Flow cytometry applications in the food industry

Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry

The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

The characterization and isolation of plant heterokaryons by flow cytometry

Summary Plant heterokaryons were identified and isolated from a population of protoplasts following fusion. Endogenous chlorophyll autofluorescence and exogenously supplied fluorochromes were utilized to differentiate between parental and heterokaryon populations. Flow cytometric analysis detected heterokaryons based upon the simultaneous presence of both chlorophyll and an exogenously supplied fluorochrome within one cell. These parameters were utilized to sort large numbers of heterokaryons from the fusion mixture using modified flow instrumentation. Modifications to the instrumentation which allowed this sorting are discussed.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

Systematic misestimation of cell subpopulations by flow cytometry: A mathematical analysis

Various sources of variability in flow cytometric determination of cell concentration have previously been investigated with respect to andrologic applications. Although common aspects related to the variation between samples, variation between operators, and accuracy have been extensively studied, specific sources of false-count estimation have found less attention. In particular, a major and well-recognized source of misestimation of cell counts (i.e., contamination of the sample by non-sperm particles) has not to date been characterized in detail. We show here by means of original mathematical research that not only the cell counts but also the percentages of cells expressing different fluorescence patterns are affected by the presence of alien particles often neglected in studies involving flow cytometric characterization. We demonstrate that there is a systematic overestimation in the proportion of unstained (viable) cells detected by flow cytometry in cases where the non-sperm particles are not excluded from analysis by additional identification other than light-scatter characteristics. Moreover, we provide an exact mathematical estimate for the magnitude of this overestimation, and we discuss the consequences for diagnostic applications and studies on sperm physiology, specifically for studies on sperm capacitation and evaluation of cryopreserved semen. Finally, equations are derived for the correction of the flow cytometric values for use in practical applications.     

A simple protocol to purify fresh nuclei from milligram amounts of meristematic pea root tissue for biochemical and flow cytometry applications

A simple protocol to purify fresh nuclei from very small amounts (mg) of Pisum sativum (cv. Lincoln) root tissue is presented. The protocol is reliable and has been repeated many times; it can be performed within a few hours and needs only a simple modification of common glassware for tissue homogenization. Similar yields of purified nuclei are obtained both with meristematic and adult root tissues using a detergent-free glyeerol buffer. Purified nuclei can be stored at –20°C for several weeks without any appreciable loss of integrity. The high yield of purified nuclei enables us to use our preparations for biochemical and cytometric investigations.     

Advances in flow cytometry for sperm sexing

This review presents the key technological developments that have been implemented in the 20 years since the first reports of successful measurement, sorting, insemination and live births using flow cytometry as a proven physical sperm separation technique. Since the first reports of sexed sperm, flow technology efforts have been largely focused on improving sample throughput by increasing the rate at which sperm are introduced to the sorter, and on improving measurement resolution, which has increased the proportion of cells that can be reliably measured and sorted. Today, routine high-purity sorting of X- or Y-chromosome-bearing sperm can be achieved at rates up to 8000 s⁻¹ for an input rate of 40,000 X- and Y- sperm s⁻¹. With current protocols, straws of sex-sorted sperm intended for use in artificial insemination contain approximately 2 × 10⁶ sperm. The sort rate of 8000 sperm s⁻¹ mentioned above corresponds to a production capacity of approximately 14 straws of each sex per hour per instrument.     

流式细胞仪检测纳帕海高原湿地浮游病毒和浮游细菌丰度

【背景】浮游病毒是水体微生物群落中重要的组成成分,深入研究浮游病毒的时空分布有助于更好地保护和开发当地的微生物资源。【目的】对采集到的纳帕海高原湿地水样中的浮游病毒和浮游细菌进行计数,揭示纳帕海高原湿地浮游病毒的分布规律。【方法】采用流式细胞仪检测2013年12月和2014年9月纳帕海高原湿地7个水样的浮游病毒与浮游细菌丰度,并对影响浮游病毒丰度的因素,如细菌丰度、叶绿素a含量以及其他环境因子进行了相关性分析。【结果】季节分布上,雨季浮游病毒和浮游细菌丰度高于旱季;水平分布上,原水样品的浮游病毒高于湿地水和淤泥水。旱季水样的浮游病毒丰度受到细菌丰度及叶绿素a浓度的影响较大;雨季水样的浮游病毒丰度受到水体的p H值和温度的影响较大。【结论】纳帕海高原湿地的浮游病毒和浮游细菌是比较活跃的。浮游病毒丰度在不同季节、不同采样点受到细菌丰度和叶绿素a浓度等因素的不同影响。在旱季噬菌体而非噬藻体或浮游植物病毒是纳帕海高原湿地中浮游病毒的优势种群。     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

Application of flow cytometry to the study of antiphagocytic properties of Klebsiella pneumoniae capsular polysaccharide

Abstract We used flow cytometry to compare the effects of whole cells and capsular polysaccharides of Klebsiella pneumoniae on the phagocytic ability ot polymorphonuclear leukocytes. Our results showed a light descrease in phagocytic activity in the presence of capsular polysaccharides, but a marked decrease with whole cells. Our findings suggest that the resistance to phagocytosis in these microorganisms is not due exclusively to their capsule, as claimed by other authors.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.