A novel two‐color flow cytometric assay for the detection of Cryptosporidium in environmental water samples

Ferrari, Belinda C.; Vesey, Graham; Davis, Kenneth A.; Gauci, Mark; Veal, Duncan

doi:10.1002/1097-0320(20001101)41:3<216::aid-cyto9>3.3.co;2-i

Cited by 16 publications

(22 citation statements)

References 11 publications

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“…Clumps of cells from different taxonomic groups in concentrated environmental samples, together with cell losses and low signal-to-background noise, have been pointed out as major problems for routine application of this combination of techniques (3). Inherent difficulties also arise from the presence of autofluorescent particles, such as minerals and algae, in environmental samples and nonspecific binding of fluorescent probes to detritus particulates (8). In applying our protocols to E. coli-spiked seawater samples, we also encountered significant "unwanted" signals coming from both autofluorescence and nonspecific binding that interfered with the signals of the hybridized E. coli.…”

Section: Discussionmentioning

confidence: 99%

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Tang

Gin

Lim

2005

Appl Environ Microbiol

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Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

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Section: Discussionmentioning

confidence: 99%

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Tang

Gin

Lim

2005

Appl Environ Microbiol

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“…These include both flow cytometry-based techniques and solid-phase image scanning cytometry techniques (3,4,(6)(7)(8). The latter usually uses a planar multielement photodetector, such as a charge-coupled device (CCD) camera, to make images from adjacent elements across the specimen, followed by scene segmentation and use of pattern recognition algorithms to locate the particular targets (9)(10)(11)(12)(13)(14)(15).…”

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confidence: 99%

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Lu¹,

Jin²,

Leif³

et al. 2011

Cytometry Pt A

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Many microorganisms have a very low threshold (\10 cells) to trigger infectious diseases, and, in these cases, it is important to determine the absolute cell count in a lowcost and speedy fashion. Fluorescent microscopy is a routine method; however, one fundamental problem has been associated with the existence in the sample of large numbers of nontarget particles, which are naturally autofluorescent, thereby obscuring the visibility of target organisms. This severely affects both direct visual inspection and the automated microscopy based on computer pattern recognition. We report a novel strategy of time-gated luminescent scanning for accurate counting of rare-event cells, which exploits the large difference in luminescence lifetimes between the lanthanide biolabels, [100 ls, and the autofluorescence backgrounds, \0.1 ls, to render background autofluorescence invisible to the detector. Rather than having to resort to sophisticated imaging analysis, the background-free feature allows a single-element photomultiplier to locate rare-event cells, so that requirements for data storage and analysis are minimized to the level of image confirmation only at the final step. We have evaluated this concept in a prototype instrument using a 2D scanning stage and applied it to rare-event Giardia detection labeled by a europium complex. For a slide area of 225 mm 2 , the time-gated scanning method easily reduced the original 40,000 adjacent elements (0.075 mm 3 0.075 mm) down to a few ''elements of interest'' containing the Giardia cysts. We achieved an averaged signal-to-background ratio of 41.2 (minimum ratio of 12.1). Such high contrasts ensured the accurate mapping of all the potential Giardia cysts free of false positives or negatives. This was confirmed by the automatic retrieving and time-gated luminescence bioimaging of these Giardia cysts. Such automated microscopy based on time-gated scanning can provide novel solutions for quantitative diagnostics in advanced biological, environmental, and medical sciences. ' 2011 International Society for Advancement of Cytometry

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“…Single-color flow cytometry has been used to identify microsporidia belonging to the genus Encephalitozoon (21), and two-color flow cytometry has been used to identify C. parvum oocysts in environmental water samples (8) and to quantify C. parvum oocysts in the feces of infected mice using TruCount tubes (16). Specific antibodies conjugated with fluorochrome have been used in flow cytometry to quantify trophozoites of Giardia duodenalis (4).…”

Section: Discussionmentioning

confidence: 99%

Purification of Enterocytozoon bieneusi Spores from Stool Specimens by Gradient and Cell Sorting Techniques

et al. 2004

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A three-step method for the purification of Enterocytozoon bieneusi spores from stool specimens was developed. The primary process of purification of the spores from bacterial contaminants involved Percoll gradient centrifugation followed by additional separation using cesium chloride density gradient centrifugation. The cesium chloride-isolated spores were further purified using a flow cytometer with cell sorting capabilities. Sorting was performed without the use of antibodies, fluorochromes, or dyes, leaving the sorted spores in their native state, which appears to be less destructive for spores. When quantified by flow cytometry using tubes with known numbers of highly fluorescent polystyrene beads, the sorted material showed a slight decrease in light scatter characteristics compared with the slightly larger Encephalitozoon species spores. Although the overall recovery of the E. bieneusi spores was low, calcofluor and Gram chromotrope staining, indirect immunofluorescence assay, and transmission electron microscopy revealed that the sorted material was highly purified and contained large numbers of E. bieneusi spores and relatively few bacteria and other debris. The sorted material appeared to be sufficiently pure and could be used for in vitro culture and for the development of a variety of diagnostic reagents as well as in studying the genome of E. bieneusi and host-parasite interactions.Microsporidia are opportunistic intracellular parasites of the phylum Microspora. Of the more than 1,000 species and as many as 100 genera of microsporidia, only 12 well-defined species (Brachiola vesicularum, Brachiola algerae, Brachiola conori, Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Enterocytozoon bieneusi, Nosema ocularum, Trachipleistophora hominis, Trachipleistophora anthropophthera, Pleistophora ronneafiei, and Vittaforma corneae)

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A novel two‐color flow cytometric assay for the detection of Cryptosporidium in environmental water samples

Abstract: A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium.

Cited by 16 publications

References 11 publications

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Automated detection of rare‐event pathogens through time‐gated luminescence scanning microscopy

Purification of Enterocytozoon bieneusi Spores from Stool Specimens by Gradient and Cell Sorting Techniques

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