Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies

McClelland, Rosemary G.; Pinder, Andrew C.

doi:10.1128/aem.60.12.4255-4262.1994

Cited by 93 publications

(37 citation statements)

References 39 publications

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“…Few studies have addressed the minimum concentration of bacteria detectable by flow cytometry, but limits in the region of 10 3 to 10 4 bacterial cells ml −1 are reportedly detectable using viability stains such as rhodamine 123 (Diaper and Edwards 1994) and nucleic acid stains such as ethidium bromide (Pinder et al 1990). The present authors have previously reported immunofluorescent labelling techniques which allow the detection of two species of salmonellas simultaneously, down to levels of 1×10 3 ml −1 in pure cultures (McClelland and Pinder 1994a), and 1×10 3 and 1×10 4 cells ml −1 in milk and egg, respectively (McClelland and Pinder 1994b ;Pinder and McClelland 1994). The total analysis time in all these cases was about 30 min.…”

Section: Introductionmentioning

confidence: 80%

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers

Clarke

Pinder

1998

J Appl Microbiol

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A proprietary fluorogenic marker for cell viability (Chemchrome) was investigated for the detection of bacteria using flow cytometry. This marker was used in combination with fluorescently labelled monoclonal antibodies (against Salmonella typhimurium). Owing to the former's broad band emission spectrum, it was necessary to use the novel dye RED613 for the antibodies. This combined protocol, being sensitive only to the live Salm. typhimurium cells, reduced errors due to intrinsic fluorescence and non‐specific binding. Detection of the order of 100 cells ml−1 was achieved in 30 min. This level was achieved even in the presence of large numbers of non‐target or dead organisms.

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Section: Introductionmentioning

confidence: 80%

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers

Clarke

Pinder

1998

J Appl Microbiol

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“…This threshold, which occurred in the range of 10 4 organisms ml 31 , was similar to that reported in studies using FC to detect Salmonella spp. in eggs and milk [8]. Interestingly, the problem appeared to be unrelated to the fact that the samples were derived from a food matrix with a heterogeneous background £ora, since studies with mixtures of pure cultures in PBS had a similar lower limit of E. coli O157:H7 measurement.…”

Section: Discussionmentioning

confidence: 99%

“…One approach to this problem has been the use of multiparameter analysis in which a strong £uorescence signal for all bacteria has been provided by nucleic acid binding dyes or by labeled antibody. A second £uorescence signal such as FITC was then used to identify the organism of interest [8]. Through the use of log-ampli¢ed FLS signals together with careful alignment and cleaning of optical and £uidic systems of the stream in quartz FC system we were able to achieve acceptable light scatter sensitivity without using nucleic acid binding dyes which would been incompatible with viable cell sorting.…”

Section: Discussionmentioning

confidence: 99%

“…While FC has been frequently used to analyze bacteria in pure cultures [4,5] it has seen more limited use with heterogeneous natural samples such as those encountered in food microbiology [6]. Foodrelated applications include detection of Listeria monocytogenes in milk [7] and Salmonella typhimurium in milk and eggs [8]. The latter investigators also used enrichment cultures and a custom-designed £ow cytometer to achieve a higher level of sensitivity [9].…”

Section: Introductionmentioning

confidence: 99%

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Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting

Tortorello¹

1997

FEMS Immunology and Medical Microbiology

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Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

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“…With experiments involving spiked milk samples, a Promega). The effect was to remove fat micelles from the milk, improving resolution of the bacteria (McClelland & Pinder, 1994b).…”

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confidence: 99%

Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry

Pinder

McClelland

1994

Journal of Microscopy

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Summary Multi‐parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full‐fat milk). In all cases, the method was accurate to levels of below 104 target cells per ml for a total assay time of about 30 min. After 6 h non‐selective enrichment in the presence of a 10 000‐fold excess of competing micro‐organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml−1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro‐organisms in the food industry.

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Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies

Cited by 93 publications

References 39 publications

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers

Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers

Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting

Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry

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