阴道毛滴虫在两种培养基中的生长与形态观察

目的通过对改良肝浸汤培养液及RPM11640培养基内阴道毛滴虫生长情况的观察，选择适合教学和科研的培养基。方法用临床分离的阴道毛滴虫标本，分别接种至改良肝浸液及RPM11640两种培养基内进行培养，pH值为5．6～5．8，通过计数了解生长情况并观察阴道毛滴虫的形态。结果肝浸汤培养液中培养48h，阴道毛滴虫达到生长高峰，虫体呈现出多分裂相，虫体变大、呈圆形，内含4～12个细胞核，细胞膜外缘可见数丛鞭毛。RPM11640培养基中阴道毛滴虫72h达到生长高峰，虫体密度约为肝浸液高峰期的2／3，未见多分裂现象，虫体与生理盐水涂片中阴道毛滴虫形态、大小相近。结论改良的肝浸液培养基适于阴道毛滴虫的药物试验及其他实验项目的研究，RPM11640培养基更适用于教学、标本的制作及实验室保种工作。     

不同浓度胎牛血清肝浸汤培养基培养阴道毛滴虫效果观察

3株阴道毛滴虫用肝浸汤培养48 h,虫体密度分别为0.7×10^5/ml、1.5×10^5/ml和2.0×10^5/ml;用含终浓度为1%、5%、10%的胎牛血清肝浸汤培养48 h,虫株Ⅰ的密度分别为6.0×10^5/ml、640.0×10^5/ml和1 280.0×10^5/ml,虫株Ⅱ分别为16.0×10^5/ml、1 600.0×10^5/ml和2 400.0×10^5/ml,虫株Ⅲ分别为65.0×10^5/ml、840.0×10^5/ml和520.0×10^5/ml。表明肝浸汤添加适量胎牛血清后培养效果良好。     

阴道毛滴虫在两种培养基中的生长与形态观察

目的 通过对改良肝浸汤培养液及RPMI1640培养基内阴道毛滴虫生长情况的观察,选择适合教学和科研的培养基。 方法 用临床分离的阴道毛滴虫标本,分别接种至改良肝浸液及RPMI1640两种培养基内进行培养,pH值为5.6~5.8,通过计数了解生长情况并观察阴道毛滴虫的形态。 结果 肝浸汤培养液中培养48 h,阴道毛滴虫达到生长高峰,虫体呈现出多分裂相,虫体变大、呈圆形,内含4~12个细胞核,细胞膜外缘可见数丛鞭毛。RPMI1640培养基中阴道毛滴虫72 h达到生长高峰,虫体密度约为肝浸液高峰期的2／3,未见多分裂现象,虫体与生理盐水涂片中阴道毛滴虫形态、大小相近。 结论 改良的肝浸液培养基适于阴道毛滴虫的药物试验及其他实验项目的研究,RPMI1640培养基更适用于教学、标本的制作及实验室保种工作。     

不同pH肝浸汤培养基培养阴道毛滴虫的观察

目的 观察不同pH值对阴道毛滴虫生长发育的影响，为实验室培养或临床诊断培养阴道毛滴虫提供参考。方法 配制pH值分别为2、2．5、3、3．5、4、4．5、5、5．5、6、6．5、7、7．5、8、8．5的肝浸汤培养基，分别接种相同数量的阴道毛滴虫，37℃恒温培养48h，用血球计数板计数滴虫数量。以pH值为横坐标，滴虫密度为纵坐标作曲线图，找出培养滴虫的最适pH值范围。同时，连续观察各pH值管滴虫生长情况，直至滴虫死亡，记录各pH值培养基中滴虫的最长存活时间。结果 阴道毛滴虫能在pH3～8的肝浸汤培养基中生长繁殖，最适pH范围为5～7．5，最适pH值是6。在pH4和pH8的培养基中连续不转代培养时，滴虫存活时间最长，分别达180h和189h。结论 肝浸汤培养基pH值为6时，滴虫生长良好，运动活泼，繁殖速度快，适用于快速繁殖滴虫，提高滴虫收获量；在pH4和pH8时，滴虫生长缓慢，但存活时间显著延长，适合保种培养。     

MTT染色法计数阴道毛滴虫体外增殖密度的试验

目的评价四氮唑蓝(5-’diphenyl tetrazolium bromide,MTT)染色法计数阴道毛滴虫增殖密度的可行性。方法在已接种了阴道毛滴虫的肝浸汤(含小牛血清)、RPMI-1640(含小牛血清)、RPMI-1640(无血清)培养基中加入MTT,与血球计数板法进行比较。结果加入MTT后4、244、8 h,上述3种培养基中大部分阴道毛滴虫虫体内部未见蓝色结晶体形成,有血清的培养液镜下观察视野不清晰。结论MTT法不宜用于计数体外培养阴道毛滴虫的增殖密度。     

阴道毛滴虫低温保存及其影响因素

观察不同浓度二甲亚砜(DMSO)和甘油、不同密度滴虫及不同冻存时间对阴道毛滴虫在-78℃低温保存的影响。阴道毛滴虫的最适冻存密度为(1～2)×106/ml。10%甘油和10%二甲亚砜的冻存效果最好,复苏存活率分别为38.0%和31.7%,两者差异无统计学意义(P0.05)。短期冻存(1～16周)的效果良好,均可在37℃重悬培养至对数生长期。     

中性粒细胞杀灭阴道毛滴虫作用的研究

目的 研究中性粒细胞对阴道毛滴虫的杀灭作用。 方法 将滴虫性阴道炎患者的阴道分泌物接种于肝浸汤培养基，获阴道毛滴虫。取患者静脉血分离血清，取其1 ml 于56 ℃ 30 min获补体失活血清。另取1 ml患者血清于0 ℃以阴道毛滴虫吸附3次，获去除抗体血清。用密度梯度离心法及聚合物加速沉降法分离、纯化患者静脉血中性粒细胞。用氮蓝四唑（NBT）和沙黄O（safranin O）染色，显微镜观察中性粒细胞与阴道毛滴虫相互作用及甲臢（NBT还原产物)颗粒（formazan）沉积。取300个阴道毛滴虫和3×104个中性粒细胞，分别在有氧或厌氧、有或无超氧化物岐化酶（SOD）及过氧化氢酶（CAT）、有或无补体等不同条件下，培养10、20、30、40、50及60 min，再接种于固态琼脂培养基，在37 ℃厌氧条件下继续培养5 d。观察计数阴道毛滴虫存活率。 结果 显微镜下可见几个中性粒细胞同时围攻杀灭1个阴道毛滴虫。含有中性粒细胞时培养的虫体存活率，厌氧条件下为85%，有氧条件为3%（P﹤0.01）。SOD及CAT可明显降低其杀虫作用，培养60 min虫体存活率分别为98%及94%，而无SOD及CAT时虫体存活率为2%（P值均<0.05）。加入去除抗体血清，可将虫体全部杀灭。加入补体失活血清则无杀虫作用。 结论 中性粒细胞杀灭阴道毛滴虫作用依赖于氧及患者血清中补体的存在。     

双氢青蒿素对阴道毛滴虫微丝作用的观察

目的　探讨双氢青蒿素对阴道毛滴虫的杀伤效果及作用机制。　方法　用含双氢青蒿素的肝浸汤培养基培养阴道毛滴虫 ,观察药物对滴虫的杀灭效果 ;用激光共聚焦显微镜观察双氢青蒿素作用前后滴虫微丝的变化。　结果　随药物作用时间延长和药物浓度增加 ,阴道毛滴虫死亡率增高。同一时间随着药物浓度的增高 ,虫体死亡率升高 (P <0 .0 1)。作用 6h ,双氢青蒿素 0 .6mg ml虫体死亡率是 2 0 % ,而 1.0mg ml时高达 85 % ;同一药物浓度随着作用时间延长 ,滴虫死亡率也升高 (P <0 .0 5 ) ,药物 0 .8mg ml时 ,6h、8h、10h、12h和 14h死亡率分别是 43 %、68%、86%、97%和 10 0 %。药物作用后滴虫微丝排列疏松 ,有空隙生成。排列杂乱无序。　结论　双氢青蒿素可作用于滴虫微丝结构 ,破坏微丝 ,具有较强的杀滴虫作用。     

中药体外抗阴道毛滴虫的试验研究

为开辟新的抗阴道毛滴虫药物 ,本实验选择了 3种具有杀虫、消炎作用的中药进行了体外抗阴道毛滴虫的效果观察。1　材料和方法1.1　培养基　选用肝浸汤培养基 ,p H5 .5～ 6 .0。1.2　试验虫种　取初次确诊为滴虫性阴道炎患者阴道后穹窿处分泌物 ,接种于培养管内 ,每 3d转种传代 ,稳定培养三代开始试验。1.3　药物制备　取仙鹤草、苦参和雷丸各 10 g,置 2 0 0 ml蒸馏水中浸泡 6 h,煮沸 30 min取出药液 ,再加水 10 0 ml,微火煎沸2 0 min取出液体并与前者药液混合 ,8层纱布滤过 ,加热浓缩成2 0 ml,即为 5 0 %的药液。设对照组为培养液。1.4　药…     

中草药体外抗阴道毛滴虫的实验研究

本文用蛇床子、苦参、苍耳子、黄柏及仙鹤草嫩茎叶等中草药混合加工提纯的黄色晶体粉和混合渗出液进行了体外抗阴道毛滴虫的实验研究。 材料与方法 1 虫种 阴道毛滴虫A、B分另来源于经灭滴灵多次治疗和初次诊断的患者阴道分泌物,分别接种于预先加入无菌兔血清1～2ml和抗菌素的无菌肝浸汤培养基中,每管8ml,置36±0.5℃孵箱内     

3种不同培养基体外培养阴道毛滴虫效果的比较

目的　探索阴道毛滴虫体外培养的适宜条件。　方法　用临床分离的阴道毛滴虫 ,按 9.0× 10 4 / ml的接种量转种至 3种不同培养基进行培养 ,p H值为 5 .6。　结果　经 3种培养基培养 ,96 h后阴道毛滴虫数量存在差异 ,其中以半胱氨酸 -肝 -胨 -麦芽糖培养基 培养基 )虫数较多 ,肝 -胨 -麦芽糖培养基 培养基 )次之 ,大豆 -肝 -胨 -麦芽糖培养基 培养基 )较少。培养基 与培养基 及培养基 相比较 ,滴虫存活率差异具有显著性意义 P<0 .0 1,P<0 .0 5 ) ,滴虫生长密度差异也具有显著性意义 P<0 .0 1,P<0 .0 5 ) ,生长密度高峰持续时间分别为 192 h、14 4 h和 96h,最长存活时间分别为 2 88h、2 16 h和 192 h。　结论　半胱氨酸 -肝 -胨 -麦芽糖培养基较适于阴道毛滴虫体外增殖     

人芽囊原虫保存方法的初步研究

目的 观察冻存剂、温度等因素对人芽囊原虫活力的影响，探索理想的人芽囊原虫保存方法。 方法 从患者阳性粪便中分离人芽囊原虫，分装到2 ml无菌冻存管内，分别加入10%二甲基亚砜（dimethylsulfoxide, DMSO）、40%丙三醇（GL）和15%乙二醇（EG）作冻存剂，放置于不同的温度下保存，用台盼蓝染色法和原虫培养法测定细胞的活力和增殖能力。 结果 虫体在室温（18 ℃～20 ℃）下可存活3周，在4 ℃～6 ℃存活不到1周。加入冻存剂后，置－20 ℃和液氮（－196 ℃）下可存活3个月以上。用40% GL作冻存剂，于液氮低温下冻存的虫体保存半年后复苏， 活力仍达41.7%，培养72 h后多见分裂相细胞。 结论 应用40% GL作为冻存剂， 在液氮中低温保存人芽囊原虫效果较佳。 4 ℃不适于保存人芽囊原虫。     

Effect of the silk protein sericin on cryopreserved rat islets

Background/purpose

Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori.

Methods

Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3?days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation.

Results

There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS.

Conclusion

The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.     

A practical preservation method for human diploid fibroblasts in aging research

A practical method for long-term storage of human diploid fibroblasts was investigated. The conditions of increased cell attachment (recovery) and/or viability are as follows; freezing 1?10 × 10⁶ cells/ampoule at a rate of 1°C/min to ?35°C with the culture medium containing 30% FBS and 15% glycerol, thawing the contents rapidly by agitation in a 40°C water bath, and diluting 5-fold by the addition of the growth medium at 15 min after thawing. Consequently, this recommended method shows 78.8% recovery and 90.8% viability, in contrast to 32.9% recovery (82.5% viability) obtained in the conventional method. On the other hand, cells frozen with DMSO as an additive showed 52.9% recovery and 85.9% viability. The preservation of human diploid fibroblasts with glycerol was accordingly preferred to a method with DMSO. This practical method is useful for long-term storage of not only young cells but aged cells for the research on cellular aging.     

Survival and infectivity of Brugia malayi microfilariae after cryopreservation

Methods were studied for the cryopreservation of microfilariae of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum was used as cryoprotectant. Samples were frozen slowly in the vapor phase of liquid nitrogen prior to emersion in liquid nitrogen (-196 degrees C). The freezing rate was -0.5 to -1.0 degrees C per minute, microfilariae remained viable for as long as, 212 and 375 days, survival rates were 94 to 98% and they were infective to Aedes togoi mosquitos. The infective larvae (L3) were obtained for 10-11 days after feeding at 28 degrees C room-temperature and the infection rate of L3 in test mosquitos was 22.4-30.6%. All DMSO should be removed from the freezing medium to restore microfilariae activity after freezing.     

An agar culture technique to quantitate Trichomonas vaginalis from women

In subjects with trichomoniasis the number of Trichomonas vaginalis in vaginal discharges or secretions is unknown. The presence of T. vaginalis was evaluated in 177 consecutive female patients attending an inner city sexually transmitted disease clinic by patient history, wet mount, and broth culture. T. vaginalis was quantitated by a novel agar culture technique. Of the 177 women, 86 (49%) were positive for T. vaginalis by either wet mount or culture. Clinical symptoms were not reliable for making an accurate diagnosis of trichomoniasis. Culture on modified Diamond's medium was more sensitive (98%) than the wet mount method (38%) in detecting T. vaginalis. Of the 86 patients who were positive for trichomoniasis, quantitation was obtained for 81 patients, with 70% yielding greater than 10(4) colony-forming units (cfu)/ml. The number of T. vaginalis ranged from 40 to greater than 10(6) cfu/ml. The wet mount method was very insensitive for detecting T. vaginalis and was positive only in patients yielding greater than 10(5) cfu/ml.