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.

Use of Iron Milk Medium for Enumeration of Clostridium perfringens

1982 ◽  
Vol 65 (5) ◽  
pp. 1129-1133 ◽  
Author(s):  
William D St John ◽  
Jack R Matches ◽  
Marleen M Wekell
Keyword(s):  
Clostridium Perfringens ◽  
Incubation Time ◽  
Incubation Temperature ◽  
Egg Yolk ◽  
Most Probable Number ◽  
Biochemical Tests ◽  
Probable Number ◽  
Large Numbers ◽  
Whole Milk ◽  
Short Incubation Time

Abstract A simple iron milk medium was used for isolation and enumeration of Clostridium perfringens from soil, sludge, and water samples. The whole milk contained only iron powder as a reducing agent; no other inhibitors were added. The iron milk most probable number (MPN) procedure was compared with 4 plating media: sulfite-polymyxin-sulfadiazine, Shahidi-Ferguson perfringens, tryptose-sulfite- cycloserine (both with and without egg yolk), and tryptone-sulfite-neomycin. The selectivity of the iron milk relies solely on the rapid growth of C. perfringens at 45°C and the stormy fermentation reaction within 18 h. Isolates were confirmed as C. perfringens by standard biochemical tests. The iron milk MPN procedure compared very well with the 4 plating media tested. Selectivity of incubation temperature, short incubation time, and ease of identification by the characteristic stormy fermentation make this method ideal for enumerating C. perfringens from large numbers of samples.

scholarly journals Clearance of Human-Pathogenic Viruses from Sludge: Study of Four Stabilization Processes by Real-Time Reverse Transcription-PCR and Cell Culture

2004 ◽  
Vol 70 (9) ◽  
pp. 5434-5440 ◽  
Author(s):  
S. Monpoeho ◽  
A. Maul ◽  
C. Bonnin ◽  
L. Patria ◽  
S. Ranarijaona ◽  
...  
Keyword(s):  
Cell Culture ◽  
Anaerobic Digestion ◽  
Most Probable Number ◽  
Official Method ◽  
Probable Number ◽  
Complex Samples ◽  
Reverse Transcription Pcr ◽  
Pathogenic Viruses ◽  
Agricultural Use ◽  
The One

ABSTRACT Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed.

scholarly journals A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts

1999 ◽  
Vol 65 (9) ◽  
pp. 3936-3941 ◽  
Author(s):  
Theresa R. Slifko ◽  
Debra E. Huffman ◽  
Joan B. Rose
Keyword(s):  
Cell Culture ◽  
Most Probable Number ◽  
Recreational Waters ◽  
Probable Number ◽  
Cell Monolayers ◽  
Water Testing ◽  
Before And After ◽  
A Cell ◽  
Cryptosporidium Parvum Oocysts ◽  
Focus Detection

ABSTRACT Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.

Detection of Staphylococcus aureus Within Starter Culture Preparations Containing Staphylococcus carnosus

1994 ◽  
Vol 57 (11) ◽  
pp. 981-984 ◽  
Author(s):  
DÖRTHE FEUERSENGER ◽  
HERMANN J. KNAUF ◽  
JÜRGEN BAUMGART
Keyword(s):  
Staphylococcus Aureus ◽  
Detection Limit ◽  
Incubation Temperature ◽  
Starter Culture ◽  
Microbiological Quality ◽  
Most Probable Number ◽  
Plate Method ◽  
Probable Number ◽  
Staphylococcus Carnosus ◽  
Drop Plate

A method is described that permits the detection of contamination by Staphylococcus aureus within starter culture preparations containing Staphylococcus carnosus. Using selective media and raised incubation temperature it was possible to distinguish between starter and contamination organisms. The detection limit was 103 S. aureus within 1011 S. carnosus by the drop-plate method. With a modified most probable number (MPN) technique the detection limit was even lower. The time needed varied between 24 and 72 h depending on the concentration of the contaminant and the method used. The method described therefore proved to be suitable for routine microbiological quality control regarding the microbiological status of starter preparations containing bulk starter staphylococci. The drop-plate method furthermore proved to be suitable for the detection of S. aureus within starter preparations of pediococci and lactobacilli.

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.

Inactivation Kinetics and Factors of Variability in the Pulsed Light Treatment of Listeria innocua Cells

2007 ◽  
Vol 70 (11) ◽  
pp. 2518-2525 ◽  
Author(s):  
AARON R. UESUGI ◽  
SARAH E. WOODLING ◽  
CARMEN I. MORARU
Keyword(s):  
Stainless Steel ◽  
Weibull Model ◽  
Inoculum Size ◽  
Listeria Innocua ◽  
Most Probable Number ◽  
Inactivation Kinetics ◽  
Xenon Lamp ◽  
Pulsed Light ◽  
Probable Number ◽  
Log Reduction

Pulsed light (PL) treatment can effectively reduce microbial populations in clear substrates and on surfaces, but its effectiveness varies as a function of substrate or treatment-related factors. For PL to be successfully adopted by the food industry, all factors of influence, as well as the inactivation kinetics for the microorganisms of concern, must be elucidated. In this study, the inactivation kinetics of Listeria innocua and the effect of inoculum size on PL inactivation were investigated. Stainless steel coupons (50.8 by 101.6 mm) of defined surface properties and transparent glass chamber slides (25.4 by 50.8 by 10 mm) were each inoculated with 1 ml of aqueous suspensions of L. innocua containing inoculum populations of up to 109 CFU. The thickness of the liquid layer in the glass slides was 1.16 mm. The inoculated substrates were exposed to PL treatment of up to 17 J/cm2 in a static PL chamber equipped with a pulsed Xenon lamp. Survivors were recovered and enumerated by both standard plate counting and most-probable-number procedures. The data indicated that in clear liquids, PL resulted in more than a 6-log reduction of L. innocua after a 12-J/cm2 treatment, regardless of the initial inoculum size. For the stainless steel surfaces, less than a 4-log reduction after a 12-J/cm2 treatment and a noticeable effect of substrate characteristics and inoculum size on inactivation were observed. The survivor curves showed pronounced tailing for all substrates used in the study. The Weibull model accurately predicted the survivor ratios for the PL treatment of L. innocua in clear liquids, with a shape and scale parameter of 0.33 and 3.01, respectively. The Weibull model resulted in significant overestimation of PL effectiveness for the stainless steel substrates, where the influence of various substrate properties and inoculum level on inactivation was significant.

scholarly journals The effect of concanavalin A on the replication of rotavirus (SA-11) in cell culture

2002 ◽  
Vol 45 (2) ◽  
pp. 125-135 ◽  
Author(s):  
Beatriz S. Hasenack ◽  
Maria Valéria J. Botelho ◽  
Flávio Lauretti ◽  
Fernando L. de Melo ◽  
Janaina M. Orlandi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Cell Cultures ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Cytopathic Effect ◽  
Polyacrylamide Gel Electrophoresis ◽  
Plaque Assay ◽  
Virus Growth ◽  
Kinetics Of

Rotavirus (RV) strain SA-11 was studied with respect to its infectivity in MA-104 cell cultures and the effect of concanavalin A (ConA). Receptors for ConA at the surface of MA-104 cell were determined by fluorescence assay and specifically inhibited by D-mannose. The kinetics of virus growth was carried out by plaque assay. Electron microscopy and polyacrylamide gel electrophoresis were used for monitoring the experiments. It was concluded that RV replication was not affected consistently by ConA, however it interfered with the development of cytopathic effect (CPE) without altering virus yields.

Efficacy of Petrifilm™ VRB for Enumerating Coliforms and Escherichia coli from Frozen Raw Beef

1987 ◽  
Vol 50 (12) ◽  
pp. 1017-1022 ◽  
Author(s):  
LAWRENCE RESTAINO ◽  
RICHARD H. LYON
Keyword(s):  
Escherichia Coli ◽  
Incubation Temperature ◽  
Ground Beef ◽  
Gas Bubbles ◽  
Most Probable Number ◽  
Type I ◽  
Type Ii ◽  
Probable Number ◽  
E Coli ◽  
Colony Diameter

Petrifilm™ violet red bile (PVRB) compared favorably to the most probable number method (MPN) and violet red bile agar (VRBA) methods for enumerating coliforms from frozen raw ground beef. When comparing PVRB and VRBA incubated at 35°C, coliform enumeration displayed a linear relationship (correlation coefficient of 0.932). However, by analyzing 64 ground beef samples, PVRB enumerated 41% more coliforms/g than did VRBA. Two distinct colony types were observed on PVRB: (a) type I (butterfly in appearence) with a colony diameter equal to or greater than 1 mm and gas bubbles 2–4 mm in diameter touching the associated colony; and (b) type II with a colony diameter less than 1 mm in diameter and gas bubbles of the associated colony not necessarily touching the colony but within a colony diameter. The disparity between PVRB and VRBA for enumerating coliforms was attributed to non-coliforms representing approximately 50% of the type II coliform colonies. At 35°C, 83.7% of the type I colonies were Escherichia coli, whereas only 10.9%, of the type II colonies were E. coli. By elevating the incubation temperature from 35°C to 44.5°C, over 90% of the colonies in the counting dilution were type I of which 99.2% were E. coli. At 44.5°C, 39.4% of the type II colonies were E. coli; however, this colony type represented only 9.5% of the total colonies on PVRB. Therefore, a reliable method for enumerating E. coli from raw meat was developed by counting only the type I colonies on PVRB incubated at 44.5°C.

Effect of Storage Temperature on the Survival of New Zealand Egg-Associated Salmonella Isolates in and on Eggs

2019 ◽  
Vol 82 (12) ◽  
pp. 2161-2168 ◽  
Author(s):  
JOANNE M. KINGSBURY ◽  
KIRSTIN THOM ◽  
TANYA SOBOLEVA
Keyword(s):  
New Zealand ◽  
Incubation Temperature ◽  
Storage Temperature ◽  
Limit Of Detection ◽  
Most Probable Number ◽  
Probable Number ◽  
Egg Storage ◽  
Chicken Feces ◽  
Storage Times ◽  
Storage Temperatures

ABSTRACT The influence of egg storage temperature on Salmonella contamination of eggs is a key consideration in determining storage and shelf life recommendations for eggs. In this study, experiments assessed the survival of Salmonella isolates on and in eggs at storage temperatures (15 and 22°C) currently used in New Zealand. Eggshell surfaces were inoculated with a cocktail of 10 Salmonella isolates comprising five serotypes, at a concentration of ∼106 CFU per egg (for determining shell surface survival) or ∼103 CFU per egg (for determining internalization). Additionally, a subset of eggs was artificially contaminated with sterile chicken feces prior to Salmonella inoculation. Inoculated eggs were incubated at 15 and 22°C. At 0, 21, and 35 days of incubation, eggshells were enumerated for Salmonella, and egg contents were tested for Salmonella presence or absence (yolk) or most probable number (albumen). Higher levels of Salmonella were recovered from eggshells following incubation at 15°C (31% relative humidity [RH]) compared with 22°C (45% RH) after both 21 and 35 days of incubation. Recoverable numbers of Salmonella from visibly clean eggshell surfaces declined over time at both storage temperatures and were at, or below, the limit of detection from eggs stored at 22°C and 45% RH for 35 days. A substantially higher concentration of viable Salmonella was recovered from eggshells that were experimentally contaminated with chicken feces compared with those without, particularly from eggs stored at 15°C and 31% RH for 35 days (2.38 log higher CFU from eggs containing feces). No Salmonella was detected in egg contents (albumen or yolk) at any incubation temperature or time point, regardless of the presence of feces. Findings emphasize the importance of current regulations that require eggs sold at retail to be visibly clean and will inform risk management decisions regarding egg storage times and temperatures with respect to Salmonella control in and on New Zealand eggs at retail. HIGHLIGHTS

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