美国标准培养物保藏中心主任R.E.Stevenson的来信

美国标准培养物保藏中心(American Type Culture Collection,ATCC)主任R.E.Stevenson博士最近致信中国微生物学会朱既明理事长,介绍该中心的工作。特予刊登,供国内有关科学家参考,以利交流。如寄送培养物,请按照我国政府有关规定办理。     

最新专利文摘

CN102653720A 一株产石杉碱甲的蛇足石杉内生真菌ES026 本发明公开了一株从药用植物蛇足石杉中分离出的内生真菌。本发明分离的内生真菌ES026经分子生物学和形态学鉴定为炭疽属胶孢种（Colletotrichum gloeosporioides）,该菌株已保藏在中国典型培养物保藏中心,保藏编号为CCTCCM2011046。     

极端嗜酸硫杆菌高效筛选、高密度发酵及保藏方法的研究

【目的】针对嗜酸硫杆菌极端特殊的生化特性,分别建立双层平板培养高效筛选方法和补料分批高密度发酵策略,并优选最佳保藏方法,以强化对该类菌种资源的利用和储备效率。【方法】分别采用以异养型微生物Sacchromyces ellipsoideu和Rhodotorula sp.为底层培养物的双层平板培养嗜酸硫杆菌,并结合透射电子显微镜技术(TEM)考察细胞形态差异。结合硫化矿培养基设计及单质硫补料培养策略,延长Acidithiobacillus thiooxidans对数期,提高比生长速率。分析不同保藏方法对嗜酸硫杆菌细胞存活率的影响。【结果】采用异养微生物——Rhodotorula sp.作为底层培养物的双层平板培养法在缩减1/3检出周期的同时将Acidithiobacillus ferrooxidans和Acidithiobacillus thiooxidans的检出率提高了3倍左右。TEM结果表明双层培养中细胞形态更为规则。采用基于Starkey-硫化矿培养基的补料分批发酵策略提高了Acidithiobacillus thiooxidans平均比生长速率,硫对生物量转化率和生产强度分别比分批培养提高31.1%和187.9%。4°C低温保藏方式更适于嗜酸硫杆菌的保藏,有效保藏期1–3月。【结论】Rhodotorula sp.为辅助培养物的双层平板培养法可有效提高嗜酸硫杆菌的筛选效率。设计的Starkey-硫化矿培养基结合补料分批培养策略可实现Acidithiobacillus thiooxidans高密度培养。简单高效的4°C低温保藏方式更适合于嗜酸硫杆菌的中短期保藏。     

两种枝顶孢霉胞内细菌的分离鉴定及种群演替

分离、鉴定了枝顶孢霉(Acremonium strictum Gams.)2种胞内细菌,探讨了细菌在其宿主真菌菌丝细胞内的种群演替规律。结果表明,2种胞内细菌分别为不动杆菌Epbas6菌株(Acinetobacter sp.Epbas6)和地衣芽孢杆菌(Bacillus licheniformis)。镜检分析和原始分离物菌落统计表明,不动杆菌和地衣芽孢杆菌的比例为77.5∶1;然而,4℃保藏6个月的真菌分离物中不动杆菌的数量大大减少,地衣芽孢杆菌占据优势,二者的比例变为1∶50.45;而保藏12个月的真菌分离物中只有地衣芽孢杆菌。根据2种细菌的16SrDNA保守序列设计引物,分别扩增了保藏6个月和12个月的枝顶孢霉分离物,发现保藏6个月的分离物能够扩增出2种细菌的保守序列,而保藏12个月的分离物只能扩增出地衣芽孢杆菌保守序列。研究结果说明在枝顶孢霉人工培养和菌种保藏过程中,2种细菌在其宿主真菌菌丝细胞内发生着复杂的生态互作,存在着动态种群演替过程。     

神农架林区和自然保护区芽孢杆菌资源的调查研究

神农架林区和自然保护区芽孢杆菌资源的调查研究黄洋,陶天申（武汉进出口食品检测中心４３００２２）（中国典型培养物保藏中心４３００７２）芽孢杆菌属是一群好氧和兼性厌氧生括、产耐热性内生孢子的杆状细菌。它的营养细胞具有活跃的酶活性和多样的代谢类型,从而被广...     

利用培养特征和生理特性鉴别担子菌菌种的研究

担子菌菌种多是菌丝型的培养物,在长期保藏中如发生错误也较难辨别。本文报道了以培养特征和生理特性作为保藏的担子菌菌种的特征集要,并根据集要制定出鉴别他们的检索表。通过对48属、71种、142株菌种的实验结果,利用培养特征和生理特性鉴别保藏的担子菌菌种是可行的。     

幽门螺旋菌的长期保藏

常规方法保藏幽门螺旋菌极为困难。将该菌新鲜培养物接入补加小牛血清及甘油的布氏肉汤中,于-70℃保藏,可安全保藏5—13个月。     

一株纤维蛋白溶解酶产生菌的鉴定及其产酶条件初步研究

从亚洲传统发酵食品--虾酱中筛选到一株产纤维蛋白溶解酶能力较强的菌株,通过形态和常规生理生化性质鉴定,发现该菌株与芽孢杆菌属细菌的特征很相近,结合16S rDNA序列分析,构建系统发育树,确定其分类地位,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006(CCTCC No.M 205071),并优化了发酵培养基组成及培养条件,本研究为该菌株的深入研究和广泛应用提供了理论依据.     

中国一新记录属-加氏菌属

从辽宁省鞍山市采集的玉米罹病籽粒上分离得到甜菜加氏菌Gabarnaudia betae。加氏菌属Gabarnaudia为我国新记录属。本文对其培养形态特征进行了描述,菌种保藏在大连民族学院真菌菌种保藏中心(菌株号IBE000935)。     

第五届国际菌种保藏会议(ICCC—Y)

由世界菌种保藏联合会(TVFCC)主办,于1984年11月25日至12月1日在泰国曼谷将举行第五届国际菌种保藏会议。会议主题是“生物工程的国际资源”。而且将着重对工艺方法,生化转化,农业和水生培养物,氮的固定,植物组织培养,模式系统和标准菌株,毒理,抗生素测定和国际     

Establishment and characterization of a fibroblast line from Simmental cattle

A fibroblast line (named SCF36) from ear marginal tissue of Simmental cattle was established successfully by direct culture of explants and cell cryopreservation techniques. Biological analysis showed that the population doubling time of the thawed cells was 42.8 h. The average viability of the cells was 96.8% before freezing and 91.5% after thawing. Measurements of lactic dehydrogenase and malic dehydrogenase isoenzymes showed no cross-contamination of this cell line with other species. Karyotyping showed that the frequency of cells with chromosome number 2n = 60 was more than 90%. Tests for bacteria, fungi, viruses and mycoplasmas were negative. The efficiencies of expression of enhanced green, yellow and red fluorescent protein genes (pEGFP-N₃, pEYFP-N₁ and pDsRed1-N₁) were between 11.3% and 28.8% after transfection; fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. This Simmental cattle fibroblast line not only contains the germline of this important cattle breed, which is preserved at the cellular level, but valuable material has also been provided for genomic, postgenomic and somatic cloning research. Moreover, the establishment of these methods may provide both technical and theoretical support for preserving the genetic resources of other livestock and poultry at the cellular level.     

Establishment and biological characteristics of Luxi cattle fibroblast bank

A fibroblast line from ear marginal tissue of Luxi cattle (LXCEM2/2) was successfully established by direct culturing of explants. Biological analysis showed that the population doubling time (PDT) for reviving cells was approximately 24 h. Measurement of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n = 60 was 90.7–92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. The efficiencies of expression of pEGFP-N3, pEYFP-N1 and pDsRed1-N1 were between 6.3% and 31.6% at 24 h, 48 h and 72 h after transfer; at 24 h, fluorescence was well distributed in the cytoplasm and nucleus except for some cryptomeric vesicles. Every index of the Luxi cattle cell line meets the quality control standards of the American Type Culture Collection (ATCC). Not only has the germline of this important cattle breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.     

Establishment and characterization of a fibroblast cell line from the Mongolian horse

A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analysis of lactic and malic dehydrogenases. In order to study exogenous gene expression, four fluorescent proteins, pEGFP-N3, pEGFP-C1, pDsRed1-N1, and pEYFP-N1, were transfected into the cells. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 12 h after transfection. This cell line not only preserves the genetic resources of the Mongolian horse at the cellular level but also provides valuable materials for genomic, postgenomic, and somacloning research in this species.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

Establishment and biological characteristics of Ujumqin sheep fibroblast line

A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, frozen in 147 cryovials with 4 × 10⁶ cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cells were morphologically consistent with fibroblasts. The growth curve was typical S-shape and the cell population passed through a lag phase, a logarithmic phase and a plateau phase. The population doubling time (PDT) was approximately 72 h. Tests for bacteria, fungi, viruses and mycoplasma were all negative. Isoenzyme polymorphism indicated that the genetic characteristics of the cell line were stable in vitro. Karyotyping analysis indicated that the chromosome number of a normal cell was of 2n = 54 and 95.4% of the entire population was diploid. The transfection efficiencies of six fluorescent proteins (pEGFP-N3, pEGFP-C1, pDsRed-N1, pEYFP-N1, pECFP-N1 and pECFP-mito) optimal at 48 h were from 18.5% to 30.1%. The cell line met all criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somatic cloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.     

Establishment and genetic characteristics analysis of in vitro culture a fibroblast cell line derived from Wuzhishan miniature pig

Establishment of fibroblast cell lines of endangered pig breeds and research on the gene functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. The Wuzhishan miniature pig ear marginal tissue fibroblast cell line (WPF22) from 22 samples, stocking 87 cryogenically-preserved vials, was successfully established by using primary explants technique and cell cryopreservation techniques. WPF22 cells were adherent, with a population doubling time of 30.2 h. Chromosome karyotyping and G-banding analysis showed that >90.2% of cells were diploid (2n = 38) prior to the 4th generation. Neither microbial contamination nor cross-contamination was detected by isoenzyme analyses. Cell viability was 97.8% before cryopreservation and 94.9% after recovery. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, six fluorescent protein genes were transferred into fibroblasts by lipofectamine-mediated method. The transfection efficiency of six fluorescent protein genes fluctuated between 8.1% and 42.6%. ECFP and DsRed were mostly shown in cytoplasmic in dots around the nucleus, and EYFP and EGFP had a slightly stronger expression in the nucleus than in the cytoplasm, but without expression in some vacuoles. Every index of the WPF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). This research thus does not only preserve important genetic resources of Wuzhishan miniature pig at the cell level, but also serve as a valuable resource for genome, postgenome and somacloning research.     

马头山羊成纤维细胞系的建立与生物学特性分析

采用组织块贴壁培养法对马头山羊耳缘组织进行培养, 成功构建了马头山羊耳缘组织成纤维细胞系, 并对其形态学、生长动力学、细胞活力测定、中期染色体、微生物污染等特性进行了研究。结果表明: 培养细胞形态为典型的成纤维细胞, 细胞群体倍增时间(PDT)约为36 h。细胞冻存复苏后的活率为96.7%, 传代后生长状况与冻存前一致。细胞中期染色体二倍体(2n=60)占主体约为96%。微生物检测细菌、真菌、病毒支原体检测结果为阴性。细胞系各项指标均达到美国典型培养中心(ATCC)标准。此细胞库的建立在细胞水平上对马头山羊的遗传资源进行了保存, 也为今后的生物学研究以及体细胞克隆保种等研究提供了理想的实验材料。     

Establishment and characterization of a fibroblast cell line derived from Jining Black Grey goat for genetic conservation

An ear marginal fibroblast cell bank was established from the Jining Black Grey (JBG) goat using attachment culture and freezing biotechniques. This bank included 32 ear samples (15 males and 17 females) and has stocks of 168 cryogenically preserved vials, each vial contained 4.0 × 10⁶ cells per milliliter. The cells of the bank that were checked for the quality and the biological characteristics showed a typical fibroblast morphology when they cultured in vitro. The growth curve consisted of a growth curve consisting of a latent phase, logarithmic growth phase and stationary phase, cell population doubling time (PDT) of 48 h. The chromosome analysis showed that the frequency of cells having the diploid number of chromosomes (60) was 98.65 ± 2.89%, and no microbe contamination (bacteria, epiphyte, virus or mycoplasma) was detected. In addition, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) zymography indicated that this cell bank was free of cross-contamination. At 24, 48 and 72 h after transfection, the expression efficiency of pEGFP-C1, pEGFP-N3, pEYFP-N1, pECFP-N1, pECFP-mito and pDsRed1-N1 were between 11.8% and 56.3%. The fluorescence could be observed well-distributed in cytoplasm and nucleus except for some cryptomere vesicles at 24 h after transfection. These newly established cell lines meet all the quality control standards established by the American Type Culture Collection. We have employed a new method for conserving the genetic resources of an important and endangered animal breed. The fibroblast bank that we have established from the JBG goat also provides an invaluable material resource for future studies that will utilize molecular and cell biology applications.     

Establishment and biological characteristics of a Jingning chicken embryonic fibroblast bank

Using tissue explantation and cryopreservation biotechniques, a Jingning chicken embryonic fibroblast bank was successfully established, which includes 43 embryo samples and a stock of 178 cryovials, each one containing 3.0×106 cells. Most of the cells were apparently fibroblasts in their morphology, and the population doubling time (PDT) was about 48 h. The total chromosome number of a diploid cell was 78. According to karyotyping and G-banding, the diploid rate in the cell bank was 97.62±2.12%. The cells were tested for microbial contamination and found free of infections from bacteria, fungi, viruses and mycoplasms. There was no cross-contamination from other cell lines as revealed by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzyme polymorphisms. Six fluorescent proteins were transfected into the Jingning chicken embryonic fibroblasts, and the transfection efficiencies of these genes were between 10.1 and 41.9%. All the tests showed that the quality of the cell line conforms to the quality criteria of the ATCC (American type culture collection). This work succeeded not only in preserving the genetic resources of Jingning chicken, but it also established a new protocol to preserve endangered animal breeds.     

Establishment and characteristics of white ear lobe chicken embryo fibroblast line and expression of six fluorescent proteins in the cells

A white ear lobe chicken embryo (WELCE) fibroblast cell bank, containing 322 tubes of frozen cells, was successfully established from primary explants of 57 embryo samples. The cells were morphologically consistent with fibroblasts, and the growth curve was sigmoidal with a population doubling time (PDT) of 48 h. Karyotyping and G-banding indicated a total chromosome number of 2n=78; the rate of diploidy in the cell bank was 97.62%. The cells were also free from bacterial, fungal, viral and mycoplasma contamination. Analysis of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes ruled out cross-contamination between cells. In order to study exogenous gene expression, six fluorescent proteins were transfected into the WELCE cells. The transfection efficiency of these genes was between 10.1 and 41.9%. The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 24h after transfection. The results indicate that the quality of the cell line meet the quality requirements of the ATCC (American Type Culture Collection).