酵母菌的分离培养及发酵实验方法

通过分离培养得到纯种酵母菌并对其进行观察区分;用几种酵母菌进行糖发酵实验,比较酵母菌对不同糖的利用能力;利用培养所得到的酿酒酵母和假丝酵母酿造葡萄酒,测量葡萄酒的酒精度数,对比确定哪一种酵母菌的酿酒效果更好、产酒率更高。通过平板划线法及稀释涂布法获得纯种的酵母菌种,同时结合菌落形态观察法初步区分酵母菌的种类;通过压滴法区别酵母菌的细胞形态;通过美蓝染色法鉴别酵母菌的死活;采用穿刺接种法分别将酵母接种在带有指示剂的半固体培养基上。3种实验酵母都不发酵乳糖,酿酒酵母的发酵蔗糖和葡萄糖的能力远不及红酵母和假丝酵母。酿酒酵母所酿造出的葡萄酒酒精度数高于假丝酵母。     

口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

医院内假丝酵母菌感染菌种的分布及耐药分析

目的了解医院内假丝酵母菌感染菌种的分布及抗真菌药物的耐药谱,为临床抗真菌治疗提供正确数据。方法采用VITEK-32微生物自动鉴定仪进行假丝酵母菌菌种的鉴定,同时用ATB FUNGUS真菌药敏条作药敏试验。结果从住院患者的血液、痰、尿、分泌物、胆汁等标本中共检出真菌505株。其中白假丝酵母菌281株,热带假丝酵母菌124株,光滑假丝酵母菌21株,季也蒙假丝酵母菌17株,近平滑假丝酵母菌16株,克柔假丝酵母菌10株,皱折假丝酵母菌8株,异常汉逊假丝酵母菌6株,5氟-胞嘧啶和两性霉素抑菌效果最好,其耐药率分别为2.8%和3.4%。其他4种抗真菌药物制霉菌素、氟康唑、伊曲康唑和酮康唑的耐药率分别为6.1%、13.7%、9.3%和28.3%。结论医院假丝酵母菌感染耐药率逐年上升,非白假丝酵母菌感染明显增加。氟康唑对白假丝酵母菌有很强的抗菌活性且毒副作用小。假丝酵母菌感染的迅速增加同广泛应用超广谱抗菌药物、激素及免疫抑制剂等有关。假丝酵母菌感染死亡率高,因此早期诊断及有效性治疗是减少死亡率的关键。     

慢性前列腺炎患者假丝酵母菌感染及耐药性分析

目的 了解慢性前列腺炎与假丝酵母菌感染的相关性及假丝酵母菌的耐药性。方法 采用常规沙堡平板分离360例慢性前列腺炎患者的前列腺液标本中的假丝酵母菌,疑似菌落用ATB Expression鉴定仪进行鉴定。采用ATB-Fungus真菌药敏板,对假丝酵母菌株进行药敏试验。结果 11．7％前列腺液标本（42／360）似丝酵母菌阳性,其中自假丝酵母菌23例（54．7％）,近平滑假丝酵母菌13例（30．9％）,其它6例（14．3％）。假丝酵母菌菌株对两性霉素B和制霉菌素敏感率均为100％,其次为酮康唑,敏感率为97．0％;对5-氟胞嘧啶耐药性最强,其耐药率为56．5％。结论 白假丝酵母菌和近平滑假丝酵母菌是慢性前列腺炎假丝酵母菌感染的优势菌种,假丝酵母菌株最敏感的药物是两性霉素B和制霉菌素。     

白假丝酵母菌突变株ORF19.2500的功能研究

目的筛选50株白假丝酵母菌基因缺失菌,寻找出对白假丝酵母菌生物被膜形成相关基因,进一步探究白假丝酵母菌生物膜的致病机制。方法利用培养生物被膜的方法筛选50株基因缺失菌;利用XTT法验证所筛选出的白假丝酵母菌突变株ORF19.2500生物被膜形成缺陷;进一步观察ORF19.2500基因缺失菌生长、菌丝形成。结果用XTT法证明白假丝酵母菌突变株orf19.2500生物膜形成缺陷,且生长曲线和滴琼脂平板的方法均提示其生长速率减慢。在spider培养基上白假丝酵母菌突变株orf19.2500不能诱导菌丝形成,但在YPD+10%小牛血清则菌丝形成正常。结论白假丝酵母菌突变株orf19.2500可利用spider培养基缺陷而影响其酵母、菌丝二态性的转化,及其生物被膜的形成。     

皮肤性病门诊生殖道酵母菌阳性结果分析

摘要：目的 探讨生殖道假丝酵母菌阳性率、药敏情况及耐药趋势,为临床合理选用抗真菌药物提供最新的依据。方法 按照《全国临床检验操作规程》,采用法国梅里埃生物公司API-20C酵母菌鉴定板、ATB-Funguns酵母菌药敏板对2012年1月至2014年12月培养生殖道酵母菌阳性的标本进行鉴定及药敏试验。结果 1 419例生殖道分泌物分离出114例假丝酵母菌,感染率为8.0%,其中白假丝酵母菌101例占88.60%,感染率最高,其他非白假丝酵母菌13例占11.40%;药敏结果表明,假丝酵母菌对5-氟胞嘧啶、两性霉素B具有较高敏感性（92.11%、100.00%）,对氟康唑、伊曲康唑、伏立康唑呈现不同程度的耐药;101例白假丝酵母菌对5-氟胞嘧啶、两性霉素B的敏感性分别为93.07%、100.00%,未出现对两性霉素B的耐药菌株。结论 皮肤性病门诊生殖道酵母菌分离株仍以白假丝酵母菌为主,白假丝酵母菌对氟康唑、伊曲康唑、伏立康唑的耐药率呈上升趋势;微生物实验室应加强对假丝酵母菌株的分离鉴定及耐药性监测,为临床医生准确合理地使用抗真菌药物提供病原学依据。     

皮肤性病门诊生殖道酵母菌阳性结果分析

目的探讨生殖道假丝酵母菌阳性率、药敏情况及耐药趋势,为临床合理选用抗真菌药物提供最新的依据。方法按照《全国临床检验操作规程》,采用法国梅里埃生物公司API-20C酵母菌鉴定板、ATB-Fungus酵母菌药敏板对2012年1月至2014年12月培养生殖道酵母菌阳性的标本进行鉴定及药敏试验。结果 1 419例生殖道分泌物分离出114例假丝酵母菌,感染率为8.0%,其中白假丝酵母菌101例占88.60%,感染率最高,其他非白假丝酵母菌13例占11.40%;药敏结果表明,假丝酵母菌对5-氟胞嘧啶、两性霉素B具有较高敏感性(92.11%、100.00%),对氟康唑、伊曲康唑、伏立康唑呈现不同程度的耐药;101例白假丝酵母菌对5-氟胞嘧啶、两性霉素B的敏感性分别为93.07%、100.00%,未出现对两性霉素B的耐药菌株。结论皮肤性病门诊生殖道酵母菌分离株仍以白假丝酵母菌为主,白假丝酵母菌对氟康唑、伊曲康唑、伏立康唑的耐药率呈上升趋势;微生物实验室应加强对假丝酵母菌的分离鉴定及耐药性监测,为临床医生准确合理地使用抗真菌药物提供病原学依据。     

497例临床假丝酵母菌菌种分布及耐药性分析

目的了解沈阳地区住院患者标本中分离出假丝酵母菌感染的类型、分布及耐药情况,为指导临床预防假丝酵母菌感染及合理用药提供理论依据。方法回顾性收集中国医科大学附属盛京医院2015～2018年住院患者送检标本中分离出假丝酵母菌菌株的临床资料,统计菌株鉴定及药敏实验数据及结果。结果共检出假丝酵母菌497株,其中以近平滑假丝酵母菌为主(175, 35.2%),其次依次为白假丝酵母菌(127, 25.6%)、菌膜假丝酵母菌(51, 10.3%)、光滑假丝酵母菌(42, 8.5%)和热带假丝酵母菌(36, 7.2%),菌种分布存在统计学差异(P=0.004);菌株宿主主要来源于0～9岁及50～69岁年龄段患者;检出标本来源以血液标本最多(405, 81.5%),胸腹水(21, 4.2%)和引流液(21, 4.2%)次之,不同标本类型之间菌株分布有统计学差异(P=0.003);临床常见假丝酵母菌对氟康唑(FLU)、伏立康唑(VRC)、氟胞嘧啶(FC)、伊曲康唑(ITR)和两性霉素B(Amb)的耐药率分别为3.0%、2.2%、4.0%、5.0%和4.0%。结论院内假丝酵母菌感染以近平滑假丝酵母菌最为常见。在治疗新生儿、老年患者和消化系统疾病患者,要警惕发生假丝酵母菌的二重感染。     

致念珠菌性包皮龟头炎白假丝酵母菌对9种抗真菌药物最低抑菌浓度测定

目的检测引起念珠菌性包皮龟头炎的白假丝酵母菌对9种抗真菌药物MIC,为临床治疗念珠菌性包皮龟头炎提供参考依据。方法常规培养分真菌,并鉴定到种,采用最低抑菌浓度法对白假丝酵母菌进行体外药物敏感试验。结果培养分离的12株菌中,白假丝酵母菌占75.00%,白假丝酵母菌生物变种占16.67%,近平滑假丝酵母菌占8.33%;11株白假丝酵母菌耐药率由高到低依次是氟康唑,伊曲康唑,咪康唑,酮康唑,克霉唑,益康唑,5-氟胞嘧啶啶,两性霉素B,制霉菌素,其中6种药物耐药率大于50%。结论白假丝酵母菌仍是造成念珠菌性包皮龟头炎最常见的致病菌,耐多药现象较为普遍,应根据临床实验室的体外药敏试验结果,指导临床合理用药。     

健康人群口腔酵母菌的分离及菌种分布

目的调查健康人群口腔酵母菌的分布。方法将来源于健康人的口腔拭子接种于改良SDA培养基,37℃培养14d,分到的酵母菌通过菌落形态、革兰染色作初步鉴定．芽管形成试验、厚膜孢子生成及假菌丝产生试验、YBC酵母鉴定卡作菌种鉴定。结果酵母菌在健康人口腔中总分离率为8．78％(86／979),其中较多的为白色假丝酵母菌37株,占43．02％,近平滑假丝酵母菌和季也蒙假丝酵母菌各9株,各占10．47％,葡萄牙假丝酵母菌6株,占6．98％,其他25株。结论健康人口腔中有多种条件致病酵母菌的寄生。     

甲真菌病多点平皿培养法与常规试管培养法的比较

目的比较甲真菌病病原体检测中多点平皿培养法与常规试管培养法的差异。方法选择2004年10月～12月及2005年10月～12月病甲标本,分别采用多点平皿培养法(试验组)和常规试管培养法(对照组)分离病原体并作菌种鉴定。结果多点平皿培养法和常规试管培养法之间比较,阳性率分别为68.4%和49.3%(P=0.0029),污染率分别为13.2%和4.6%(P=0.0246)。致病菌分离结果:对照组皮肤癣菌75.7%,酵母菌18.9%,非皮肤癣菌霉菌5.4%;试验组则分别为46.2%,34.6%和16.7%。两组分离菌种比较,P值均<0.05。试验组共检出2例混合感染,对照组则无。结论多点平皿培养法阳性率高,可以提高对甲真菌病的病原菌的检出率,尤其是对非皮肤癣菌酵母菌和霉菌的检出率,且有助于发现甲真菌病的混合感染。但多点平皿培养法污染率亦高,应规范无菌操作。     

Biological control of maize seed pathogenic fungi by use of actinomycetes

The effectiveness of twoStreptomyces spp. strains to controlpathogenic fungi was studied in stored maizegrain. The treatments included seeddisinfection and inoculation withStreptomyces spp. strains previously isolatedfrom maize rhizosphere. Actinomycete inoculumconsisted of filtered suspension and totalsuspension of fermentor-producedStreptomyces spp. strains biomass. Treatmentswith Streptomyces spp. strains aloneeffectively suppressed the development ofAspergillus spp., Curvularia lunata, andDrechslera maydis and significantly(p < 0,05) reduced the incidence ofFusarium subglutinans and Cephalosporiumacremonium. Among the inoculation treatments,nondisinfested seed inoculated with filteredsuspension was the only treatment that did notsuppress the development of Penicilliumspp. Maize seed inoculation with totalsuspension of strains was the most effectivetreatment to control the incidence of seedpathogenic fungi. The development of theDiplodia maydis was only suppressed by thecombination of seed disinfection andinoculation with total suspension of strains.Although, the strain DAUFPE 11470 showed thegreatest effectiveness for controlling thefungi pathogenic to seed, root and shootdevelopment was reduced by treatment with thisstrain.The results indicate thatStreptomyces spp. strains reduce the incidenceof seed pathogenic fungi and have potential asa biological control agent. However, an efficient methodof seed treatment with the biological controlagent must be developed before it can become anagricultural practice.     

Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension

Streaking is a severe problem when narrow range basic immobilized pH gradient strips are used as the first dimension of two-dimensional (2-D) electrophoresis. It is demonstrated that this cysteinyl related streaking is eliminated when focusing is done in the presence of hydroxyethyl disulfide (DeStreak). Use of DeStreak also results in 2-D maps with simplified spot patterns and improved reproducibility.     

Microculture des champignons sur disques de polythène

The microculture of fungi on polythene discs, simple, rapid and economic method, could be used as a variant of the slide culture method. It may also be used for the direct isolation of fungi from various specimens (hair, skin, etc.).     

Comparison of methods for isolation of dematiaceous fungi from nature

Direct plating and animal inoculation techniques were compared for effectiveness in isolating dematiaceous fungi from nature. The direct plating technique involved comparison of aqueous and mineral oil extraction of the samples with subsequent plating on Mycobiotic and Sabhi agar. Twenty four different organisms were recovered from 19 samples using both extraction procedures and both media. Dematiaceous fungi isolated by direct plating were Alternaria alternata, Cladosporium spp., Exophiala jeanselmei, Wangiella dermatitidis and 6 unidentified organisms. Direct plating resulted in more isolations of dematiaceous fungi partly because of the frequent isolations of A. alternata and W. dermatitidis. Both more and a larger variety of organisms were recovered on Mycobiotic than on Sabhi agar. The aqueous extraction of samples resulted in more direct plating isolations of fungi than oil extraction.Only dematiaceous fungi were isolated with the animal inoculation techniques. Fungi isolated from hamsters included Cladosporium spp., Exophiala spinifera, Phialophora verrucosa, Rhinocladiella sp. and 3 unidentified organisms. Fungi isolated by the mouse inoculation technique included Bispora betulina, Cladosporium spp., W. dermatitidis and 1 unidentified organism. In general there was variation in the types of organisms isolated from the same samples depending on the isolation procedure used.     

新生隐球菌交配型分析试验方法和培养基的研究

比较观察了Hayagar(HA)、Matingagar (MA)、Haycubeagar (HCA)、稻壳琼脂(RSA)、富营养Hayagar(IHA)以及沙保弱培养基 (SDA) 6种培养基的新生隐球菌交配试验效果。按 2周内产生阳性结果排列培养基的次序是HCA(95% )、RSA(95% )、HA(86% )、MA(38% )、SDA(38% )和IHA(2 4 % )。采用点状可以替代划线接种方法用于处理大批量标本并节省材料。此外 ,先将诱导菌株和受试菌株混合预培养可以使原方法不能交配的菌株产生阳性结果     

Clinico-experimental study of a dried selective medium for isolating fungi in the genus Candida

The trial of a newly developed dried selective medium for the isolation of fungi of the genus Candida, involving the inoculation of 103 museum yeast strains and 542 specimens of pathological material, has been carried out. The data obtained in this trial indicate that the proposed medium has advantages over wort agar and Sabouraud medium with antibiotics in the germination index, in the ability to ensure fungal growth after the inoculation of pathological material, and in selective properties. The medium ensures the intactness of the morpho-cultural, biochemical and serological properties of the fungi. The results of the trial recommend the newly developed preparation for laboratory practice for the isolation of yeast-like fungi from clinical material in the diagnosis of candidiasis, intestinal microflora disturbances, as well as for isolation of the fungi from various environmental objects.     

A simple method of isolation and purification of cultures of wood-rotting fungi

A simple method of isolation and purification of cultures of higher fungi, mainly wood-rotting fungi, without special requirements for the presence of nitrogencontaining nutrients is described. Parts of fruiting bodies, spores or infected wood is inoculated on Petri dishes with an agar medium of the following composition: 1.0 g KH2PO4, 0.2 g MgSO4 - 7 H2O, 0.1 g caSO4, 0.01 g Fe2(SO4)3, 10.0 g glucose, tap water to 1 litre; 20.0 g agar. This medium does not suit most of the contaminants but fungal hyphae overgrow the whole surface of the dish so that a purified culture can be obtained from parts distant from the inoculation site.     

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.     

Comparison of disk diffusion test and Etest for voriconazole and fluconazole susceptibility testing

The distribution for voriconazole and fluconazole susceptibility was determined by Etest and disk diffusion test in 143 clinical isolates. The majority of the strains of Aspergillus spp., Candida krusei, C. inconspicua, C norvegensis and Saccharomyces cerevisiae displayed resistance or decreased susceptibility to fluconazole in contrast to voriconazole. The absolute categorical agreement for voriconazole and fluconazole susceptibility results by the disk method and Etest was 90.5 and 74.8 % respectively. The error rate bounding analysis showed only 0.7 % of false susceptible results ( very major error) with voriconazole, but 2.8 % with fluconazole. Fluconazole can be used as a surrogate factor to predict voriconazole susceptibility but with lower reliability for susceptible-dose dependent and resistance category, especially in Candida glabrata isolates. The results of the disk method were not substantially influenced by the composition of media (Mueller-Hinton agar vs antimycotic Sensitivity Test agar), even if with the latter the results had fewer tendencies to produce false susceptibility of C.glabrata isolates to both of the triazole drugs. Disk test as well as Etest were shown to represent suitable methods for routine evaluation of susceptibility of clinical isolates of pathogenic fungi, including aspergilli, to fluconazole and voriconazole.