Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders

AIMS: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. METHODS AND RESULTS: The BactiFlow flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. CONCLUSIONS: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.     

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

Optimization of flow-cytometric discrimination between reticulocytes and erythrocytes

An automatized technique to count reticulocytes by means of flow cytometry is described. Blood samples were stained by the fluorescent dye acridine orange without the use of fixative. Scatter and red fluorescence of the blood cells were measured in a flow cytometer. A discrimination between reticulocytes and erythrocytes was only achieved by using logarithmic amplification. The discrimination was better in peak mode than in area mode. The optimum dye concentration was 0.5 mg/liter acridine orange. At lower dye concentrations, not all reticulocytes were measured, whereas at higher dye concentrations the degree of discrimination between reticulocytes and erythrocytes decreased. There was a suitable discrimination between reticulocytes and erythrocytes. The reticulocyte numbers were scored by flow cytometry as well as by microscope for blood samples with 0.1-14% reticulocytes. The correlation between both methods was close.     

Evaluation of media and techniques to enumerate heterotrophic microbes from karst and sand aquifer springs

Several media and techniques were compared for their efficiency to enumerate viable heterotrophs from both a karst and sand aquifer spring. A medium designed to enumerate bacteria from nutrient-poor waters (HCFU) as well as R2A medium proved superior to tryptic soy agar; however, the difference was always less than one order of magnitude. Membrane filtration resulted in lower counts of microbes than the spread plate, multitube turbidity, or drop plate methods from samples of both sand and karst springs. The drop plate technique yielded higher viable counts from the sand spring and basin of the karst spring, with a precision of 21% (coefficient of variation) and a maximum plating efficiency of 3.4% (viable count/direct count × 100). Subsequently, 63% of isolates from drop plates were recovered on HCFU. Microcolonies were visible by epifluorescence microscopy, acridine orange staining, and subsequent examination of excised agar sections containing drops. Correspondence to: A.T. Mikell, Jr.     

Binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) analyzed by flow cytometry

We have developed a method for quantitatively assessing binding, ingestion, and growth of Chlamydia trachomatis (L2 serovar) in several mammalian cell lines using fluorescence staining and flow cytometry. Cells were incubated with chlamydia at 4 degrees C to monitor binding; ingestion was determined by raising the temperature to 37 degrees C for 1-4 h and removing extracellular bacteria with pronase. Growth of bacteria was measured by assessing brightly stained intracellular inclusions. Fixation with methanol prior to fluorescent staining provided the most intense specific staining with minimal background, as well as preserving cell morphology. Our data reveal relatively slow ingestion of L2 by McCoy fibroblasts (maximum ingestion by 4 h) and a sizeable population of McCoy cells (30-40% of total cells) that ingest L2 but do not permit its growth under certain infectious conditions. It was possible to correlate specific histogram patterns on the flow cytometer with fluorescent microscope observations. This system provides a means of analyzing quantitative interactions between chlamydia and individual host cells.     

Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.     

Zymomonas mobilis cell viability: measurement method comparison

Comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied onZymomonas mobilis cultures, was performed. The slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated theZymomonas cell viability. The slide technique is advantageous also because it gives information on the cell morphology changes, notably the abnormal cell elongation, in the ethanol fermentation.     

Susceptibility of Fusobacterium nucleatum to killing by peroxidase-iodide-hydrogen peroxide combination in buffer solution and in human whole saliva

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.     

Flow cytometric discrimination between Acinetobacter calcoaceticus 69-V and Alcaligenes eutrophus JMP134 by fluorescently labelled rRNA-targeted oligonucleotide probes and DNA staining

In situ hybridization with fluorescently monolabelled rRNA-targeted oligonucleotide probes (17 to 18 nucleotides) was used to discriminate between Alcaligenes eutrophus JMP 134 and Acinetobacter calcoaceticus 69-V by flow cytometry. The strains were grown in batch experiments in a mixed population. The forward light scatter and fluorescence of each bacterial cell were measured with a single laser cytometer. The intensity of fluorescence after rRNA staining depended on the content of ribosomes, which correlated with the growth rate of bacteria. Therefore exponentially growing cells could be clearly detected. For other growth phases, signal amplification was necessary using multiple probes. The two bacterial strains were identified with differently labelled probes under an epifluorescent microscope. Using a single laser cytometer, rRNA based identification was possible nut not ideal. Better discrimination between the two strains of the mixed population was achieved by DNA staining, combined with the different forward light scatter signals. Due to the significantly different cellular DNA and GC content of both strains, the fluorescent dye DAPI (4′, 6-diamidino-2-phenylindole), preferring AT-rich regions of DNA, was found to be a supplementary tool for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.     

二烯丙基二硫上调Beclin1 介导白血病K562 细胞自噬性死亡的研究

目的：通过二烯丙基二硫诱导白血病K562 细胞发生自噬性死亡,探讨其作用机制。方法：40 mg/LDADS 作用K562 细胞12 小时后,透射电镜观察K562 细胞超微结构,MDC 染色荧光显微镜观察自噬泡及流式细胞仪定量检测自噬率,RT-PCR 检测Beclin1mRNA 的表达水平。结果：DADS 作用后的K562 细胞后,透射电镜可观察到胞质内出现大量自噬体;MDC染色荧光显微镜观 察显示,K562 细胞胞浆中的自噬泡明显增多,而空白组与溶媒组胞浆中的自噬泡很少;流式细胞术定量测定空白对照组、溶媒对 照组、DADS药物组自噬率分别为(7.27± 5.60)%、(7.10± 5.13)%、(27.39± 6.51)%（P<0.05）;空白对照组为0.658± 0.007,溶媒对 照组为0.671± 0.012,两者的Beclin1mRNA 的表达强度无明显差异（P>0.05）,DADS 药物组为0.911± 0.008,高于对照组（P<0. 05）。结论：二烯丙基二硫可诱导白血病k562 细胞发生自噬性死亡,其机制可能与Beclin1 的上调有关。     

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

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 3.0 x 10(4) CFU ml(-1) were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0.71. 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 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.     

长爪沙鼠发情周期规律与判定方法

目的研究长爪沙鼠发情周期,揭示发情规律,优化判定方法。方法连续18 d采集50只长爪沙鼠阴道上皮脱落细胞涂片,采用角化细胞计数法研究长爪沙鼠发情周期规律。比较瑞氏染色、HE染色和直接镜检判定发情周期4个时相的优缺点。结果长爪沙鼠的发情周期有稳定型、不稳定型、假孕三种类型。其中稳定型占68.6%,发情周期为(106.3±35.0)h,可分为4个时相。4个时相角化细胞的比例分别为发情前期(13.5±7.8)%、发情期(86.7±9.9)%、发情后期(27.9±12.8)%和发情间期(3.3±2.8)%。结论角化细胞计数能准确地判定长爪沙鼠的发情周期及各个时相。直接镜检法能快速反映阴道脱落细胞的形态。     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

姜黄素对人食管癌EC9706细胞凋亡的诱导作用

目的:应用姜黄素处理人食管癌EC9706细胞,研究姜黄素对人食管癌EC9706细胞凋亡的诱导作用。方法:应用细胞计数、流式细胞仪、琼脂糖凝胶电泳、Hoechst染色、H.E染色和透射电镜检测经姜黄素诱导处理后人食管癌EC9706细胞的凋亡。结果:经姜黄素诱导处理后,人食管癌EC9706细胞生长抑制率达69.9%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达23%;琼脂糖凝胶电泳显示出细胞凋亡典型的180-200 bp及其倍体的DNA"梯状"条带;Hoechst染色显示细胞核内出现浓染致密的固缩形态或颗粒状荧光;光镜和电镜下可见典型的细胞凋亡特征:细胞体积缩小,染色体凝集,可见有成群或单独存在的凋亡细胞,电镜下可见凋亡小体存在。结论:姜黄素能够有效诱导人食管癌EC9706细胞的凋亡,从而进一步为食管癌等恶性肿瘤疾病的治疗和凋亡机理的研究提供重要基础和科学依据.     

Flow cytometry of Escherichia coli for process monitoring

A laser flow cytometer was used to study different Escherichia coli populations under various cultivation conditions. A host strain E. coli 5K was analyzed for cell size, protein and DNA-content during continuous cultivation. Also, a recombinant E. coli 5K(pHM12) strain (used for the intracellular production of penicillin-G acylase) was studied in regard to gene expression using different cytometric techniques. An argon ion laser (30 mW) and a 100 W high-pressure mercury lamp were used as light source in the cytometer. A new fluorogenic staining technique for intracellular penicillin-G acylase is described.Recombinant E. coli temperature sensitive cells were analyzed for intracellular fusion protein production due to temperature induction.     

Interferometry in flow to sort unstained X- and Y-chromosome-bearing bull spermatozoa

BACKGROUND: It was found earlier that the difference in volume between unstained X- and Y-chromosome-bearing sperm heads could be detected using interference microscopy in visible light. This could be the basis for an alternative to the conventional method to sort X and Y sperm, which uses DNA staining and ultraviolet (UV)-excitation that may be harmful to sperm cells. A novel technique is introduced combining interferometry with flow cytometry. MATERIALS AND METHODS: Interference optics were built into an existing flow cytometer/cell sorter and used to sort fresh unstained bull sperm cells on the basis of their head volume. Sorted fractions were stained with a DNA stain, and reanalyzed using the conventional method. RESULTS: Purities between 60-66% were found in both X- and Y-enriched fractions. It was possible to sort up to 300 cells per second. The system was found to be less sensitive to the orientation of sperm cells than the conventional method. CONCLUSIONS: Interferometry can be combined with flow cytometry and used to obtain significantly enriched fractions of X- and Y-bearing sperm without staining and UV light. Sorting speeds and purities at this point, however, are much lower than with the conventional method.     

Use of the direct epifluorescent filter technique for the enumeration of bacterial spores

Heat treatment at 80°C for 10 min effectively destroyed all vegetative cells (except for Gram-positive cocci) and made easier the counting of bacterial spores, which stained orange, green or rarely transparent/black with a dull green halo, in the direct epifluorescent filter technique. The numbers of both orange- or green-staining spores were lower than the plate count. A variety of physiological conditions were used to investigate the relationship of the different staining patterns with germination status. It was concluded that orange-staining spores had germinated and their number agreed with the plate count after incubation in yeast glucose broth at 30°C for 4 h. This observation was unreliable, however, but it was found that a total spore count in the DEFT gave a good agreement with the plate count.