丁胺卡那霉素对耳蜗外毛细胞运动及胞内游离钙的影响

本文运用连续录像手段和荧光测钙技术研究丁胺卡那霉素对离体耳蜗外毛细胞运动及胞内游离钙浓度的影响。结果显示：（１）正常状态下外毛细胞内游离钙的荧光比值为１．３９±０．０９（ｘ±ｓｘ,ｎ＝１５）;从等渗环境进入低渗环境外毛细胞变短变粗（Ｐ＜０．０５,ｎ＝７。（２）丁胺卡那霉素（６．６７ｍｇ／ｍｌ）可使外毛细胞胞膜皱缩内陷,局部直径明显变细（Ｐ＜０．０１,ｎ＝１９）,即使在低渗环境中也难以恢复原状。（３）丁胺卡那霉素（３．２３ｍｇ／ｍｌ）可瞬间降低细胞内钙离子浓度,使荧光比值降低１４．６８±２．０５％（Ｐ＜０．０１,ｎ＝１２）。提示丁胺卡那霉素的耳毒性作用机制可能通过降低细胞内游离钙浓度,进而影响外毛细胞钙依赖性的“慢能动性”运动     

依地酸钠对黏液型铜绿假单胞菌生物膜的作用

目的探讨金属螯合剂依地酸钠（EDTA）对黏液型铜绿假单胞菌（PA）成熟生物膜的杀菌作用和对其结构的影响。方法平板法培养成熟铜绿假单胞菌生物膜,微量肉汤稀释法测量EDTA、环丙沙星的最低抑菌浓度,平板计数法计算EDTA、环丙沙星单独及联合对生物膜菌落数的影响,荧光探针FITC-ConA染细菌胞外多糖、荧光显微镜下观察EDTA作用前后多糖差别,荧光探针SYT09／H标记生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（ISA）对EDTA作用前后的生物膜结构参数进行定量分析。结果当EDTA浓度为5MIC时达到对PA生物膜的最大杀菌效应,可使菌落数由10^7CFU／ml降至10^4CFU／ml,0．1MIC、5 MIC的EDTA均可增强环丙沙星对生物膜的杀菌作用,高浓度组效果更明显、使菌落数降至10^2CFU／ml。EDTA作用后荧光显微镜下可见多糖被破坏,明显减少。激光共聚焦显微镜下可见EDTA作用后生物膜死茵比例增加,菌落变稀疏。ISA软件分析结果显示：5MIC的EDTA作用后生物膜厚度（d）由（22．59±4．13）μm降至（8．97±2．45）μm,t=8．515,P〈0．05;AP（区域孔率）由0．89±0．07增加至0．97±0．02,t=-2．653,P〈0．05;ADD（平均扩散距离）由3．08±0．96降至1．59±0．24,t=4．510,P〈0．05;TE（结构熵）由6．25±0．79降至3．02±0．67,t=9．375,P〈0．05;0．1MIC的EDTA效果没有5MIC明显。结论EDTA可以破坏铜绿假单胞菌生物膜的结构,增强抗生素对生物膜杀菌活性。     

铜绿假单胞菌3-氧十二烷酰高丝氨酸内酯信号分子对单核细胞Toll样受体2和4的影响

目的研究铜绿假单胞菌群体感应系统信号分子3-氧十二烷酰高丝氨酸内酯（3-O-C12-HSL）对人外周血单核细胞凋亡以及1]LR2／4mRNA表达的影响。方法自健康献血者血液中分离单核细胞进行体外培养,并以0、1、10、50和100μmol／L3-O-C12-HSL作用,采用MTT法,观察不同浓度的3-O-C12-HSL分别作用对人单核细胞生长增殖的影响。用RT-PCR测定单核细胞TLR2／4mRNA表达。结果3-O-C12-HSL刺激单核细胞12h后TLR2／4mRNA表达降低,且呈剂量依赖关系,TLR2／4mRNA在100μmol／L时最为显著（0．3494±0．0979,0．3351±0．1121）。3-O-C12-HSL对单核细胞增殖有抑制作用,呈剂量依赖关系,差异有显著性（P≤0．05）。结论3-O-C12-HSL可以激发单核细胞细胞凋亡,下调Toll样受体的表达水平,从而削弱单核细胞对铜绿假单胞菌的应答能力,这可能是铜绿假单胞菌逃逸免疫清除的重要原因之一。     

芸豆植物凝集素对细胞生长和增殖的影响

目的:以乳酸菌及盘基网柄菌为代表研究芸豆PHA对其增殖的影响。方法:采用纸片扩散法观察芸豆PHA对乳酸菌增殖的影响;采用液体振荡和贴壁培养观察芸豆PHA对盘基网柄菌生长增殖的影响。结果:1)乳酸菌培养12 h,1 mg/ml组滤纸片周围出现大的菌落,继续培养各浓度含芸豆PHA滤纸片周围均出现生长圈,对照组无生长圈;2)振荡培养24h时,低剂量各芸豆PHA组细胞密度比值最大,1mg/ml组达2.25,高于2.5mg/ml组细胞密度不随时间而增加,5.0和10 mg/ml组贴壁培养12h,盘基网柄菌不运动为死亡状态。结论:芸豆PHA对不同种类生物的生长增殖具有广泛的调节作用,其作用呈明显的剂量-效果关系。     

单种群菌落分形结构的形成及其机制研究

在变形杆菌、绿脓杆菌单种群菌落分形结构的形成过程中,细菌的运动、潜－生序变化及环境的营养水平是菌落形成扩散限制聚合模型（ＤＬＡ）分数维图样的关键。对菌落环境的分析结果显示单种群菌落中非平衡环境的形成是菌落分形结构产生的根本机制     

大蒜素对铜绿假单胞菌生物膜群体密度感应系统调控毒力因子表达的影响

目的通过体外测定铜绿假单胞菌（Pseudomonas aeruginosa）群体密度感应（Quorum Sensing,QS）系统调控的毒力因子表达,比较大蒜素干预前后毒性因子表达的差异以及对铜绿假单胞菌PAO1成熟生物膜（biofilm,BF）的影响。方法应用ELISA法比较处理前后外毒素A的含量差异;利用弹性蛋白一刚果红染色的方法,测定处理前后弹性蛋白酶活性;用蒽酮一硫酸法测定鼠李糖脂;利用荧光酶标仪检测青脓素含量变化;利用激光共聚焦显微镜观察干预前后大蒜素对铜绿假单胞菌成熟BF的影响。结果大蒜素干预后与生理盐水对照组比较,外毒素A、弹性蛋白酶、鼠李糖脂和青脓素表达分别由（19．630±0．573）pg/μl、（0．467±0．003）、（2．009±0．063）g／L、（9325．833±367．675）下降到（6．529±0．289）pg／μl、（0．032±0．001）、（0．269±0．009）g／L、（7819．167±111．800）。激光共聚焦显微镜观察可见生理盐水对照组细菌菌落云集呈蘑菇状分布,干预组黏附的细菌稀疏散在平坦分布。结论大蒜素可抑制铜绿假单胞菌群体密度感应系统调控的毒力因子表达,减弱其毒力,干扰BF分化成熟。     

三种抗菌织物对红色毛癣菌和须癣毛癣菌生长抑制效应的实验研究

目的通过分析汉麻织物、含纳米银锦纶纤维织物和无机银抗菌整理后棉布对红色毛癣菌、须癣毛癣菌生长的影响,探讨抗菌织物对皮肤癣菌的抑制作用。方法制备红色毛癣菌、须癣毛癣菌的菌悬液,分别接种于上述抗菌织物,并紧贴在PDA培养基表面培养,每12 h观察菌落形态及大小,根据菌落平均直径绘制生长曲线。结果三种抗菌织物上24 h内均未见菌落生长。①汉麻布样上,红色毛癣菌和须癣毛癣菌菌落直径分别在36-84 h、36-72 h时间区间内小于棉布组,差异有显著性（P〈0.05）,之后与棉布组渐趋一致。②含纳米银锦纶织物上,在36-108 h内红色毛癣菌菌落直径小于棉布组,差异有显著性（P〈0.05）,120 h时已无显著差异（P〉0.05）,须癣毛癣菌在48 h后才开始生长,在60-108 h区间内菌落直径小于棉布对照（P〈0.05）。③无机银抗菌整理后棉布样上,红色毛癣菌和须癣毛癣菌分别在48 h和60 h后才开始生长,在120 h以内两种皮肤癣菌菌落直径均小于棉布组,差异有显著性（P〈0.05）。结论三种抗菌织物与棉布相比较,在一定时间区间内,均可显著抑制红色毛癣菌和须癣毛癣菌的生长,尤其是经无机银抗菌整理后棉布的抑菌更有效和持久。     

北极海洋沉积物中锰细菌的分离与系统发育

对中国第二次北极科学考察采集的北极海洋沉积物中的锰细菌进行了筛选、分离和系统发育分析。根据其在筛选平板上菌落的形态学特征,分别从站位P11和S11采集的沉积物中分离到了21株和19株锰细菌。系统发育分析表明,两个站位的锰细菌群落组成有着明显的差别。站位P11分离的可培养锰细菌主要由细菌域（Bacteria）中变形杆菌门的γ-变形杆菌纲（γ-Proteobacteria）和放线菌纲（Actinobacteria）组成,二者分别占86％和14％;γ-变形杆菌纲主要包括嗜冷杆菌属（Psychrobacter）、希瓦氏菌属（Shewanella）、假交替单胞菌属（Pseudoaheromonas）、不动杆菌属（Acinetobacter）、海杆菌属（Marinobacter）,其中以嗜冷杆菌属为主,其比例可达67％。从站位S11分离到的可培养锰细菌主要包括细菌域中变形杆菌门的α-变形杆菌纲（α-Proteobacteria）和γ-变形杆菌纲以及拟杆菌门（Bacteroides）中的黄杆菌纲（Flavobaeteria）;γ-变形杆菌纲主要包括希瓦氏菌属、海单胞菌属（Marinomonas）和交替单胞菌属（Aheromonas）,α-变形杆菌纲主要为鞘氨醇单胞菌属（Sphingomonas）。实验菌株均对Mn^2＋有着较强的抗性,其中以菌株Marinomonas sp．S11-S-4耐受性最高。     

全反式维甲酸联合替莫唑胺对胶质瘤U251细胞增殖与凋亡的影响

目的：研究全反式维甲酸（ATRA）联合替莫唑胺（TMZ）对胶质瘤细胞株U251增殖及凋亡的影响。方法：体外培养胶质瘤细胞株U251,分为ATRA组、TMZ组、ATRA＋TMZ组和空白对照4组,应用MTT法测定U251细胞生长抑制率,流式细胞仪检测细胞周期分布和细胞凋亡率,westernblot法检测细胞维甲酸受体β（RARβ）蛋白和凋亡相关蛋白Caspase．3蛋白的表达情况。结果：对照组、ATRA组、TMZ组及ATRA＋TMZ组的细胞生长抑制率分别为（1．72±0．12）％、（9．87±0．87）％、（23．87＋1．32）％及（35．74±1．44）％,ATRA＋TMZ组的细胞生长抑制率显著高于单独应用TMZ（P〈0．01）。对照组、ATRA组、TMZ组及ATRA＋TMZ组的细胞凋亡率分别为（1．32±0．11）％、（4．16±0．35）％、（8．44±0149）％、（15．27±1．03）％,ATRA＋TMZ组的凋亡率显著高于单独应用TMZ有更高的凋亡率（P〈0．01）。对照组、ATRA组、TMZ组及ATRA＋TMZ组RARp蛋白相对表达量分别0．452±0．054、0．837±0．068、0．195±0．021、0．376±0．039,ATRA＋TMZ组RARβ蛋白相对表达量显著高于TMZ组（P〈0．01）。ATRA＋TMZ组Caspase．的3蛋白表达相对水平为（0．832±0．059）,明显高于ATRA组（0．334±0．041）及TMZ组（0．521＋0．032）,差异具有统计学意义（P〈0．01）。结论：全反式维甲酸联合替莫唑胺能更有效抑制U251细胞的增殖,增加其凋亡率,这可能与其增加RARβ和Caspase-3蛋白的表达抑制U251细胞增殖、诱导细胞凋亡有关。     

纳米银水凝胶涂膜干预气管导管表面铜绿假单胞菌黏附及生物膜形成的实验研究

目的研究纳米银水凝胶涂膜对气管导管（endotracheal tube,ETT）表面铜绿假单胞菌粘附及细菌生物膜（biofilm,BF）形成的干预作用。方法实验共设6组,分别为空白对照组,涂纳米银3．5、7．0、10．5、14．0和17．5μg／cm^2组。参考Brown平板法,制备ETT、表面铜绿假单胞菌BF模型。通过超声振荡-平板菌落计数法检测体外培养6、12、18h时各组ETT表面BF中的活细菌粘附数量。借助激光共聚焦显微镜观察BF中的活死菌分布情况,并测量BF厚度。结果（1）与空白对照组相比,最小量涂膜组（3．5μg／cm^2）体外培养6h时,导管表面活细菌的粘附量显著减少（P〈0．05）,12h时差异无显著性（P〉0．05）;最大量涂膜组（17．5μg／cm^2）体外培养6h时,ETT表面几乎未见细菌粘附（P〈0．05）,18h时BF中的活菌数量及BF厚度均显著减少（P〈0．05）。（2）激光共聚焦显微镜观察可见,培养6h时,空白对照组ETT表面粘附的死活菌呈不规则散点样分布,未见明显的细菌菌落形成,而各实验组ETT表面仅有细菌零星分布,其数量少于空白组。18h时空白对照组表面可见大量活死菌堆积粘连,有小菌落形成并相互交通成地图状,可见典型BF结构,而此时最大量涂膜组（17．5μg／cm^2）表面仅见数量不等的菌落形成,菌落周围可见数量不等的细菌分布。结论纳米银水凝胶涂膜可有效减少ETT表面铜绿假单胞菌的粘附数量,延缓导管表面细菌BF形成,其作用强弱随培养时间及单位面积中的纳米银剂量的变化而变化。     

马氏珠母贝(Pinctada fucata martensii)PmHEX基因的功能研究

几丁质是软体动物贝壳有机框架的重要成分,其代谢在贝壳矿化中发挥重要作用。β-N-乙酰-己糖胺酶(HEX, EC3.2.1.52)是几丁质代谢的关键水解酶。为了探究马氏珠母贝β-N-乙酰-己糖胺酶(Pm HEX)(登录号:MF555152)在贝壳形成中的作用,本研究利用原位杂交(ISH)技术检测Pm HEX基因在外套膜的定位,结果显示Pm HEX的mRNA主要分布于外侧褶的外上皮细胞、中褶的内侧上皮细胞和内褶上皮细胞。利用RNAi技术抑制Pm HEX表达后,Pm HEX在边缘区和套膜区的表达量均显著下调;SEM观察发现实验组的棱柱层和珍珠层的微观结构都出现不同程度的紊乱。综上所述,Pm HEX可能通过影响几丁质代谢,参与马氏珠母贝贝壳棱柱层和珍珠层的矿化过程。     

MINIATURIZED ANAEROBIC CULTIVATION METHODS FOR RECOVERY OF CLOSTRIDIUM SPOROGENES FROM MEAT

Two new miniaturized methods (sandwiched microtiter plate [SMP] and mini-tube) for recovery of Clostridium sporogenes from food samples were developed and evaluated. One hundred microliter of SFP (Shahidi Ferguson Perfringens) agar and 10 üL of diluted food sample were used in the SMP anaerobic system. The samples were sandwiched between two sterile microtiter plates to create an anaerobic environment. Black colonies in the sandwiched wells were counted as C. sporogenes. In case of mini-tube system, 1 mL of SFP agar (45C) containing diluted food sample was aspirated into a 1.2 mL sterile pipet and allowed to solidify in place. The two ends are sealed to create an anaerobic environment. Black colonies were counted directly through the plastic tube. C. sporogenes was inoculated into ground beef samples for recovery study. The recovery rates in SFP agar, using the SMP and mini-tube method were compared with the double-tube method. Organisms were recovered more in the double-tube than with SMP method and mini-tube method after 24–30 h incubation. However, the final counts at 48 h were similar in all methods from the food samples. These new simple methods have potential use for recovery of Clostridium spp.     

The Interaction of Humidity and Resistance Elicitors on Expression of Polygenic Resistance of Barley to Mildew

Elicitor-active glucan was applied to leaves of genotypes of barley expressing different levels of resistance to powdery mildew ( Erysiphe graminis f. sp. hordei )which was assessed by measuring mildew colonies per leaf or speed of papilla induction response to mildew challenge. The influence of humidity was also assessed by treating leaves with glucan, inoculating with mildew and immediately sealing the leaves in Perspex containers in an apparatus which enabled temperature, air speed and humidity to be controlled accurately. In water-treated controls low humidity alone reduced colonies per leaf, and at 60% r.h. genotype 7204 showed significantly less effect of humidity compared with other genotypes, although at 40% it responded as other genotypes. At low humidity glucan stimulated resistance for genotypes Proctor and 7204 but not for Golden Promise or 9855; the effect of humidity or elicitor alone was only significant on genotype 7204, Chitin also proved an effective elicitor, but at high and not at low humidity. Time of pre-treatment before mildew challenge, leaf stage, type of elicitor, concentration of elicitor, post-inoculation light regime, mildew isolate and barley genotype all had major influences on papilla response. Other factors were less important. Based on papilla formation, the barley cultivar Proctor was most and Golden Promise the least responsive to glucan as an elicitor. The potential for exploiting the genetic basis of inducible resistance is discussed.     

用显微穿刺技术直接测量微血管压力的伺服零方法研究

本实验制作了尖端的直径为０５－５０μｍ和斜面为２５°的玻璃微插管,成功地建立了用显微穿刺（ｍｉｃｒｏｐｕｎｃｔｕｒｅ）技术直接测量微血管压力（Ｐｍ）的伺服零方法（ｓｅｒｖｏｎｕｌｍｅｔｈｏｄ）,对自发高血压大鼠（ＳＨＲ）和正常血压大鼠（ＷＫＹ）肠系膜Ｐｍ进行了测量研究。结果表明,ＳＨＲ的平均动脉压（ＰＡ）和微动脉的Ｐｍ均明显大于ＷＫＹ的ＰＡ和Ｐｍ;ＰＡ在微动脉中明显降低,最大压降在直径小于５０μｍ的微动脉以及毛细血管中。     

Effects of coral colony morphologies on mass transfer and susceptibility to thermal stress

This study tested the hypothesis that corals of the same species, but of varying size and shape, may respond differently to thermal stress because of different mass transfer capacities. High mass transfer rates are an advantage under thermal stress, and mass transfer rates are assumed to scale with size. Yet large, corymbose Acropora colonies are more vulnerable to thermal stress than small corymbose Acropora colonies. We took a two-tiered approach to examine the differences in the susceptibility of different coral morphologies to thermal stress. Firstly, the response of several coral species of different sizes and shapes were measured in the field through a thermal stress event. Secondly, diffusion experiments were conducted using gypsum-coral models of different morphologies to estimate mass transfer rates, to test whether dissolution rates differed in accordance with colony morphology and colony size. Coral colonies with a high height to diameter ratio were subjected to more partial mortality than flat colonies. These results agree with mass transfer theory. The diffusion experiments showed that in a low-flow environment, small encrusting colonies had higher rates of dissolution than large flat or small branched colonies. These results, however, disagree with mass transfer theory. We show that the volume of space between colony branches predicts the response to thermal stress in the field. Small encrusting colonies were most likely to maintain mass transfer and were therefore more likely to survive thermal stress than large branched colonies. We predict that an increase in the frequency and intensity of thermal stresses may see a shift from large branched coral colonies to both small colonies, and flat-massive colonies with low aspect ratios.     

Flight activity and colony strength in the stingless bee Melipona bicolor bicolor (Apidae, Meliponinae)

Flight activity of Melipona bicolor bicolor, coming from Cunha (23o05'S, 44o55'W), Atlantic Forest, was studied in ten colonies, and in two periods: from July to September 1993 and from August to September 1995. The colonies were grouped in weak, medium and strong, according to the diameter of the combs, which can provide a good idea of the number of cells built. 855 observations were accomplished for 5 minutes, every half-hour, from 8 to 18 hours. The total number of bees that entered and left the hive and the number of bees that arrived with mud, pollen and resin, besides the number that went out with debris in that period were counted. It was also registered the temperature and the relative humidity of the air. The total external activity, as well as pollen collection, was maximum in the first hours of the morning, mainly in strong colonies. Weak colonies moved their maximum activity approximately to 12 hours. Pollen collection declined gradually, while mud and resin collection rose; removal of debris was greater in the beginning of the morning and in the end of the afternoon. Flight activity increased as relative humidity of the air rose, being optimum for strong colonies in the range between 80%-89%, and for the weakest colonies between 70%-79%. The minimum temperature observed for exit of the bees was 11oC, with optimum temperatures ranging between 17oC and 22oC. The results showed that the general state of the colony influences the different strategies of food collection and that these bees should be adapted to environments of high relative humidity as the Atlantic forest.     

The sensitivity of mesenchymal stromal cells subpopulations with different adhesion properties and derived from hemopoietic organs to growth factors EGF,bFGF, and PDGF

The action of three growth factors (EGF, bFGF, and PDGF) on mesenchymal stromal cell (MSC) subpopulations from rat adult bone marrow (BM) and embryo liver (EL) was investigated. These cells are plastic-adhesive and have different rates of adhesion (AS1–AS4 subpopulations). The efficiency of colony-formation, the size of colonies, and the number of early osteogenic progenitors with alkaline phosphatase activity in colonies and induced osteogenesis were analyzed. It was shown that EGF increased the number of bone marrow (BM) MSC colonies, but it had no influence on osteogenic differentiation. bFGF suppressed colony formation, but it stimulated both early and late stages of osteogenesis. PDGF increased the size and the number of colonies in AS2 and AS3 subpopulations, but it stimulated only the terminal stage of ostegenesis. The distinction between MSC subpopulations from two organs was found: MSC from EL had small osteogenic capacities and low sensitivity to grow factors; MSC from BM had no such characteristics. MSC subpopulations with different adhesion properties and from different tissues had compatible sensitivity to growth factors.     

Colonial Morphology of Treponemes Observed by Electron Microscopy

The colonial morphology of three strains of cultivable, nonpathogenic treponemes including a human oral treponeme was examined by light and electron microscopy. Treponema phagedenis strains Kazan and Reiter produced large white colonies on the surface of solid media composed of sterility test broth, 0.9 to 3.1% agar, rifampin, and 12.5% rabbit or horse serum. A human oral treponeme, strain G7201, grew as diffused white zones on 0.9 to 3.1% agar plates. Under the cultural conditions employed agar concentrations slightly affected the time of appearance of colonies of the three strains of treponemes. When the colonies of these three strains were viewed by scanning electron microscopy, differences in their colonial morphology were observed. The 11-day-old colonies of human oral strain G7201 were very small, 5 to 15 μm in diameter, and had a slight irregular border. Kazan treponemes developed circular, entire and low convex colonies. Scanning and transmission electron microscopy revealed that the colonies of Reiter treponemes contained spherical forms almost up to 5 μm in diameter, each consisting of an outer membrane and a treponemal main body. They were very similar to the spherical bodies produced by strain G7201 in sucrose-containing broth.     

Immunohistochemical localization of senescence marker protein-30 (SMP30) in the submandibular gland and ultrastructural changes of the granular duct cells in SMP30 knockout mice

Senescence Marker Protein-30 (SMP30) is a calcium-regulating protein that decreases in an androgen-independent manner as aging occurs. An enzyme-labeled antibody technique has demonstrated that SMP30 localized to the ducts (granular, intercalated, and striated ducts) of mouse submandibular glands. Immunoelectronmicroscopy demonstrated that the granular duct cells were strongly positive for SMP30, but that pillar cells in the granular duct were negative for the protein. In SMP30-knockout (KO) mice, the granular ducts were smaller in diameter. Swelling of mitochondria in the granular duct cells was observed; however, this phenomenon was not observed in the pillar cells. After administration of alpha-isoproterenol, a beta-adrenergic stimulant, a large numbers of small secretory granules were present in the granular duct cells and an expansion of the rough endoplasmic reticulum in SMP30-wild type (WT) mice; in contrast, little change was observed in SMP30-KO mice. These results suggest that SMP30 may be closely related to a signal transduction pathway in the granular duct cells of submandibular glands.     

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.