Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

Vanhee, Lies M. E.; Nelis, Hans; Coenye, Tom

doi:10.1038/nprot.2008.228

Cited by 14 publications

(8 citation statements)

References 29 publications

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“…Solid phase cytometry‐based assays address these issues by automated counting of stained cells collected on membrane filters (Aurell et al ., 2004 ; Parthuisot et al ., 2011 ). However, this technique is hampered by low sensitivity (Hambsch et al ., 2010 ) and low throughput of a maximum of 15–20 samples per day (Aurell et al ., 2004 ) due to the time‐consuming and often difficult manual validation of potentially identified targets (Aurell et al ., 2004 ; Vanhee et al ., 2009 ). Also the scanning procedure may fail completely due to low signal intensity (Hambsch et al ., 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of total and viableLegionella pneumophilain tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

Keserue

Baumgärtner

Felleisen

et al. 2012

Microbial Biotechnology

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SummaryWe developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS‐FCM method). The method requires 120 min and can discriminate ‘viable’ and ‘membrane‐damaged’ cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also ‘viable but not culturable’ (VNBC) cells are detected by our method, this result was expected. The IMS‐FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1–12. This and the fact that no Lp‐containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS‐FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.

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Section: Introductionmentioning

confidence: 99%

Rapid detection of total and viableLegionella pneumophilain tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

Keserue

Baumgärtner

Felleisen

et al. 2012

Microbial Biotechnology

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“…In order to ensure the sterility of injectable pharmaceutical products, the sterility test must be completed and, in addition, it is necessary to have control of the environmental monitoring of air, surfaces, people and product pre-sterilization bioburden. Despite the fact that the traditional method is very efficient and has often been used for the release of injectable products, it has the disadvantages mentioned above (Pinto et al, 2010;Vanhee et al, 2009a). Thus, with the need to simplify the work involved, reduce the time consumed, and increase analytical capacity, reliability and accuracy, it is believed that rapid methods can yield results with the same degree of security and speed (Silveira, 2006).…”

Section: Discussionmentioning

confidence: 99%

Solid phase cytometry applied to sterility tests for injecting 0.9% sodium chloride

Silva¹,

Silva²,

Sã³

et al. 2015

Afr. J. Pharm. Pharmacol.

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Sterility tests described in official compendia are carried out by membrane filtration or by direct inoculation into suitable culture media. About 14 days are needed to provide results and release products for sale, so speed is of the essence in rapid microbiological methods. Solid phase cytometry is a fast innovative method for testing the sterility of injectable medications. It is based on the detection of viable cells by using reagent viability markers which permeate the cell membrane, and are cleaved by non-specific sterases to form fluorochrome, which is detected by a Chem Scan RDI®. This study set out to evaluate this technology when applied to the sterility test in a 0.9% sodium chloride injection solution, using Chem Scan RDI® equipment. Microorganisms recommended by the official compendia Clostridium sporogenes NCTC12935 (ATCC 11437), Pseudomonas aeruginosa NCTC12924 (ATCC 9027), Staphylococcus aureus NCTC10788 (ATCC 6538), Bacillus subtilis NCTC10400(ATCC 6633), Aspergillus brasiliensis NCPF2275 (ATCC16404) and Candida albicans NCPF3179(ATCC 10231), and two "in house" microorganisms, Micrococcus luteus and Staphylococcus epidermidis, obtained from monitoring the pre-sterilization bioburden, were evaluated in order to validate the proposed method. When the solid phase cytometry method was compared to the traditional membrane filtration sterility test for all the microorganisms tested, it was found to be significantly faster in that it reduced analysis time from 14 days to approximately 3 h.

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“…Subsequent highlighting of a green spot in the secondary scan map on the PC resulted in the direction of the microscope to the respective position on the membrane filter (2, 12), which allowed rapid visual inspection of all fluorescent spots. The red fluorescence of C. albicans cells can easily be observed using a 590-nm-cutoff emission filter (20). Preparation of spiking solutions.…”

Section: Methodsmentioning

confidence: 99%

“…These fungi were cultured overnight at 30°C in Sabouraud liquid medium, and serial dilutions were made in physiological saline. Subsequently, the number of cells was determined by SPC prior to spiking (20), and 1 ml of the obtained cell suspensions was used to spike the blood samples. For all spiking experiments, blood was freshly drawn from healthy volunteers, maintained at 4°C, and used in experiments the same day.…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, the data for each fluorescent spot are analyzed by a computer to differentiate between fluorescent microorganisms and particles. Each retained spot can be inspected visually by an epifluorescence microscope (11,20). Due to its low detection limit, speed, and possible use of taxonomic probes for identification, SPC has the potential to overcome the shortcomings of other methods for quantification of Candida species in blood samples (7,14).…”

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confidence: 99%

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Rapid and Direct Quantification of Viable Candida Species in Whole Blood by Use of Immunomagnetic Separation and Solid-Phase Cytometry

et al. 2010

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Candida species are a common source of nosocomial bloodstream infections in critically ill patients. The sensitivity of the traditional diagnostic procedure based on blood culture is variable, and it usually takes 2 to 4 days before growth of Candida species is detected. We developed a 4-h method for the quantification of Candida species in blood, combining immunomagnetic separation (IMS) with solid-phase cytometry (SPC) using viability labeling. Additionally, Candida albicans cells could be identified in real time by using fluorescent in situ hybridization. By analysis of spiked blood samples, our method was shown to be sensitive and specific, with a low detection limit (1 cell/ml of blood). In a proof-of-concept study, we applied the IMS/SPC method to 16 clinical samples and compared it to traditional blood culture. Our method proved more sensitive than culture (seven samples were positive with IMS/SPC but negative with blood culture), and identification results were in agreement. The IMS/SPC data also suggest that mixed infections might occur frequently, as C. albicans and at least one other Candida species were found in five samples. Additionally, in two cases, high numbers of cells (175 to 480 cells/ml of blood) were associated with an endovascular source of infection.

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Detection and quantification of viable airborne bacteria and fungi using solid-phase cytometry

Cited by 14 publications

References 29 publications

Rapid detection of total and viableLegionella pneumophilain tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

Rapid detection of total and viableLegionella pneumophilain tap water by immunomagnetic separation, double fluorescent staining and flow cytometry

Solid phase cytometry applied to sterility tests for injecting 0.9% sodium chloride

Rapid and Direct Quantification of Viable Candida Species in Whole Blood by Use of Immunomagnetic Separation and Solid-Phase Cytometry

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