  
Cell Lines ◽  
Cytopathic Effect ◽  
Plaque Assay ◽  
Suspended Cell ◽  
Naturally Occurring ◽  
Routine Monitoring ◽  
Rd Cells ◽  
Coxsackie B Viruses ◽  
Simian Cell ◽  
Coxsackie B

The routine monitoring of waters and wastewaters using the BGM - suspended cell plaque assay system has resulted in high isolation rates for polio and coxsackie B viruses with echoviruses only rarely being detected. Ten cell lines (five of human origin, five of simian derivation) were examined for their susceptibility to twenty six enteroviruses and their ability to isolate naturally occurring enteroviruses in wastewater effluent determined. All the viruses produced cytopathic effect in the simian cell lines but only in the FL amnion line of human cells. The detection of naturally occurring enteroviruses was most efficient when BGM cells were used with 82% of samples being positive. RD cells gave 73% positive whilst chimpanzee liver gave 64%. However, the number of plaques formed in the presence of each cell line showed that BGM detected 50% of all plaques observed with RD and chimpanzee liver detecting 21% and 9% respectively. The serotypes detected in this study were of the polio and coxsackie B groups, no echoviruses being identified.

Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems

1984 ◽  
Vol 30 (3) ◽  
pp. 334-338 ◽  
Author(s):  
D. B. Roszak ◽  
D. J. Grimes ◽  
R. R. Colwell
Keyword(s):  
Salmonella Enteritidis ◽  
Fluorescent Antibody ◽  
Viable Count ◽  
Plate Count ◽  
Most Probable Number ◽  
Potomac River ◽  
Direct Count ◽  
Probable Number ◽  
Solid Media ◽  
Order Of Magnitude

An environmental isolate (13-1BB) of Salmonella enteritidis serogroup C1 was inoculated into sterile Potomac River water microcosms to observe survival and culturability of the organism by employing acridine orange direct count, fluorescent antibody direct count, direct viable count, plate count on veal infusion agar and xylose lysine decarboxylase agar, and indirect enumeration by the most-probable-number method (MPN), using media selective for Salmonella. Loss of culturability on laboratory media was observed within 48 h. However, cultures could be "resuscitated" and cultured on solid media, following addition of nutrients to the microcosms. Cells, resuscitated 4 days after apparent "die-off" (0 colony-forming units (cfu)/mL) using plate count techniques, yielded numbers of cfu in the same order of magnitude as had been observed before the onset of nutrient limitation. Microscopic techniques for direct viable counting indicated that viability is maintained for as long as 60 days after depletion of nutrients, although attempts to culture these cells, by addition of nutrient, after 21 days yielded apparently sterile plates. Thus, longer periods of "dormancy" appear to require conditions other than simple nutrient addition for resumption of cell growth and division.

Effect of source water quality on solar disinfection rate under multiple experimental conditions

2014 ◽  
Vol 4 (4) ◽  
pp. 714-719
Author(s):  
M. Mansoor Ahammed ◽  
Shilpa Dave
Keyword(s):  
Natural Waters ◽  
Municipal Wastewater ◽  
Heterotrophic Bacteria ◽  
Exposure Period ◽  
Most Probable Number ◽  
Experimental Conditions ◽  
Probable Number ◽  
Solar Disinfection ◽  
Complete Inactivation ◽  
Naturally Occurring

Water samples from four different sources of varying physico-chemical and microbial quality with their naturally occurring microorganisms were exposed to sunlight in polyethylene terephthalate bottles under similar conditions. Up to 3-log10 reduction of total coliforms (TC) was observed during a 6-h exposure period under weak/moderate radiation conditions (<600 W/m2). Complete inactivation of TC was not achieved in 6 h of exposure for waters with larger initial TC such as river water (1 × 103 most probable number [MPN]/100 mL) and treated municipal wastewater (2 × 105 MPN/100 mL) under these conditions. Heterotrophic bacteria showed lower inactivation rates than did TC. The inactivation rate for spiked Escherichia coli was faster than for naturally occurring coliforms. Further tests with compound parabolic collectors showed that complete inactivation of naturally occurring TC could be achieved within 6 h of exposure for all the natural waters tested. The results of the study thus indicate the need to use naturally occurring organisms in testing the effectiveness of solar disinfection, and the importance of source quality on the inactivation rates of microorganisms.

The Anderson–Baird-Parker direct plating method versus the most probable number procedure for enumerating Escherichia coli in meats

1981 ◽  
Vol 27 (1) ◽  
pp. 147-149 ◽  
Author(s):  
M. K. Rayman ◽  
B. Aris
Keyword(s):  
Escherichia Coli ◽  
North American ◽  
Most Probable Number ◽  
Probable Number ◽  
Direct Plating ◽  
E Coli ◽  
Membrane Filters ◽  
The North ◽  
Plating Method

Comparison of the Anderson–Baird-Parker direct plating method (DP) and the North American most probable number procedure (MPN) for enumerating Escherichia coli in frozen meats revealed that the DP method is more precise and yields higher counts of E. coli than the MPN procedure. Any of three brands of membrane filters tested was suitable for use in the DP method.

Plaque Assay for Adenovirus Type 41 Using the PLC/PRF/5 Liver Cell Line

1993 ◽  
Vol 27 (3-4) ◽  
pp. 321-327 ◽  
Author(s):  
W. O K. Grabow ◽  
D. L. Puttergill ◽  
A. Bosch
Keyword(s):  
Cell Line ◽  
Plaque Assay ◽  
Laboratory Strain ◽  
Adenovirus Type ◽  
The Other ◽  
Most Probable Number ◽  
Probable Number ◽  
Cytopathic Effects ◽  
Viral Particles ◽  
Conjunctival Cells

In research on methods for the detection and enumeration of the fastidious adenovirus types 40 and 41 (Ad40/41), the PLC/PRF/5 cell line derived from a primary human hepatocellular carcinoma proved substantially more sensitive than the Graham 293 (human embryonic kidney), A549 (human lung), KB (human oral epidermoid) and Chang conjunctival cells generally used for propagation of these viruses. In comparative tests based on the detection of a cytopathic effect in microtitre plate monolayers inoculated with tenfold dilutions of virus suspensions and quantitation by most probable number calculation, the PLC/PRF/5 cell line yielded a hundred times higher count for a laboratory strain of Ad41, and ten times higher counts for a laboratory strain of Ad40 and two Ad41 stool isolates, than any of the other cells. The PLC/PRF/5 cells also retained an optimal condition for longer and displayed cytopathic effects earlier and more clearly than the other cell lines. Efforts to develop plaque assays for Ad40/41 by using various modifications of established plaque techniques were successful only for the laboratory strain and the two stool isolates of Ad41 using PLC/PRF/5 cells. The reason for the exceptional susceptibility of PLC/PRF/5 cells to Ad40/41, and the ability of these cells to plaque Ad41 but not Ad40, cannot be explained. Quantitative PLC/PRF/5 cell culture assays on viral suspensions in which numbers of viral particles were counted by electron microscopy using a calibrated colloidal gold particle suspension as reference, indicated that the minimal infective dose of Ad40/41 for humans may be in the same range as that of many other enteric viruses.

Analysis of Coliform and Colifecal Total Pollution Test on Various Types of Drinking Water Using the MPN (Most Probable Number) Method

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Wanda Aulya ◽  
Fadhliani Fadhliani ◽  
Vivi Mardina
Keyword(s):  
Drinking Water ◽  
Fecal Coliform ◽  
Pathogenic Bacteria ◽  
Microbiological Quality ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Inspection Method ◽  
Sample Number ◽  
Probable Number ◽  
Fecal Coliform Bacteria

Water is the main source for life and also the most severe substance caused by pollution. The mandatory parameters for determining microbiological quality of drinking water are total non-fecal Coliform bacteria and Coliform fecal (Escherichia coli). Coliform bacteria are a group of microorganisms commonly used as indicators, where these bacteria can be a signal to determine whether a water source has been contaminated by bacteria or not, while fecal Coliform bacteria are indicator bacteria polluting pathogenic bacteria originating from human feces and warm-blooded animals (mammals) . The water inspection method in this study uses the MPN (Most Probable Number) method which consists of 3 tests, namely, the presumption test, the affirmation test, and the reinforcement test. The results showed that of 15 drinking water samples 8 samples were tested positive for Coliform bacteria with the highest total bacterial value of sample number 1, 15 (210/100 ml), while 7 other samples were negative. From 8 positive Coliform samples only 1 sample was stated to be negative fecal Coliform bacteria and 7 other samples were positive for Coliform fecal bacteria with the highest total bacterial value of sample number 1 (210/100 ml).

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