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Observation of Pneumococcal Phase Variation in Colony Morphology
肺炎球菌菌落形态学相位变异的观察   

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Abstract

Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae is correlated with a reversible switch between the opaque and transparent colony forms on agar plates, a phenomenon referred to as phase variation. The opaque variants appear to be more virulent in animal models of bacteremia but are deficient in nasopharyngeal colonization animal models. In contrast, the transparent variants display higher levels of nasopharyngeal colonization but relatively lower virulence in animal models. We have recently demonstrated that pneumococcal phase variation between these two colony types is caused by a reversible switch of genome DNA methylation (or epigenetic) patterns, which is driven by DNA inversions in the DNA methyltransferase genes. Observation of colony morphology is a simple and useful method to differentiate colonies with different characteristics, such as size, color, and opacity. This protocol describes how to study pneumococcal phase variation in colony morphology with a dissection microscope.

Keywords: Streptococcus pneumoniae(肺炎链球菌), Colony morphology(集落形态学), Phase variation(相位变异), Epigenetic switch(表观遗传开关), DNA inversion(DNA倒位), Dissection microscope(解剖显微镜)

Background

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis in children worldwide (Walker et al., 2013). The success of this pathogen in its adaptation to various ecological niches of human host depends on its remarkable phenotypic plasticity (Croucher et al., 2013; Johnston et al., 2014a), which has been reflected by the inter-strain antigenic variation in the capsular polysaccharides and surface proteins (Croucher et al., 2013 and 2011), acquisition of new virulence factors (Park et al., 2012), extensive drug resistance (Croucher et al., 2014) within the species. Natural genetic transformation is a well-known mechanism contributing to this phenotypic plasticity (Johnston et al., 2014b). Moreover, S. pneumoniae is also capable of spontaneous phase variation between opaque and transparent colony phenotypes, a widespread phenomenon in microbial pathogens (van der Woude, 2011). The opaque variants, which have more capsule and less teichoic acid, are more virulent in the lung and bloodstream; the transparent counterparts, which express less capsule and have more teichoic acid, are more adaptive to the nasopharynx (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Our recent studies have revealed that pneumococcal phase variation between two colony phenotypes is determined by DNA inversion between the methyltransferase hsdS genes in the colony opacity determinant (cod) locus (Feng et al., 2014; Li et al., 2016).

The protocol we present here describes the complete experimental procedure from preparing a bacterial stock to obtaining an image of colony morphology as described in our recent study (Li et al., 2016). Animal blood is commonly added to agar medium to promote growth of S. pneumoniae, but the color of the blood makes it difficult to differentiate opaque and transparent colonies by microscopic approach. Instead, catalase is supplemented to agar plates to culture S. pneumoniae by neutralizing the inhibition effect of hydrogen peroxide (produced by the pneumococci themselves) on pneumococcal growth when it comes to observe and document colony morphologies by dissection microscope (Kim and Weiser, 1998; Weiser et al., 1994; Manso et al., 2014; Li et al., 2016). Since colony morphology phenotypes can be indicative of bacterial physiological and pathogenic properties, this protocol may offer a valuable method to study the impact of genetic and epigenetic elements or environmental conditions on bacterial biology and disease pathogenesis.

Materials and Reagents

  1. Pipette tips
  2. Petri dishes (100 mm) (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 263991 )
  3. 50 ml tubes (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 339652 )
  4. 0.2 μm filter (Pall, catalog number: 4612 )
  5. 1.5 ml tubes (Eppendorf, catalog number: 022363204 )
  6. Spreading rods (Sigma-Aldrich, catalog number: Z376779 )
  7. S. pneumoniae strain ST556 (Li et al., 2012)
  8. Todd Hewitt broth (Sigma-Aldrich, catalog number: T1438 )
  9. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  11. Tryptic soy agar (TSA) (BD, DifcoTM, catalog number: 236950 )
  12. Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
  13. Phosphate buffered saline (PBS) (Mediatech, catalog number: 21-040-CV )
  14. Todd Hewitt broth with yeast extract (THY) medium (see Recipes)
  15. TSA medium (see Recipes)
  16. Catalase working solution (see Recipes)

Equipment

  1. Pipettes
  2. Dissection microscope (ZEISS, model: Stemi 2000-C )
  3. 37 °C water bath
  4. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: GENESYSTM 30 )
  5. 37 °C 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  6. Autoclave (SANYO, model: MLS-3780 )
  7. Analytical balance (Mettler-Toledo International, model: NewClassic ML802 )
  8. Vortex mixer  
  9. Centrifuge (Eppendorf, model: 5417 R )
  10. Class II biological safety cabinet (Thermo Fisher Scientific, Thermo ScientificTM, model: MSC-AdvantageTM Class II )
  11. Digital single lens reflex camera (Canon, model: EOS 550D )
  12. Small mirror with a diameter of ~5 cm

Procedure

  1. Setup of the microscope (Video 1)
    1. Position the dissection microscope ~5 cm off the bench by placing two boxes under both sides of the dissection microscope base.
    2. Take down the illuminator from the microscope.
    3. Fix the illuminator to the surface of the bench between the two boxes using a piece of tape, and make the direction of the light source vertically towards the observer.
    4. Remove the lens of the camera and install the camera on the top of the microscope.
    5. Remove the object stage that is embedded in the base of the microscope.

      Video 1. Setup of the dissection microscope

  2. Preparation of pneumococcal colonies for observing colony morphology
    1. Thaw a frozen stock of pneumococcal strain ST556 in a water bath at 37 °C.
    2. Inoculate 5 ml THY medium with 50 μl of the thawed frozen stock.
    3. Incubate the culture in a 5% CO2 incubator at 37 °C for about 5 h.
    4. Check the optical density (OD) of the culture at a wavelength of 620 nm using a spectrophotometer until the culture reaches mid-log phase (OD620 = 0.4-0.6).
      Note: The phase-variable colonies can be formed by either bacterial cells from fresh cultures or frozen stocks. If you want to use the freshly grown culture as described in 2d to make colonies, proceed to step 2g.
    5. Mix the bacterial culture with an equal volume of 30% glycerol in THY, and store the stocks at -80 °C for future use.
    6. Thaw a frozen stock of S. pneumoniae strain ST556 in a water bath at 37 °C.
    7. Dilute the culture or thawed stock with PBS at 1:10,000 dilution to approximately 104 colony forming unit (CFU)/ml.
    8. Thaw a frozen stock of catalase working solution at room temperature.
    9. Mix 50 μl of bacterial diluted stock with 100 μl of the catalase working solution, and spread the mixture on a TSA plate.
    10. Incubate the plate in the 5% CO2 incubator at 37 °C for 16 h.
      Note: In this protocol, we only detected the phase variation in colony opacity of pneumococcal strain ST556. Based on our previous observations (Li et al., 2016), 6 out of 8 strains showed opaque and transparent colony phenotypes at the 16-h time point, such as ST556 (serotype 19F), TH2901 (serotype 6B), and TH2835 (serotype 14). However, TIGR4 (serotype 4) and D39 (serotype 2) displayed different colony phenotypes at the 24-h time point. The incubation time of the plates should be adjusted according to the strain you use.
  3. Observation of pneumococcal colonies (Video 2)
    1. Open the lid of the plate (Procedure 2), and put the plate with its open side down on the stage of the dissection microscope.
    2. Turn on the illuminator, and use a small mirror under the stage of the microscope to reflect the light onto the plate.
    3. Adjust the intensity of the light and angle of reflected light by the mirror until you can see the colonies.

      Video 2. Observation of pneumococcal colonies

    4. Adjust the magnification of the microscope based on your needs.
    5. Adjust the focus of the microscope to make your field clear.
    6. Turn on the camera, and turn off the flash and automatic brightness adjustment system of the camera.
    7. Fix the angle of the mirror by holding it steadily.
    8. Photograph the colonies.

Data analysis

The representative image (Figure 1) illustrates that pneumococcal strain ST556 is capable of spontaneous phase variation in colony opacity, resulting in the formation of opaque and transparent two types of colony morphologies on the TSA plate supplemented with catalase.


Figure 1. A photograph of the phase variation between opaque and transparent colonies in a clonal population of S. pneumoniae strain ST556. Colonies were produced on a TSA plate supplemented with catalase after 16-h incubation and observed under a dissection microscope as described above. The representative opaque and transparent colonies are indicated by red and blue arrowheads, respectively. The scale bar was shown at the bottom right of this image (Li et al., 2016).

Recipes

  1. THY medium
    1. Dissolve the powder of Todd Hewitt broth (TH) and yeast extract (Y) in ddH2O (30 g TH and 5 g Y/L)
    2. Autoclave the medium at 121 °C for 15 min
    3. Store the medium at 4 °C
  2. TSA agar plates
    1. Dissolve the TSA powder in ddH2O (40 g/L)
    2. Autoclave the medium at 121 °C for 15 min
    3. Pour 15-20 ml TSA medium into each dish
    4. After solidification, use plates directly or store at 4 °C
  3. Catalase working solution (40,000-100,000 U/ml)
    1. Weigh 0.2 g catalase using an analytical balance
    2. Dissolve the catalase from 3a in 10 ml PBS in a 50 ml tube by vortex for 1 min
    3. Centrifuge the solution at 12,000 x g for 10 min at 4 °C
    4. Sterilize the supernatant from 3c by filtration using a 0.2 μm filter
    5. Aliquot the solution into 1.5 ml tubes
    6. Store the tubes at -80 °C
      Note: Avoid using the freeze-thawed catalase solution.

Acknowledgments

This work was supported by grants from National Natural Science Foundation of China (No. 31530082; No. 31530082) and the Grand Challenges Exploration of the Bill and Melinda Gates Foundation (No. OPP1021992). We thank Dr. Jeffrey N. Weiser (Department of Microbiology, New York University School of Medicine, New York, USA) for sharing his experience about observation of pneumococcal colony morphology.

References

  1. Croucher, N. J., Chewapreecha, C., Hanage, W. P., Harris, S. R., McGee, L., van der Linden, M., Song, J. H., Ko, K. S., de Lencastre, H., Turner, C., Yang, F., Sa-Leao, R., Beall, B., Klugman, K. P., Parkhill, J., Turner, P. and Bentley, S. D. (2014). Evidence for soft selective sweeps in the evolution of pneumococcal multidrug resistance and vaccine escape. Genome Biol Evol 6(7): 1589-1602.
  2. Croucher, N. J., Finkelstein, J. A., Pelton, S. I., Mitchell, P. K., Lee, G. M., Parkhill, J., Bentley, S. D., Hanage, W. P. and Lipsitch, M. (2013). Population genomics of post-vaccine changes in pneumococcal epidemiology. Nat Genet 45(6): 656-663.
  3. Croucher, N. J., Harris, S. R., Fraser, C., Quail, M. A., Burton, J., van der Linden, M., McGee, L., von Gottberg, A., Song, J. H., Ko, K. S., Pichon, B., Baker, S., Parry, C. M., Lambertsen, L. M., Shahinas, D., Pillai, D. R., Mitchell, T. J., Dougan, G., Tomasz, A., Klugman, K. P., Parkhill, J., Hanage, W. P. and Bentley, S. D. (2011). Rapid pneumococcal evolution in response to clinical interventions. Science 331(6016): 430-434.
  4. Feng, Z., Li, J., Zhang, J. R. and Zhang, X. (2014). qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data. Nucleic Acids Res 42(22): 13488-13499.
  5. Johnston, C., Campo, N., Berge, M. J., Polard, P. and Claverys, J. P. (2014a). Streptococcus pneumoniae, le transformiste. Trends Microbiol 22(3): 113-119.
  6. Johnston, C., Martin, B., Fichant, G., Polard, P. and Claverys, J. P. (2014b). Bacterial transformation: distribution, shared mechanisms and divergent control. Nat Rev Microbiol 12(3): 181-196.
  7. Kim, J. O. and Weiser, J. N. (1998). Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J Infect Dis 177(2): 368-377.
  8. Li, G., Hu, F. Z., Yang, X., Cui, Y., Yang, J., Qu, F., Gao, G. F. and Zhang, J. R. (2012). Complete genome sequence of Streptococcus pneumoniae strain ST556, a multidrug-resistant isolate from an otitis media patient. J Bacteriol 194(12): 3294-3295.
  9. Li, J., Li, J. W., Feng, Z., Wang, J., An, H., Liu, Y., Wang, Y., Wang, K., Zhang, X., Miao, Z., Liang, W., Sebra, R., Wang, G., Wang, W. C. and Zhang, J. R. (2016). Epigenetic switch driven by DNA inversions dictates phase variation in Streptococcus pneumoniae. PLoS Pathog 12(7): e1005762.
  10. Manso, A. S., Chai, M. H., Atack, J. M., Furi, L., De Ste Croix, M., Haigh, R., Trappetti, C., Ogunniyi, A. D., Shewell, L. K., Boitano, M., Clark, T. A., Korlach, J., Blades, M., Mirkes, E., Gorban, A. N., Paton, J. C., Jennings, M. P. and Oggioni, M. R. (2014). A random six-phase switch regulates pneumococcal virulence via global epigenetic changes. Nat Commun 5: 5055.
  11. Park, I. H., Kim, K. H., Andrade, A. L., Briles, D. E., McDaniel, L. S. and Nahm, M. H. (2012). Nontypeable pneumococci can be divided into multiple cps types, including one type expressing the novel gene pspK. MBio 3.
  12. van der Woude, M. W. (2011). Phase variation: how to create and coordinate population diversity. Curr Opin Microbiol 14(2): 205-211.
  13. Walker, C. L., Rudan, I., Liu, L., Nair, H., Theodoratou, E., Bhutta, Z. A., O'Brien, K. L., Campbell, H. and Black, R. E. (2013). Global burden of childhood pneumonia and diarrhoea. Lancet 381(9875): 1405-1416.
  14. Weiser, J. N., Austrian, R., Sreenivasan, P. K. and Masure, H. R. (1994). Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization. Infect Immun 62(6): 2582-2589.

简介

肺炎链球菌(肺炎球菌)是导致肺炎,脑膜炎,败血症和中耳炎的重要人类病原体。这种细菌通常作为共生体存在于鼻咽中,但有时会传播到人类的无菌部位并导致局部或全身炎症。这种双相行为。肺炎支原体与琼脂平板上的不透明和透明集落形式之间的可逆转换相关,这称为相变。不透明变体在菌血症的动物模型中似乎更具毒性,但在鼻咽定殖动物模型中是缺陷的。相比之下,透明变体在动物模型中显示较高水平的鼻咽定植,但相对较低的毒力。我们最近证实,这两种菌落类型之间的肺炎球菌相变是由基因组DNA甲基化(或表观遗传)模式的可逆转换引起的,由DNA甲基转移酶基因的DNA反转驱动。菌落形态的观察是区分具有不同特征(如大小,颜色和不透明度)的菌落的简单且有用的方法。该方案描述了如何利用解剖显微镜研究集落形态的肺炎球菌相变。
【背景】肺炎链球菌是全球儿童细菌性肺炎,脑膜炎和败血症的主要原因(Walker等人,2013)。这种病原体在适应人类宿主的各种生态环境中的成功取决于其显着的表型可塑性(Croucher等人,2013; Johnston等人,,2014a) ,其已被荚膜多糖和表面蛋白质中的菌株间抗原变异反映(Croucher等人,2013和2011),获得新的毒力因子(Park等人, ,2012),广泛的耐药性(Croucher等人,2014)。自然遗传转化是有助于这种表型可塑性的一种众所周知的机制(Johnston等,,2014b)。而且,肺炎支原体也能够在不透明和透明的集落表型之间自发相变,这是微生物病原体中广泛存在的现象(van der Woude,2011)。具有更多胶囊和较少磷酸的不透明变体在肺和血液中更具毒性;表达较少胶囊并具有更多磷壁酸的透明对应物更适应于鼻咽(Kim和Weiser,1998; Weiser等人,1994; Manso等人, ,2014; Li 等人,2016)。我们最近的研究表明,两个菌落表型之间的肺炎球菌相变可以通过菌落不透明度决定簇( cod cod cod cod))locus locus locus the et al。,2014; Li等人,2016)。
我们在这里介绍的协议描述了从我们最近的研究(Li et al。,2016)中所述的制备细菌原料以获得集落形态图像的完整实验步骤。通常将动物血添加到琼脂培养基中以促进S的生长。肺炎支原体,但是血液的颜色使得通过显微镜方法难以区分不透明和透明的菌落。相反,将过氧化氢酶补充至琼脂平板培养。肺炎链球菌通过中和过氧化氢(由肺炎球菌本身产生)对肺炎球菌生长的抑制作用,当通过解剖显微镜观察和记录菌落形态时(Kim和Weiser,1998; Weiser等人, ,1994; Manso等人,2014; Li等人,2016)。由于菌落形态表型可以指示细菌的生理和致病性质,因此该方案可以提供一种有价值的方法来研究遗传和表观遗传元件或环境条件对细菌生物学和疾病发病机理的影响。

关键字:肺炎链球菌, 集落形态学, 相位变异, 表观遗传开关, DNA倒位, 解剖显微镜

材料和试剂

  1. 移液器提示
  2. 培养皿(100毫米)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:263991)
  3. (Thermo Fisher Scientific,Thermo Scientific TM ,目录号:339652)
  4. 0.2μm过滤器(Pall,目录号:4612)
  5. (Eppendorf,目录号:022363204)
  6. 扩展棒(Sigma-Aldrich,目录号:Z376779)
  7. S上。肺炎衣原体ST556(Li et al。,2012)
  8. Todd Hewitt肉汤(Sigma-Aldrich,目录号:T1438)
  9. 酵母提取物(Sigma-Aldrich,目录号:Y1625)
  10. 甘油(Sigma-Aldrich,目录号:G5516)
  11. 胰蛋白酶大豆琼脂(TSA)(BD,Difco TM,目录号:236950)
  12. 来自牛肝的过氧化氢酶(Sigma-Aldrich,目录号:C9322)
  13. 磷酸盐缓冲盐水(PBS)(Mediatech,目录号:21-040-CV)
  14. Todd Hewitt肉汤用酵母提取物(THY)培养基(见食谱)
  15. TSA培养基(见食谱)
  16. 过氧化氢酶工作溶液(见配方)

设备

  1. 移液器
  2. 解剖显微镜(ZEISS,型号:Stemi 2000-C)
  3. 37°C水浴
  4. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:GENESYS TM 30)
  5. 37℃5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell< 150> 150i)
  6. 高压灭菌器(SANYO,型号:MLS-3780)
  7. 分析天平(Mettler-Toledo International,型号:NewClassic ML802)
  8. 涡街搅拌机
  9. 离心机(Eppendorf,型号:5417 R)
  10. II类生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM ,型号:MSC-Advantage TM II类)
  11. 数码单反相机(佳能,型号:EOS 550D)
  12. 直径约5厘米的小镜子

程序

  1. 显微镜的设置(视频1)
    1. 将夹层显微镜放置在5厘米的长凳上,将两个盒子放在解剖显微镜底座的两侧。
    2. 从显微镜下取下照明器。
    3. 使用一块胶带将照明器固定在两个盒子之间的工作台表面,并使光源的方向垂直朝向观察者。
    4. 取下相机的镜头,并将相机安装在显微镜的顶部。
    5. 移除嵌入显微镜底座的物体台。

      视频1

  2. 制备用于观察菌落形态的肺炎球菌菌落
    1. 在37℃的水浴中解冻冷冻的肺炎球菌菌株ST556菌株。
    2. 接种5 ml THY培养基,加入50μl解冻的冷冻原料。
    3. 将培养物在5%CO 2培养箱中于37℃孵育约5小时。
    4. 使用分光光度计检查波长为620nm的培养物的光密度(OD),直到培养物达到对数中期(OD> 620 = 0.4-0.6)。
      注意:相变菌落可以由来自新鲜培养物或冷冻原种的细菌细胞形成。如果要使用2d中描述的新鲜培养物进行菌落处理,请转到步骤2g。
    5. 将细菌培养物与THY中相同体积的30%甘油混合,并将库存储存在-80℃以备将来使用。
    6. 解冻冷冻库存。肺炎链球菌菌株ST556在37℃的水浴中。
    7. 用1:100稀释的PBS稀释培养物或解冻的原液至约10μg/ ml集落形成单位(CFU)/ ml。
    8. 在室温下解冻冷冻储存的过氧化氢酶工作溶液。
    9. 将50μl细菌稀释的液体与100μl过氧化氢酶工作溶液混合,并将混合物铺在TSA板上。
    10. 将板在5%CO 2培养箱中于37℃孵育16小时。
      注意:在本协议中,我们只检测到肺炎球菌菌株ST556的菌落不透明度的相变。根据我们以前的观察(Li et al。,2016),8个菌株中有6个菌株在16 h时间点显示不透明和透明的菌落表型,如ST556(血清型19F),TH2901(血清型6B)和TH2835(血清型14)。然而,TIGR4(血清型4)和D39(血清型2)在24小时时间点显示不同的集落表型。板的孵育时间应根据您使用的菌株进行调整。
  3. 观察肺炎球菌殖民地(视频2)
    1. 打开板的盖子(步骤2),将其开口侧的板放在解剖显微镜的台上。
    2. 打开照明器,并在显微镜的舞台下使用一个小镜子将光线反射到板上。
    3. 调整镜子的光线强度和反射光角度,直到看到殖民地为止。

      视频2

    4. 根据您的需要调整显微镜的倍率。
    5. 调整显微镜的焦点,使您的领域清晰。
    6. 打开相机,关闭相机的闪光灯和自动亮度调节系统。
    7. 通过稳定地固定镜子的角度。
    8. 拍摄殖民地。

数据分析

代表性的图像(图1)说明,肺炎球菌菌株ST556能够在菌落不透明度中自发相变,导致在补充有过氧化氢酶的TSA板上形成不透明和透明的两种类型的菌落形态。


图1.在 S的克隆群体中的不透明和透明菌落之间的相位变化的照片。肺炎支原体菌株ST556 菌株ST556 菌株ST556 。在培养16小时后,代表性的不透明和透明的殖民地分别由红色和蓝色箭头表示。比例尺显示在该图像的右下角(Li 等人,2016)。

食谱

  1. THY中等
    1. 将Todd Hewitt肉汤(TH)和酵母提取物(Y)的粉末溶解于ddH 2 O(30g TH和5g Y / L)中,/ /
    2. 在121℃高压灭菌15分钟
    3. 将介质储存在4°C
  2. TSA琼脂平板
    1. 将TSA粉末溶解于ddH 2 O(40g / L)
    2. 在121℃高压灭菌15分钟
    3. 将15-20毫升TSA培养基倒入每个盘中
    4. 固化后,直接使用板材或在4℃下储存
  3. 过氧化氢酶工作溶液(40,000-100,000 U / ml)
    1. 称量0.2 g过氧化氢酶使用分析天平
    2. 将来自3a的过氧化氢酶通过旋转1分钟溶解在50ml管中的10ml PBS中
    3. 在4℃下将溶液以12,000 x g离心10分钟
    4. 通过使用0.2μm过滤器过滤从3c灭菌上清液
    5. 将溶液等分至1.5 ml管中
    6. 将管储存在-80°C
      注意:避免使用冻融过氧化氢酶溶液。

致谢

这项工作得到了中国国家自然科学基金(31530082; 31530082)和比尔和梅琳达·盖茨基金会大挑战探索(编号OPP1021992)的资助。我们感谢Jeffrey N. Weiser博士(纽约大学医学院微生物系,美国纽约),分享了他对观察肺炎球菌集落形态的经验。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, J., Wang, J., Jiao, F. and Zhang, J. (2017). Observation of Pneumococcal Phase Variation in Colony Morphology. Bio-protocol 7(15): e2434. DOI: 10.21769/BioProtoc.2434.
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