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.

scholarly journals Efficacy of a solar still in destroying virus and indicator bacteria in water for human consumption

2018 ◽  
Vol 13 (4) ◽  
pp. 1
Author(s):  
Felipe Tiago do Nascimento ◽  
Carlos Augusto do Nascimento ◽  
Fernando Rosado Spilki ◽  
Rodrigo Staggemeier ◽  
Cláudio Marcos Lauer Júnior
Keyword(s):  
Escherichia Coli ◽  
Human Adenovirus ◽  
Adenovirus Type ◽  
Pilot Scale ◽  
Total Coliform ◽  
Water Disinfection ◽  
Most Probable Number ◽  
Human Consumption ◽  
Solar Still ◽  
Probable Number

Natural water distillation can destroy and/or inactivate microorganisms that are sensitive to heat and ultraviolet radiation (UV). This method is currently used to provide fresh water in ships and in the desalination of brackish water. For the development of this research, a pilot-scale solar still was built and installed in the southern region of Brazil, in order to assess its efficiency in water disinfection, which was based on the most probable number (MPN) of total coliforms and  Escherichia coli, in addition to the DNA copy number of human adenovirus type 5 (HAdV-5) in raw, undistilled samples and in treated distilled water. Results showed that the distillation process removed 100% of total coliform and Escherichia coli and 4.5 log (99.997%) of HAdV-5, which meets the microbiological standards for drinking water according to national Brazilian regulations, as well as USEPA and HEALTH CANADA requirements.

Differentiation of Clostridium perfringens from Related Clostridia in Iron Milk Medium

1985 ◽  
Vol 48 (2) ◽  
pp. 130-134 ◽  
Author(s):  
CARLOS ABEYTA ◽  
ANITA MICHALOVSKIS ◽  
MARLEEN M. WEKELL
Keyword(s):  
Clostridium Perfringens ◽  
High Selectivity ◽  
Positive Response ◽  
Food Samples ◽  
Gas Production ◽  
The Other ◽  
Plate Count ◽  
Most Probable Number ◽  
Probable Number ◽  
Positive Results

The stormy fermentation reaction of Clostridium perfringens in iron milk medium was compared to that of several C. perfringens-like strains. These clostridia, C. barati, C. perenne, C. absonum, and C. paraperfringens are very similar to C. perfringens on the basis of certain biochemical reactions and, consequently, are often difficult to distinguish from C. perfringens. Furthermore, these related clostridia may also be present in foods. Results of this study demonstrate that after 18 h of incubation at 45°C, only C. perfringens gave a positive reaction in iron milk with inocula as low as 22 cells/g. Some of the other strains began to show only gas production at 18 h. After 24 to 42 h some strains gave positive results and after 72 h all were positive. Enumeration of C. perfringens from food samples in iron milk medium by a 3-tube most probable number (MPN) technique gave similar results to enumeration by plate count using Shahidi-Ferguson Perfringens (SFP) agar. Furthermore, a rapid positive response occurred after only 2 and 3 h incubation of iron milk inoculated with 108 and 107 cells/ml, respectively. The high selectivity, ease of identification and rapid growth of C. perfringens in iron milk make the iron milk MPN procedure a valuable assay for accurate enumeration and differentiation of C. perfringens from related Clostridia in food products.

ICMSF Methods Studies. XI. Collaborative/Comparative Studies on Determination of Coliforms Using the Most Probable Number Procedure1

1979 ◽  
Vol 42 (8) ◽  
pp. 638-644 ◽  
Author(s):  
J. H. SILLIKER ◽  
D. A. GABIS ◽  
A. MAY
Keyword(s):  
Comparative Studies ◽  
Confidence Limit ◽  
Collaborative Study ◽  
The Other ◽  
Most Probable Number ◽  
Probable Number ◽  
Collaborative Studies ◽  
Different Types ◽  
Confirmatory Tests

Results of two international collaborative studies on the MPN technique for determination of coliforms in foods are reported. Three methods involving use of different presumptive and confirmatory media were compared. Results of one collaborative study conducted among 15 laboratories using eight different types of inoculated foods showed differences among the laboratories as great as 3.3 log units. The greatest difference between confirmatory tests using different media was 0.5 log units. Results of the other collaborative study conducted among five laboratories using three types of naturally contaminated foods showed differences among the laboratories as great as 1.4 log units. The greatest difference between tests using different media was 0.2 log unit. Both studies showed that the 95% confidence limit for a single value reported by a given laboratory was ± 1 log unit or ± 0.45 log unit for a mean of five values. The second study showed that a major source of variation within laboratories was between replicate aliquots. The findings are discussed in terms of their significance with respect to the monitoring of microbiological specifications for food.

Enumeration of Enterobacteriaceae in Various Foods with a New Automated Most-Probable-Number Method Compared with Petrifilm and International Organization for Standardization Procedures

2008 ◽  
Vol 71 (2) ◽  
pp. 376-379 ◽  
Author(s):  
P. PAULSEN ◽  
C. BORGETTI ◽  
E. SCHOPF ◽  
F. J. M. SMULDERS
Keyword(s):  
International Organization ◽  
The Other ◽  
Most Probable Number ◽  
Average Difference ◽  
Detection Range ◽  
Probable Number ◽  
International Organization For Standardization ◽  
Glucose Agar ◽  
The Mean ◽  
Mean Differences

An automated most-probable-number (MPN) system (TEMPO, bioMérieux, Marcy l'Etoile, France) for enumeration of Enterobacteriaceae (EB) was compared with methods involving violet red bile glucose agar (VRBG) (International Organization for Standardization [ISO] method 21528-2) (ISO-VRBG) and Petrifilm (PF-EB). The MPN partitioning (three different volumes with 16 replicates of each) is done automatically in a disposable card. Bacterial growth is indicated by acid production from sugars, lowering the pH of the medium, and quenching the fluorescence of 4-methylumbelliferone. After incubation, the number of nonfluorescent wells is read in a separate device, and the MPN is calculated automatically. A total of 411 naturally contaminated foods were tested, and 190 were in the detection range for all methods. For these results, the mean (±standard deviation) counts were 2.540 ± 1.026, 2.547 ± 0.995, and 2.456 ± 1.014 log CFU/g for the ISO-VRBG, PF-EB, and automated MPN methods, respectively. Mean differences were −0.084 ± 0.460 log units for the automated MPN results compared with the ISO-VRBG and 0.007 ± 0.450 for the PF-EB results compared with the ISO-VRBG results. The automated MPN method tends to yield lower numbers and the PF-EB method tends to yield higher numbers than does the ISO-VRBG method (difference not significant; Kruskal-Wallis test, P = 0.102). Thus, the average difference was highest between the automated MPN method and the PF-EB method (−0.091 ± 0.512 log units). Differences between the automated MPN and ISO-VRBG results of >1 log unit were detected in 3.4% of all samples. For 3.9% of the samples, one comparison yielded differences of <1 log CFU/g and the other yielded >1 but <2 log CFU/g, which means that the differences are possibly >1 log CFU/g. For the ISO-VRBG method, confirmation of isolates was necessary to avoid a bias due to the presence of oxidase-positive glucose-fermenting colonies. The automated MPN system yielded results comparable to those of the confirmed Enterobacteriaceae ISO-VRBG method but required only 24 h of analysis time.

Evaluation of Four Pea (Pisum sativum) Cultivars in PE-2 Medium for the MPN Enumeration of Anaerobic Spore-Forming Organisms

1997 ◽  
Vol 60 (12) ◽  
pp. 1574-1576 ◽  
Author(s):  
O. A. OGUNRINOLA ◽  
C. G. EDWARDS ◽  
P. M. DAVIDSON
Keyword(s):  
Pisum Sativum ◽  
Anaerobic Bacteria ◽  
False Positives ◽  
Clostridium Butyricum ◽  
The Other ◽  
Most Probable Number ◽  
Clostridium Sporogenes ◽  
Probable Number ◽  
Cooked Meat

Untreated ‘Alaska’ seed peas have traditionally been used to prepare PE-2 medium, a medium used to recover anaerobic bacteria. Three cultivars of seed peas, ‘Columbian’, ‘Yellow’, and ‘Scotch’ were compared to the cultivar ‘Alaska’ peas in PE-2 for recovery of Clostridium butyricum ATCC 860, Clostridium sporogenes ATCC 7955/NCA 3679, and Thermoanerobacterium thermosaccharolyticum ATCC 25773 determined by using the most probable number (MPN) technique. Organisms were grown in cooked meat medium (CMM) and enumerated by the three-tube MPN method in PE-2 media incubated at 37°C for 48 h. Recovery of C. butyricum and T. thermosaccharolyticum grown in homogenates of commercial cream-style corn and vegetable beef soup were also evaluated. Similar recovery of organisms from stock cultures or from foodstuffs were observed in PE-2 prepared with three of the pea cultivars (‘Alaska’, ‘Columbian’, and ‘Yellow’). While the ‘Scotch’ cultivar PE-2 medium yielded recoveries comparable to PE-2 prepared with the other pea cultivars, turbid tubes were occasionally observed from which viable microorganisms could not be recovered. This cultivar is therefore not recommended due to the probability of yielding false positives in the MPN technique.

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.

Growth of Salmonella during Sprouting of Alfalfa Seeds Associated with Salmonellosis Outbreaks

2001 ◽  
Vol 64 (5) ◽  
pp. 618-622 ◽  
Author(s):  
D. S. STEWART ◽  
K. F. REINEKE ◽  
J. M. ULASZEK ◽  
M. L. TORTORELLO
Keyword(s):  
Enzyme Immunoassay ◽  
Water Samples ◽  
The Other ◽  
Most Probable Number ◽  
Calcium Hypochlorite ◽  
Probable Number ◽  
Two Samples ◽  
Rinse Water ◽  
Commercial Enzyme Immunoassay ◽  
Alfalfa Seeds

Growth of Salmonella was assessed during sprouting of naturally contaminated alfalfa seeds associated with two outbreaks of salmonellosis. Salmonella was determined daily in sprouts and sprout rinse water samples by a three-tube most probable number (MPN) procedure and a commercial enzyme immunoassay (EIA). Growth of Salmonella in the sprouts was reflected in the rinse water, and the MPNs of the two samples were generally in agreement within approximately 1 log. The results from EIA testing of sprouts and water samples were also in agreement. The pathogen was present in the seed at less than 1 MPN/g, and it increased in number to maximum population levels of 102 to 103 MPN/g in one seed lot and 102 to 104 MPN/g in the other seed lot. Maximum populations of the pathogen were apparent by day 2 of sprouting. These results show the ability of the pathogen to grow to detectable levels during the sprouting process, and they provide support for the recommendation to test the sprout water for the presence of pathogens 48 h after starting seed sprouting. The effectiveness of a 10-min, 20,0000-μg/ml (ppm) calcium hypochlorite treatment of the outbreak-associated seeds was studied. For both seed lots, the hypochlorite treatment caused a reduction, but not elimination, of Salmonella contamination in the finished sprouts. These results confirm the need to test each production batch for the presence of pathogens, even after 20,000 μg/ml (ppm) hypochlorite treatment of seeds, so that contaminated product is not distributed.

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.

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.

Behavior of A Cell Line Derived from A Mouse Submaxillary Adenocarcinoma during the Initial 480 Days in Vitro

1978 ◽  
Vol 64 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Ida Blotta ◽  
Pacifico Meo ◽  
M. Cristina Giuliano ◽  
Marinella Bellocci ◽  
Rosella Ritrovato ◽  
...  
Keyword(s):  
Cell Line ◽  
Male Mouse ◽  
The Other ◽  
Plant Lectins ◽  
Viral Expression ◽  
Viral Particles ◽  
A Cell

A cell line was established from a transplantable adenocarcinoma, containing viral particles of the A and B type, derived from a tumor appearing spontaneously in the submaxillary region of a male mouse of the C3H/He strain. This line, after 480 days in vitro, did not change the original epithelial-like morphology, the viral expression, the membrane immunofluorescence and the degree of agglutination by various plant lectins. After 208 days of culture, the presence of up to 3 pairs of metacentric chromosomes appeared in about 55% of the cells. However, this change in the chromosomal pattern was not sufficient, at least within the limits of our observation, to modify significantly the other parameters investigated, with the possible exception of the oncogenicity, which showed a modest decrease after 296 days of culture.

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