A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk

Gunasekera, Thusitha S.; Attfield, Paul V.; Veal, Duncan

doi:10.1128/aem.66.3.1228-1232.2000

Cited by 242 publications

(171 citation statements)

References 25 publications

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“…10 4 and the dynamic range was from 10 4 to 10 6 cells per mL. Similar results have been found for detection of Salmonella typhimurium in eggs and milk with immuno-FCM (McClelland & Pinder, 1994;Gunasekera et al, 2000). The detection level of immunofluorescence microscopy (IF) was ten times lower, whereas dilution plating was even more sensitive.…”

Section: Discussionsupporting

confidence: 74%

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Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Tebaldi¹,

Peters²,

Souza³

et al. 2010

Trop. plant pathol.

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Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 10 3 -10 7 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 10 3 -10 6 CFU/mL and immuno-FCM from 10 4 -10 6 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds. Key words: seed pathology, flow sorting, PCR-amplification, viability probes, immunofluorescence, bacteria. RESUMO Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidadeA combinação do uso do citômetro de fluxo (FCM) e de anticorpo policlonal (imuno-FCM) foi comparada à microscopia de imunofluorescência (IF) e ao plaqueamento em meio de cultura semi-seletivo, para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijão. Concentrações de Xap variando de 10 3 a 10 7 CFU/mL foram adicionados aos extratos de sementes. Um método de separação pelo citômetro de fluxo foi desenvolvido para a detecção de Xap em extratos de semente e posterior confirmação por PCR. Para avaliação da viabilidade das células foram usadas sondas fluorescentes, iodeto de propídio (PI)/carboxi diacetato de fluoresceína (cFDA) e PI/SYTO 9 e também, a combinação de imuno-FCM e PI. Em meio semi-seletivo e IF foram detectadas 10 3 -10 6 UFC/mL e no FCM 10 4 -10 6 UFC/mL, em extratos de sementes artificialmente infestados. Xap somente foi detectada em extratos de sementes por PCR, após o processo de separação pelo FCM. Foi possível pelo FCM a quantificação e identificação de células viáveis (verde fluorescente) e células mortas (vermelho fluorescente) de Xap, pelas sondas cFDA/SYTO 9 e PI, respectivamente. A combinação de immuno-FCM e PI poderá ser uma técnica promissora e segura para a detecção deste patógeno em sementes. Palavras-chave: patologia de sementes, PCR, sondas de viabilidade, imuno-fluorescência, bactéria. INTRODUCTIONXanthomonas axonopodis pv. phaseoli (Smith) Vauterin, Hoste, Kersters & Swinges (Xap) is a seedtransmitted plant-pathogenic bacterium w...

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Section: Discussionsupporting

confidence: 74%

“…The combination of FCM and DVC methods with fluorescent probes has been used for the detection of bacteria in water (Lebaron et al, 1998), food (Gunasekera et al, 2000) and phytopathogenic bacteria such as C. michiganensis subsp. michiganenis and X. campestris pv.…”

Section: Introductionmentioning

confidence: 99%

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Tebaldi¹,

Peters²,

Souza³

et al. 2010

Trop. plant pathol.

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“…The quality of the bulk tank milk is normally assessed by numerous parameters and among these is the total bacterial count, which is considered to reflect the general hygienic condition at the farm. Techniques based on flow cytometric principles are routinely applied in the dairy industry for measuring the total bacterial count within few minutes (Suhren and Walte 1998;Gunasekera et al 1999;Bolzoni et al 2000). However, these techniques do not differentiate between different species and therefore do not provide information on the cause of an elevated bacterial count.…”

Section: Introductionmentioning

confidence: 99%

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

Holm

Mathiasen²,

Jespersen³

2004

J Appl Microbiol

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Aims: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. Methods and Results: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3AE0 · 10 4 CFU ml )1 were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0AE71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows: staining with Oregon Green Ò conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. Conclusions:The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. Significance and Impact of the Study: This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.

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“…Current research illustrates a poor correlation between standard plate counts and cytometric screening due to non-specific binding of fluorescent dyes to protein particles (Gunasekera et al, 2000), which failed to be adequately removed by enzymatic treatments or commercial protein-clearing agents (McClelland and Pinder, 1994a, b). Meanwhile, Doherty et al, (2010) further resolved the negative effect of environmental proteins by introducing a mild homogenisation step in order to break-down cellular chains for the provision of true cytometric cell counts.…”

Section: Cell Release Mechanism For Enhanced Cytometric Screeningmentioning

confidence: 99%

New Insights into Cell Encapsulation and the Role of Proteins During Flow Cytometry

Doherty¹,

Brodkorb²

2012

Flow Cytometry - Recent Perspectives

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A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk

Cited by 242 publications

References 25 publications

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

New Insights into Cell Encapsulation and the Role of Proteins During Flow Cytometry

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