Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes.

Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.     

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye–DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THO1 locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.     

Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters

Steady-state fluorescence polarization measurements obtained with a flow cytometer were compared with those obtained with an SLM subnanosecond fluorometer. Measurements were made over time after exposure of HeLa cells to the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), or [12-(9:anthroyloxy) stearate (12-AS). After 1 min, anisotropy values of 0.28 (DPH), 0.28 (TMA-DPH), and 0.21 (12-AS) were obtained. Thereafter, the anisotropy of DPH- and 12-AS-labelled cells rapidly decreased (0.18 and 0.12 after 5 min), while that of TMA-DPH-labelled cells changed only slightly (0.27 after 30 min), suggesting that DPH and 12-AS, unlike TMA-DPH, do not remain anchored in the HeLa plasma membrane, but translocate to more fluid environments inside the cell. These suggestions were confirmed by visual observation with fluorescence microscopy. There was no significant difference between the results obtained with the flow cytometer and those obtained with the fluorometer.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

Optimisation of flow cytometric measurement of parasitaemia in plasmodium-infected mice

Mouse malaria is often used as a model for drug testing. The results of drug trials are monitored by tedious (and consequently, sometimes inaccurate) microscopic counting of blood smears, or by flow cytometry. We suggest an improved, accurate and time-saving flow cytometric method for determination of parasitaemias in mice infected with Plasmodium vinckei petteri or Plasmodium berghei. The method involves collection of drops of blood from the tail vein, fixation, storage, permeabilisation, staining and analysis with a visible range flow cytometer. Three nucleic acid dyes, YOYO-1, propidium iodide and acridine orange were compared. YOYO-1 was found to be the best stain for the discrimination of parasitised erythrocytes from non-infected ones. A good direct correlation was obtained between parasitaemia determined by conventional microscopy and parasitaemia measured by flow cytometry. Drug effects could be assessed by the cytometric method. For the detection of low level of parasitemia, parasitised cells were treated with RNAse to completely cancel RNA-derived signals originating from host reticulocytes. This procedure also revealed discrete peaks arising from red cells infected with multiple parasites or from parasites with different numbers of nuclei.     

一种基于荧光强度分析的高通量定量检测细胞凋亡方法

根据正常细胞、凋亡细胞和坏死细胞的细胞膜对核酸荧光染料的不同选择通透性,用4μmol/L YO-PRO-1（YP）和4μg/ml 碘化丙啶（Propidium iodide, PI）染色96孔板中的细胞样品。分别在485/538 (Ex/Em, nm) 和530/590 (Ex/Em, nm) 的检测波长下借助荧光分光光度计检测细胞样品孔的YP和PI荧光强度。将YP和PI荧光强度值与用荧光显微镜对同一细胞样品细胞凋亡和坏死的定量分析结果相对应,通过对YP荧光强度值与凋亡细胞数的直线回归分析 (r = 0.999,P<0.01),得到依据YP荧光强度值求得凋亡细胞数的直线相关方程。该方法可检测出样品中少至180个的凋亡细胞,具有灵敏度高和快速高效的特点。     

Increased sensitivity of a direct fluorescent antibody test for Legionella pneumophila in bronchoalveolar lavage samples by immunomagnetic separation based on BioMags

In the present study, immunomagnetic separation of Legionella pneumophila from mock bronchoalveolar lavage (BAL) fluid samples, which were artificially spiked with L. pneumophila, and culture positive patient BAL fluid samples, was achieved using BioMags (superparamagnetic particles) loaded with purified rabbit immunoglobulin G specific for L. pneumophila. Bacteria binding onto BioMag-immunomatrix were directly stained with a L. pneumophila species-specific DFA reagent and examined under a fluorescence microscope. BioMag-based immunomagnetic separation (BIMS) followed by DFA staining (BIMS-DFA) could correctly identify all the 20 (100%) BAL samples which were spiked with low numbers (2x10(2) CFU) of L. pneumophila. Cultures could be recovered from 15 (75%) of these 20 spiked BAL samples, 5 (25%) of the samples failed to yield positive cultures. Both culture and BIMS-DFA methods showed 100% positive results when spiked BAL samples containing high bacterial load (10(4) CFU) were tested. The findings with true patient culture positive BAL specimens which were examined retrospectively indicated that BIMS-DFA is significantly more sensitive for detecting L. pneumophila than conventional cytospin method of DFA staining (cytospin-DFA). Out of the 25 culture positive BAL specimens tested, 7 (28%) proved negative by cytospin-DFA whereas BIMS-DFA correctly detected all the 25 (100%) specimens. It is suggested that the BIMS-DFA procedure increases the sensitivity of DFA testing for L. pneumophila in large volume samples such as BAL fluids.     

Flow microsphere immunoassay-based method of virus quantitation.

A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach. Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples. After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer. The adsorption of virus to microspheres was examined under different experimental conditions. The flow cytometric assay was determined to be as accurate in detecting adenovirus as titering on 293 cells. The proposed method can be used to quantify virus in viral stocks and in biological samples.     

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.     

A comparison of methods for counting viruses in aquatic systems

In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1, 3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10(8) particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.     

TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation.     

A Comparison of Methods for Counting Viruses in Aquatic Systems

In this study, we compared different methods—including transmission electron microscopy—and various nucleic acid labeling methods in which we used the fluorochromes 4′,6′-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3′-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10⁸ particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.     

The flow cytometry of Bacillus anthracis spores revisited

BACKGROUND: The potential use of Bacillus anthracis spores as a weapon of terror has rekindled interest in the rapid detection and identification of the spores of these bacteria. Prior efforts to utilize flow cytometry (FCM) for this purpose resulted in tedious and time-consuming protocols. Advances in rapid immunoassays suggest a reinvestigation of the use of FCM because this may allow for the development of a rapid and sensitive system for detection and/or identification of spores in suspect samples. METHODS: In this study, antiserum was raised in goats using three different strains of B. anthracis spores as the immunogen. The resultant antibodies were purified, labeled with fluorescein, and evaluated for use in an immunoassay on a Coulter Epics XL flow cytometer. In the protocol that was developed, fluorescein-labeled antibodies are simply mixed with the sample, allowed to incubate, and then analyzed on the flow cytometer. Washes and centrifugation were eliminated. RESULTS: The results showed that a rapid (5 min) and sensitive immunological analysis was feasible. The detection limit (approximately 10(3) colony-forming units [CFU]/ ml) varied with strain, but there was no difference in the detection limit between live and irradiated spores. In addition, the power of FCM was utilized to minimize false-positive reactions among similar species of Bacillus by placing constraints on scatter and fluorescence intensity. The data also suggest that scatter might be useful to determine spore viability. CONCLUSION: This study shows that FCM may be an effective platform on which to perform immunological analysis for the detection and/or presumptive identification of B. anthracis spores. Published 2000 Wiley-Liss, Inc.     

Fluorescence spectra of cells stained with a DNA-specific dye, measured by flow cytometry

Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.     

PCR-BASED FLUORESCENT METHOD FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN POULTRY SAMPLES

A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 10⁵ CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

Evaluation of a leukocyte esterase and nitrite test strip for detection of bacteriuria

The LN Chemstrip^® leukocyte esterase (LE) spot and the nitrite (N) spot were compared separately and in combination with colony-forming units (CFU/ml) in 628 urines. Using either a positive LE or a positive N as an indicator of significant bacteriuria (10⁵/ml), the sensitivity, specificity, and the negative predictive values were high, but the positive predictive value was low. When LE and N were both negative, there was a close correlation with CFU/ml of less than 20,000. For reactions other than LE-N-, a bimodal response was observed with most bacterial counts split between 20,000 CFU/ml and 10⁵ CFU/ml.     

Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry

BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer. RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure. CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.     

Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity

By use of a flow cytometer, the fluorescence of cells stained with hematoporphyrin derivative and the fluorescence of plastic beads stained with different dyes were analysed as a function of the intensity of the exciting laser light. The ratios of the fluorescence values of stained and unstained cells as well as of stained cells and beads were sensitively dependent on excitation intensities. As a consequence of this finding, the normalization of cellular fluorescence by use of reference particles needs to be made on a well-defined and reproduced intensity of the exciting laser light.