酿酒微生物计数方法探讨

采用琼脂倾注法接种不同菌株,并对平板菌落分别进行人工计数与自动计数,对两种方法的计数结果进行比较分析,以验证全自动菌落计数仪的菌落识别功能和计数准确性。结果表明,全自动菌落计数仪对于菌落较小的细菌类能准确地快速计数,重现性好,最大误差率为3.95%;但对于菌落形态较大的霉菌类、酵母类等计数准确率低,最大误差率分别为42.86%和35.50%。因此,在实际应用中应以自动计数为主,通过合理设置菌落直径、颜色、亮度、背景偏差等参数以方便自动计数,同时采用人工计数对结果进行修正,以提高计数准确率。     

基于图像识别技术的海参分级与计数设备的设计

针对海参不同规格分级和计数的需求,以图像识别技术为基础,设计了一套机电一体化高精度自动分级与计数系统。该设备通过图像识别、采集、计算和处理海参在传送带上的投影面积大小来确定海参的规格,进而对海参进行相应等级的分选和计数。结果表明：该设备具有对单体海参分辨误差率低、分选精度高、分选速度快、计数准确等特点,达到了预期效果。     

用于水生病原菌的3种高通量活菌计数方法的比较

以一株副溶血弧菌(Vibrio parahaemolyticus)作为研究对象,比较了MTT [3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]比色法、ATP生物发光法和高通量生长曲线法在活细菌高通量计数上的应用效果。用96孔培养板进行不同浓度细菌活菌计数,确定了上述3种方法在副溶血弧菌活菌计数的标准曲线和线性范围。结果显示,副溶血弧菌的MTT比色法以DMSO溶解的MTT产物甲瓒在555 nm的吸光度(OD555 nm)为计数依据,活菌数的对数(LgC)与LgOD555 nm线性关系的标准曲线为LgC=(1.0439±0.0200)LgOD555 nm+(8.0565±0.0125),相关系数R²=0.9965,线性检测范围为7.8×106~2.5×108 CFU/ml;ATP生物发光法以ATP产生的相对发光度值(RLU)为计数依据,LgC与LgRUL线性关系的标准曲线为LgC=(0.9590±0.0065)LgRLU+(0.9949±0.0366),相关系数R²=0.9994,线性检测范围为1.0×104~3.0×108 CFU/ml;高通量生长曲线法以生长曲线达到拐点的时间(Ts)为计数依据,LgC与Ts线性关系的标准曲线为LgC=?(0.8727±0.0230)Ts+(9.0128±0.1572),相关系数R²=0.9924,线性检测范围为1.0×100~1.0×107 CFU/ml。用3种方法对实际菌液测量并与平板计数法比较表明,ATP生物发光法与高通量生长曲线法有很好的准确性,MTT比色法准确度稍差,而高通量生长曲线法有最宽的线性范围,也最适合高通量测定。     

布氏菌病活疫苗活菌计数项目能力验证结果分析

为了解全国布氏菌病活疫苗生产企业活菌计数能力,2015年对该项目进行了能力验证分析,每名参比企业发放布氏菌活疫苗(S2株)样品3份,要求在规定时间内对其进行活菌计数并提交结果报告。结果显示,参比的17家企业中,15家结果"满意",2家企业结果"不满意",表明全国大部分布氏菌病活疫苗生产企业具备可信任的布氏菌病活疫苗活菌计数检测能力。     

基于改进Bayes抠图算法的麦穗小穗自动计数方法

小麦产量评估需人工获取田间单位面积的麦穗数和麦穗小穗数，往往耗时耗力。为了实现高效、自动地麦穗小穗计数，提出一种基于改进Bayes抠图算法的麦穗小穗自动计数方法。该方法首先利用改进Bayes抠图算法对获取地自然生长条件下的麦穗图像进行抠图，将麦穗从自然背景中分割出来。然后对该图像进行平滑滤波和二值化，运用迭代极限腐蚀运算对二值化图像进行腐蚀处理，去除麦穗图像中的麦芒，分离出麦穗上每个单独的麦穗小穗。再运用面积滤波滤除掉面积过小的区域，对剩余区域的黑洞进行填充，由此每个单独的麦穗小穗形成一个单独的连通区域，最后对连通区域进行标记和计数，完成麦穗小穗的自动计数。使用4个小麦品种的麦穗图像对麦穗上的小穗进行计数验证，结果表明，该方法在识别4个品种田间麦穗单幅图像中小穗数量的平均计数精度达到94.53%，平均相对误差为5.47%，对比已有麦穗小穗自动计数方法，计数精度显著提高，这对于小麦在线产量预估具有重要意义。     

水泵疲劳寿命预测中应力计数算法研

提出了水泵零件疲劳寿命预测的主要思路,讨论了疲劳寿命预测的关键算法——应力计数算法。针对传统应力计数算法存在应力循环提取失效的缺陷,引入临界净应力的概念,提出了对传统计数算法的改进措施。并将改进后的应力计数算法应用于一台轴流泵叶轮的疲劳寿命预测;同时结合该算例,详细说明了二维概率Miner准则疲劳寿命预测的关键步骤。     

Direct Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification (LAMP) assay is used for detecting target genes, DNA extraction is unnecessary in many cases. Simple pretreatment (e.g. heating) is enough to obtain rather sensitive responses. Even test samples without any pretreatment can be used as template. This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies. In this study, using Stx1 gene from E. coli as model, we verified that viable cells, dead cells and extracellular DNA could function as template in the LAMP assay. In the incubation at 63℃, viable bacteria in the LAMP reaction mixture lysed completely within 2 min, providing DNA template for nucleic acid amplification. The Stx1 gene in diluted culture medium, spiked tap water, spiked seawater and real seawater all could be detected, with or without the step of DNA extraction. We found that the complex substances in real sample (e.g. natural seawater) exhibited considerable inhibitory effect on the sensitivity of the LAMP assay. These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring, bio-resource surveys, food safety, etc. in particular those based on environmental DNA.     

斑节对虾性腺组织、细胞的原代培养条件优化

通过观察不同成分培养基及不同培养方法培养的斑节对虾性腺组织及细胞原代培养生长情况,分析细胞活力及生长相关基因表达情况,判断3种改良培养基的适用条件,为进一步细胞培养试验提供支持。试验结果表明,以L-15培养基为基础培养基,不同配方的培养效果差距较大,其中机械分离法可成功培养离体斑节对虾性腺组织,而酶解法与3号培养基结合效果较好;在培养过程中可见离体卵母细胞多呈悬浮生长,部分可见分裂相,离体精原细胞多呈贴壁生长,1号、2号培养基中的细胞可维持良好生长状态达一周以上;CCK测试结果表明,机械分离的性腺组织和酶解法分离的性腺细胞分别在1号、3号培养基中有更高的细胞活力,而2号培养基中的性腺组织及细胞在两种培养方法下的活力差别不大。进一步的荧光定量PCR检测了离体培养过程中MAPK信号通路相关基因的表达变化情况,结果表明,Ras、c-Mos、MEK1、ERK基因在卵母细胞中表达变化更显著,且c-Mos基因与离体细胞生长发育情况呈正相关。本试验系统性地筛选了适宜斑节对虾性腺组织、细胞的原代培养方法,为了解培养过程中的生理过程变化提供了相关参考数据。     

In vitro production of viable eggs from isolated mouse primary follicles by successive culture

To produce viable eggs from single primary follicles in vitro, primary follicles containing oocytes (average 39.0 ± 0.2 µm in diameter) were isolated from the ovaries of 1-week-old mice, and cultured in combination with culture membranes for the first 8 days up to the secondary follicle stage, followed by the next 12 days to the later stages. After culture with a combination of first and second culture membranes using high and low adhesion characteristics, the average oocyte diameters of the surviving follicles increased by almost two-fold in all four groups. Further, the oocyte maturation rate was the highest (74.1%) in the culture group with low adhesion with collagenase and high adhesion. In this culture group, when the O₂ concentration was changed from 20% in the first culture to 5% in the second culture, the cleavage rate increased to 47.5%, which was comparable to the level of the in vivo control (34.6%). Finally, 39 embryos at the 2- to 8-cell stages were transferred into the oviducts of three pseudopregnant females, and eight live pups (20.5%) were obtained. Of the eight pups, six survived for at least six months and were fertile. The present study shows successive in vitro cultures of single isolated primary follicles for the production of viable eggs. We believe that this culture system, with a combination of culture membranes under controlled O₂ conditions, is applicable to other mammalian species, including humans.     

人工感染溶藻弧菌对大黄鱼免疫功能的影响

为了解大黄鱼在抗溶藻弧菌感染时免疫功能的变化规律,将160尾健康大黄鱼分为感染组和对照组,通过腹腔分别注射0.2 mL浓度为2×107 CFU·mL-1的溶藻弧菌和灭菌生理盐水,在注射0,1,3,7,11,15,19 d后从两组各取6尾大黄鱼,尾静脉取血,进行外周血的血相、NBT阳性细胞数、血清抗菌活力、抗体效价等免疫学指标的测定。结果显示：在人工感染的初期,感染组大黄鱼外周血的红细胞、白细胞、淋巴细胞和NBT阳性细胞的数量及血清抗菌活力等指标均较对照组有显著提高;在注射后1 d外周血粒细胞的数量显著低于对照组;抗体效价在7 d开始增加,15 d达到峰值,且用间接ELISA和试管凝集两种方法所得结果具有非常高的同步性。结果表明：在感染溶藻弧菌后,大黄鱼能通过红细胞和白细胞增殖、释放抗菌物质、产生特异性抗体等方式提高其对溶藻弧菌的免疫力;在感染的早期阶段非特异性免疫因子起主要作用,在感染后期阶段特异性免疫因子起重要作用;NBT阳性细胞数可以作为细菌感染的指标。图2表1参24