流式细胞术在微生物快速检测领域的研究进展

概述了流式细胞术的发展历史、工作原理及其在微生物快速检测方面的应用。     

利用流式细胞仪筛选紫外诱变高含油小球藻

为筛选得到高含油的小球藻藻株,对若夫小球藻(Chlorella zofingensis)进行紫外诱变,尼罗红染色后再利用流式细胞仪对高含油藻株进行筛选。结果表明:紫外诱变40 min后,经流式细胞仪筛选,分选出的90%的突变藻株总脂含量都高于野生对照株。最终筛选到的藻株S16经培养总脂产量达到2.4 g/L,比野生对照株提高了50%。     

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.     

In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen-thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen-thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen-thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 mug/ml at a temperature of 37 degrees C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91+/-2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flow cytometric efficiency.     

具有典型泛醌结构成分微生物参比菌株的筛选

泛醌(Ubiquinone,UQ)是一种类异戊二烯醌,是微生物分类鉴定的主要化学分析指标之一。采用薄层层析与高效液相色谱相结合的方法对具有典型泛醌组分的微生物菌株进行测定、分析,通过比较菌株的生长性能以及泛醌类型与含量,最终得到培养条件简单、培养时间短且泛醌成分单一、含量高的一套参比菌株。该套菌株马克斯克鲁维酵母(Kluyveromyces marxianus)CICC 31691,东方伊萨酵母(Issatchenkia orientalis)CICC 1817,眼马赛菌(Massilia oculi)CICC 23887,日本假单胞菌(Pseudomonas japonica)CICC 23895,类球红细菌(Rhodobacter sphaeroides)CICC 10287分别为Q-6、Q-7、Q-8、Q-9和Q-10醌组分的参比菌株,可应用于细菌和酵母的常规鉴定及新种分类鉴定。     

Cell wall hydrolase as a surface-associated protein target for the specific detection of Lactobacillus rhamnosus using flow cytometry

Flow cytometry (FC) is a very popular technique for to the study of different eukaryotic cell phenotypes. In addition, FC has many applications in the field of food microbiology, although the few number of specific molecules that can be potentially targeted with fluorescence-conjugated antibodies limits its potential applications. The present work represents a step forward in the application of FC for detection of specific foodborne or probiotic bacteria by using fluorescent antibodies binding specific surface-associated protein. To illustrate this novel approach, we have used a monoclonal and polyclonal antibodies targeting cell-wall hydrolase (CWH), one of the major surface-associated proteins of the probiotic Lactobacillus rhamnosus GG. In order to verify the specific union of the antibodies to this surface protein, ten different bacterial strains belonging to different bacteria species taxonomically related and no related to L. rhamnosus, were tested. The optimal conditions for bacteria detection corresponded to a final suspension of 5E10⁷ bacteria in exponential phase of growth using FC buffer, without freezing, without adding extra BSA to the FC buffer and without fixing bacteria. Our results showed that GG strain was detected using the polyclonal anti-CWH serum in a higher extent (91.62%) than the rest of strains (L. casei 393: 64.49%; L. amylovorus: 46.13%, E. coli: 45.13%; L. acidophilus DSM20079^T: 35.83%; B. longum NCIMB8809: 33.68%; L. gasseri BM7/10: 32.41%; L. reuteri DSM21016: 29.26%; L. plantarum NCIMB8826: 26.33% and L. delbruekii: 22.52%). The percentage of L. rhamnosus labeled with the monoclonal antibody was 53.06, which contrasted with the 91.62% labeled with the polyclonal antibody. The low affinity of the monoclonal antibody was perhaps due to the lack of immunogenicity of the single epitope chosen for antibody generation (CWHp). The high efficiency observed for the polyclonal anti-CWH serum is maybe a consequence of being constituted by a mixture of antibodies that recognizes different epitopes in the L. rhamnosus GG protein.With this proof of concept, we have shown for the first time that foodborne or probiotic bacteria can be specifically detected using antibodies targeting adequate molecules present on their surface.     

黄曲霉毒素B1污染玉米的比重筛分研究

研究比重法对AFB_1污染玉米的筛分效果。结果表明:风量、纵向倾角和振幅对比重筛分效果具有显著影响(P0.05),且在风量为6 000m~3/h、倾角7°、振幅15mm时,不同污染程度的玉米经比重筛分后,相比原料中的AFB_1含量,重质玉米中AFB_1含量分别降低21.2%和38.4%,轻质玉米和破碎率比例适中。比重筛分机是中国粮食生产中的重要设备,通过比重筛分去除AFB_1污染严重玉米具有良好的应用前景。     

拮抗大肠杆菌的海洋微生物的筛选与鉴定

该研究采用涂布平板法、对峙培养法及琼脂扩散法从北冰洋沉积物样品中分离、筛选出对大肠杆菌（Escherichia coli）具有良好拮抗作用的一株菌株,通过对其菌株形态进行观察、分析其生理生化试验结果以及进行分子生物学技术检测,进行分类鉴定,并进行了抑菌谱研究。结果表明,从北冰洋沉积物样品中共分离纯化出97株菌株,其中21株菌株对大肠杆菌有一定的拮抗作用,且1株菌株拮抗效果最好,编号为105号,平均抑菌圈直径为（19.3±1.0） mm。该菌株被鉴定为萎缩芽孢杆菌（Bacillus atrophaeus）,抗菌谱较为广泛,对大肠杆菌、铜绿假单胞菌、鼠伤寒沙门氏菌、肺炎克雷伯氏菌都具有抑制作用。     

拮抗丁香假单胞菌海洋微生物的筛选与鉴定

该研究采用平板涂布法和平板划线法从印度洋沉积物中分离纯化海洋微生物,首先采用滤纸片法对丁香假单胞菌拮抗菌株进行初筛,然后采用牛津杯法进行复筛,并通过形态观察、生理生化试验和分子生物学技术对其进行菌种鉴定,最后研究其抑菌谱。结果表明,从印度洋沉积物中共分离纯化出115株微生物,经过初筛得到33株丁香假单胞菌拮抗菌株,再经过复筛后,筛选出一株对丁香假单胞菌抑菌效果最好的菌株,编号为NO.4.1.3-1,其抑菌圈直径达（12.63±0.06） mm。该菌被鉴定为贝莱斯芽孢杆菌（Bacillus velezensis）;除屎肠球菌外,该菌对8株致病菌均有抑制作用,且对肺炎克雷伯氏菌、金黄葡萄球菌的抑菌作用较好,具有较广的抑菌谱。     

Measurement of nuclear DNA content in fission yeast by flow cytometry

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci.97: 509–516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis (‘cut’) could be observed for the orp1 mutant after only 4 h at restrictive temperature. © 1997 John Wiley & Sons, Ltd.     

Quantification of structural properties of cell-sized individual liposomes by flow cytometry

We describe a new high-throughput method of quantifying the structural properties of individual cell-sized liposomes. An internal aqueous solution of liposomes was labeled with a green fluorescent marker and the membrane with a red marker. The double-labeled liposomes were analyzed using flow cytometry, and the internal aqueous volume and lipid membrane volume of each liposome were measured. The experimental results indicate that both the internal aqueous and lipid membrane volumes positively correlate with the intensity of forward-scatter (FS) and side-scatter (SS) signals in a logarithmic scale. In addition, liposomes in 18 small areas gated by log(FS) and log(SS) were sorted by fluorescence-activated cell sorting (FACS), and observed by optical microscopy. Structural characteristics observed in the microscopy images of heterogeneous liposomes correlated with FACS data. Because this method does not employ any particular assumption about the shape and structure of liposomes, flow cytometry is a powerful tool for estimating the internal and membrane volumes of individual cell-sized liposomes with heterogeneous shapes and structures.     

Short communication: Screening inhibition of dairy-relevant pathogens and spoilage microorganisms by lactose oxidase

The inhibitory effect of lactose oxidase on the growth of foodborne pathogens and spoilage microorganisms associated with dairy products was evaluated through an overlay inhibition assay. Lactose oxidase generates hydrogen peroxide via lactose oxidation into lactobionic acid. Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, Staphylococcus aureus, Pseudomonas fragi, and Penicillium chrysogenum were used as indicators. A commercially available solution of lactose oxidase was applied at different concentrations (0, 0.12, 1.2, and 12 g/L) in 4 types of media [brain heart infusion agar (BHI), BHI + sodium thiocyanate (NaSCN), BHI + lactose, and BHI + NaSCN + lactose] to evaluate the effect of lactose and thiocyanate on microbial inhibition. Lactose oxidase inhibited the growth of all the indicators at a concentration of 12 g/L of the enzyme solution in the presence of lactose alone and in combination with NaSCN. However, supplementation with NaSCN had no effect on the magnitude of microbial inhibition. Staphylococcus aureus was the most sensitive pathogen, and Ps. fragi was the most sensitive of all the indicators in general to lactose oxidase. Listeria monocytogenes and Ps. fragi showed higher susceptibility to the antimicrobial effect of lactose oxidase at 6°C than at their corresponding optimum growth temperature. The inhibitory effect was attributed to the generation of hydrogen peroxide from the oxidation of lactose. Findings from this study demonstrate that lactose oxidase could be used as a novel approach to inhibit the growth of mold and bacteria. It could also be applied as a label-friendly preservative in dairy foods.     

Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes

Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry. The efficiency of immunocapturing using magnetic beads was also determined. None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L. monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples. Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23%. Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L. monocytogenes cells captured by the beads.     

Screening of specific nucleic acid targets for Cronobacter sakazakii and visual detection by loop-mediated isothermal amplification and lateral flow dipstick method in powdered infant formula

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 10⁰ cfu/mL and 5.7 × 10¹ cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.     

流式细胞技术检测酸性饮料中菌落总数的研究

利用流式细胞技术(Flow Cytometry,FCM)检测酸性饮料(pH<4.6)中的菌落总数并与平板法和滤膜法进行比较分析,建立FCM快速检测酸性饮料中菌落总数的方法。选择13种常见实验菌株,通过人工加菌实验进行验证。结果表明,基质和含菌量对FCM法的检测结果影响较大,酸性饮料中的部分菌株难以直接被FCM法检出,需经过滤富集菌于中性TSB肉汤中36 ℃培养22 h排除基质和含菌量低的影响,培养后的菌溶液经FCM法与平板法检测结果均在一个数量级。运用AOAC官方分析方法对酸性饮料中不同浓度13种实验菌株滤膜法和增菌22 h后FCM法的定性检测结果进行统计学分析,卡方值X²<3.84,同时假阴性率=1.9%<2%,假阳性率=4.2%<9.6%,说明FCM法可替代滤膜法进行酸性饮料中菌落总数的定性检测,用于产品的快速放行。     

流式细胞仪快速检测酸奶中乳酸菌含量

目的建立一种使用流式细胞仪检测酸奶中乳酸菌含量的快速检测方法。方法吸取稀释后样品匀液100μL加入20 mL试管中,30 min内使用流式细胞仪检测。经过光电倍增器过滤和检测,荧光脉冲的密度和高度被记录下来并被换算为细菌的个数。结果与国标法相比,仪器检测时间约缩短了3 d,试验结果表明流式细胞仪检测结果与国标结果 R值均在1以下。结论流流式细胞仪法与国标平皿菌落计数法的结果具有很高的一致性。采用流式细胞仪对酸奶中的乳酸菌进行快速检测,培养时间为由48 h缩短至13 min,大大提高了检测速度,对于乳制品生产企业来说,意义重大。     

产耐酸性α-淀粉酶菌株的筛选及其固态发酵条件的优化

以酒糟为原料,应用锥虫蓝法,通过比较透明圈直径与菌落直径的比值,能较快选出目的菌株,经筛选得到1株产耐酸性-α淀粉酶的野生AS-Y菌株,其酶活达到64.3U/g。通过鉴定为曲霉AS-Y菌株,并对AS-Y菌株的固态发酵条件进行了优化。     

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.     

Grinding characteristics,physical, and flow specific properties of roasted maize and soybean flour

Grinding characteristics (one and two stage grinding) of roasted maize (RM) and soybean (RS) were conducted in attrition mill to find the effect of feed rate and peripheral speed on average particle size (A_PS), specific energy consumption (S_EC), size reduction ratio (SRR), and grinding constants. Carr Index (CI), Housner ratio (HR), density, water, and oil absorption capacity of flour were also studied as a function of particle size. The higher SRR, A_PS, and grinding recovery were recorded in the first stage grinding as compared with second one. S_EC was decreasing significantly (p < .05) from 0.0039 ± 0.0011 to 0.0013 ± 0.0019 kwh/kg as, feed rate increased from 9 to 15 kg/hr. The grinding constants were significantly increased in the second stage grinding. The flowability of flour such as CI and HR was poor at lower particle size. S_EC and A_PS analysis revealed that a two‐stage segregated grinding would be suitable for RM and RS grinding.

Practical applications

Roasted maize and soybean flour are highly nutritive food and prepared by roasting, grinding and sieving. Flour particle sizes and flow specific properties play an important role in addressing malnutrition and ready‐to‐eat food formulations. The digestibility and release of bioactive components also depend upon particle size and granular morphology of the food. Though, the present study is also acquainted with energy consumption during processing operation and product quality and which are key parameters for scaling‐up and designing the grinding equipment. The information generated regarding the energy consumption and health benefits may be useful for food processors and consumers as well.