BioLumix微生物荧光光电检测系统在化妆品中的应用评估

本研究将用于化妆品中Bio Lumix微生物荧光光电检测系统与《化妆品安全技术规范2015》微生物检测法进行了比较。结果显示两种方法结果基本一致,对于《化妆品安全技术规范2015》微生物检测法检测化妆品,出具可靠的检测报告需要5 d,而Bio Lumix系统快速检测化妆品细菌项目和霉菌、酵母项目可在16 h和35 h内分别发出预警,并可控制在48 h内完整地输出检测报告。Bio Lumix微生物荧光光电检测系统具有检测时间短、检测效率高、操作简单等优点。     

流式细胞术检测毕赤酵母发酵过程中胞内活性氧水平

以2′,7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)和碘化丙锭(PI)为标记探针,通过DCFH-DA/PI双染色与PI单染色的对照,检测毕赤酵母胞内活性氧(reactive oxygen species,ROS)的水平及其影响。研究发现发酵过程细胞活性下降与胞内ROS积累相关。在甘油生长期,细胞几乎没有ROS积累,细胞活性接近100%。在甲醇诱导初期,部分细胞积累少量的ROS,细胞活性仍然很高,死亡细胞所占比例只有1.5%。在甲醇诱导后期,94.0%的细胞积累了大量的ROS,高含量的ROS造成细胞损伤,引起部分细胞丧失了活性,在总共29.1%的死亡细胞中,高ROS积累的死亡细胞占了25.4%。     

应用流式细胞术检测毕赤酵母的细胞活性

选取两种细胞活性染色试剂二乙酸荧光素(fluoresceindiacetate,FDA)和碘化丙锭(propidiumiodide,PI),应用流式细胞术(flowcytometry,FCM)检测毕赤酵母细胞活性。比较FDA/PI双染色与PI单染色的FCM图谱,后者能够很好地将死活细胞区分开来并得到正确的比例。利用PI单染色检测发酵过程细胞活性的变化,甘油补料阶段几乎没有细胞死亡,进入甲醇补料阶段后,随着细胞密度的增加,细胞的活性不断降低,发酵88h时细胞活性仅为73.8%。     

FCST法与ATB法在酵母样真菌药敏试验中的对比研究

目的探讨ATB（Automatic testing bacteriology）法和FCST（Flow Cytometry Susceptibility Testing）法在检测常见几类酵母样真菌对抗生素敏感性中的一致性。方法应用ATB法和FCST法检测153株酵母样真菌氟胞嘧啶、氟康唑、伏立康唑、伊曲康唑和两性霉素的敏感性。结果白假丝酵母菌对氟胞嘧啶、氟康唑、伏立康唑、伊曲康唑和两性霉素的Kappa检验值分别为0．593、1．000、0．542、0．624、0．467（P〈0．01）;其他酵母样真菌一致性较好。结论相对于ATB常规药敏试验,FCST具有快速、准确、稳定等优点,有着广泛的临床应用前景。     

流式细胞术在细菌快速检测中的应用

流式细胞仪(Flow cytometer)是集应用流体学、光学、电子学、生物学、免疫学等多门学科和技术于一体的新型高科技仪器。它的核心技术是流式细胞术(Flow cytometry,FCM),该技术是利用流式细胞仪,使单个细胞或其他微小生物粒子处于快速直线流动状态,且逐个通过光束,从而对单个细胞或微粒进行多参数(数量、大小、核酸含量、细胞活性、特定菌群或物种等)定量分析和分选的检测技术,具有快速、灵敏、精确以及便于操作等突出优点。本文简要介绍流式细胞仪的原理,并论述流式细胞技术在实验室研究、工业生产、临床诊断、环境评估等领域的细菌快速检测应用。     

MTT法检测细菌细胞数的主要影响因素分析

MTT法是生物活细胞计数的灵敏、快速和便捷方法。报告对MTT法应用于细菌细胞数定量分析时的分析前培养时间、MTT反应时间、MTT剂量和检测波长等主要影响因素的分析结果,同时报告MTT反应产物溶解检测与直接检测,以及MTT法与平板菌落计数法定量检测的结果比较。结果如下：Formazan生成量与细菌活细胞数呈正相关,对数生长期变化最明显;MTT为0.25 mg/mL,反应2 h时吸光值与菌落数具有良好的线性关系（r=0.999 8）;Formazan的最大吸收波长为570～580 nm;MTT反应产物溶解检测和直接检测结果经统计学分析无显著性差异（P〉0.05）;细菌细胞数在107～109 cfu/mL范围内,MTT法与菌落计数法定量结果呈现良好的线性关系（r≥0.991 7）。     

微生物实验中常用细菌染色方法的改良

细菌染色方法是微生物实验中观察细菌形态结构以及掌握细菌代谢、繁殖等情况的常用普及技术。微生物实验中常用的细菌染色法包括其革兰染色法、抗酸染色法等。为进一步提高实验中细菌形态的观测质量,本文就对两种常用细菌染色法进行了改良,据实验结果显示,其改进后方法操作更加简单,更有效起到细菌染色作用及观测作用。     

阿哈湖深层水中微生物和硫酸还原菌数量及群落结构空间变化

采用荧光原位杂交法分析了贵州阿哈湖深层水环境中总微生物、真细菌和硫酸盐还原菌数量。结果表明:该湖水中微生物总量为1.6×107个.L-1,真细菌占微生物总量的52.9%,且微生物总量和真细菌数量垂直变化无明显差异。随着水体深度的增加,活性微生物数量增加,且微生物的群落结构更加复杂。阿哈湖深层水体中有一定数量的硫酸盐还原菌存在。     

圆红冬孢酵母两阶段培养法生产微生物油脂

为缩短发酵时间,减少原料消耗,采用细胞增殖和油脂积累分离的两阶段模式,培养圆红冬孢酵母Rhodosporidium toruloides AS 2.1389生产微生物油脂。结果表明,细胞增殖阶段获得的R.toruloides AS 2.1389细胞,重悬接种在葡萄糖溶液中,可快速积累油脂,菌体油脂含量超过自身干重的55%。增殖阶段细胞的菌龄越高,产油能力越强。油脂积累阶段使用高浓度葡萄糖溶液或未灭菌的葡萄糖溶液,油脂合成可以有效进行。油脂中脂肪酸成分以含16和18个碳原子的长链脂肪酸为主,可作为制备生物柴油的新型原料。     

厌氧生境体系中产氢产乙酸细菌的FISH定量解析

产氢产乙酸细菌是一类在有机物厌氧降解过程中起重要作用的细菌。以基于16S rRNA序列设计的特异性寡核苷酸探针为基础,优化FISH实验条件,确定该技术检测产氢产乙酸细菌的实验条件为样品固定19h、乙醇脱水5min,杂交缓冲液中甲酰胺浓度55%。运用建立的FISH技术检测了几种厌氧消化体系中产氢产乙酸细菌的数量,并与用传统MPN方法的结果进行了比较。结果表明,产氢产乙酸细菌分布广泛,废水处理UASB反应器和动物消化道,特别是反刍动物瘤胃中的产氢产乙酸细菌数量较高,其丰度分别为1.70×109 cells/mL样品,6.50×108 cells/mL样品。湖底沉积物中产氢产乙酸细菌数量较少,仅占整个微生物群落的0.4%,含量为1.20×108 cells/mL样品。     

Detection of spoilage-causing yeasts and bacteria in tchapalo,the Ivorian traditional sorghum beer

In this study, we aimed to analyse the spoilage potential of the isolated yeast, LAB and AAB species. Thus, 11 strains were inoculated at 0·3% (v/v) into a sterile filtered tchapalo and stored for 3 days at ambient temperature (27–30°C). All the tested strains grew well or remained stable except for Limosilactobacillus fermentum and Pediococcus acidilactici, which decreased throughout the storage time. A significant decrease of total soluble solids was observed only for Saccharomyces cerevisiae (from 7·8 to 5·8 °Brix) and Meyerozyma guilliermondii (from 7·8 to 5·5 °Brix). The tchapalo samples inoculated with the LAB strains Weissella paramesenteroides, P. acidilactici, L. fermentum and the yeast strain Candida tropicalis were judged similar to the control by the panellists. However, the strains of Lacticaseibacillus paracasei and Latilactobacillus curvatus (LAB), S. cerevisiae, M. guilliermondii and Kluyveromyces marxianus (yeasts) and Acetobacter pasteurianus and A. cerevisiae (AAB) induced the spoilage of the tchapalo appearance, smell and/or taste. In the spoiled tchapalo quantitative and qualitative modification of some volatile compounds (VOCs), such as lilac aldehyde, ethyl acetate, ethyl hexanoate, ethyl octanoate and phenethyl acetate, were observed. These results provide information about the microorganisms that need to be removed to extend the shelf life of tchapalo.     

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

Development of an apparatus and protocol for the efficient isolation of bacteria and yeasts that attach to Botrytis cinerea germlings

An apparatus and protocol for the efficient and consistent isolation of bacteria and yeasts with the ability to attach to germlings of Botrytis cinerea is described. The study focused on minimising microbial contamination by bacteria or yeasts which do not attach to the pathogen B. cinerea but which interact with materials used in the equipment and would otherwise be isolated along with target microbes. After development, the assay reduced the contamination rate to 1–2 cells per 100 added and this was found to be a satisfactory level for the selection of microbial attachers to fungal hyphae. One or more phenotypes of microbes that adhered to B. cinerea germlings were found in 97% of the 70 samples collected from phylloplane washings and processed using this assay.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR

Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable techniques such as real-time PCR (rt-PCR) and nested PCR for enumerating and detecting the presence of this bacterial group without plating. Primers were designed on the basis of the available 16S rRNA gene sequences and tested successfully with reference acetic acid bacteria strains. The usefulness of rt-PCR was demonstrated by comparing the results with traditional techniques (colony and microscope counting). The results were similar with all the techniques. Optimized rt-PCR enabled numbers between 10(7) and 10(1) cells mL(-1) to be enumerated, while nested PCR detected less than 10 cells mL(-1). Although this latter technique cannot be used for enumeration, it has several advantages in routine laboratory analysis.     

Identification and characterization of yeasts in sugarcane silages

Aims: To enumerate the micro‐organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. Method: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. Conclusions: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. Significance and Impact of the Study: Knowledge of the population of micro‐organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.     

A differential medium for lactic acid-producing bacteria in a mixed culture

Aims:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue (mMRS-BPB) was tested as a medium for counting and differentiation of each lactic acid-producing bacterium (LAB), especially in a mixed culture.
Methods and Results:  Type strains of 10 LAB species ( Lactobacillus acidophilus , L. brevis , L. bulgaricus , L. gasseri , L. paracasei , L. plantarum , L. reuteri , Weissella confusa , Bifidobacterium bifidum and B. infantis ) and five commercial yogurts were inoculated on plate count agar with bromocresol purple, mMRS, and mMRS-BPB. Each type strain showed more clearly formed colonies on the three media under anaerobic conditions than under aerobic conditions. Especially each type strain produced colonies with specific characteristics of each species on mMRS-BPB. Commercial yogurts produced the largest number of colonies with various shapes and colours on mMRS-BPB.
Conclusions:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue under anaerobic conditions is appropriate for counting and differentiating each LAB in a mixed culture.
Significance and Impact of the Study:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue will be useful in isolation and enumeration of each LAB from fermented foods as well as intestinal microflora.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.