搜索

Preparation of Candida albicans Biofilms for Transmission Electron Microscopy
制备白色念珠菌生物膜用于透射电镜分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Transmission Electron Microscopy is a form of microscopy that allows for imaging of distinct portions of an individual cell. For Candida albicans biofilms, it is often used to visualize the cell walls of fixed samples of yeast and hyphae. This protocol describes how to grow, harvest, and fix Candida albicans biofilms in preparation for Transmission Electron Microscopy.

Materials and Reagents

  1. Plate containing grown Candida albicans colonies
  2. 500 ml 0.22 μm filter (Corning, catalog number: 431118 )
  3. Aluminum foil
  4. Bacto-peptone (BD Biosciences, catalog number: 211677 )
  5. Bacto-yeast extract (BD Biosciences, catalog number: 212750 )
  6. Cell scraper (BD Biosciences, Falcon®, catalog number: 353085 )
  7. Difco Dextrose (BD Biosciences, catalog number: 215530 )
  8. Glutaraldehyde (Sigma-Aldrich, catalog number: G5882 )
  9. Lead nitrate (PbNO3) (Electron Microscopy Sciences, catalog number: 17900 )
  10. MOPS (Thermo Fisher Scientific, catalog number: BP308 )
  11. 4% Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19140 )
  12. Parafilm
  13. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  14. Propylene oxide (Electron Microscopy Sciences, catalog number: 20410 )
  15. RPMI 1640 (Sigma-Aldrich, catalog number: R6504 )
  16. Sodium cacodylate trihydrate (Sigma-Aldrich, catalog number: C0250 )
  17. Sodium chloride (Thermo Fisher Scientific, catalog number: S671-3 )
  18. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 , 500 gm)
  19. Sodium hydroxide (Sigma-Aldrich, catalog number: 59223C )
  20. Sodium phosphate dibasic anhydrous (Thermo Fisher Scientific, catalog number: BP332-500 )
  21. Sodium phosphate monobasic monohydrate (Thermo Fisher Scientific, catalog number: S369-500 )
  22. Spurr’s Resin Kit (Polyscience, catalog number: 01916-1 )
  23. Uridine (Sigma-Aldrich, catalog number: U3750 )
  24. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 )
  25. YPD + uridine (see Recipes)
  26. RPMI + MOPS (see Recipes)
  27. 1x PBS (see Recipes)
  28. Fixative (see Recipes)
  29. Reynold’s Lead Citrate Strain (see Recipes)

Equipment

  1. 15 ml conical tube (Corning, catalog number: CLS 430055 )
  2. 6 well polystyrene plate (Grenier Bio-one, catalog number: 657160 )
  3. Hemocytometer
  4. Shaking incubator with adjustable temperatures and speeds
  5. Transmission Electron Microscope facility with trained microscopist

Procedure

  1. 5 ml of YPD + uridine culture in a 15 ml conical tube is inoculated with a single colony of Candida albicans growing on a plate that is between 2 and 10 days old (age of colony may vary based on growth rate of particular strain). Most Candida strains and species should grow well in this medium, but if another medium is commonly used for a specific strain, that can be used instead.
  2. Inoculum is incubated overnight at 30 °C and 200 rpm.
  3. Cell concentration of overnight culture is determined by cell counting on a hemocytometer.
  4. Inoculum diluted to 1 x 106 cells/ml in RPMI + MOPS.
  5. 1 ml of diluted incolulum added to each well of a 6 well plate.
    Note: Inoculum spread to cover the entire bottom of the well.
  6. 6 well plate incubated at 30 °C for 60 min without shaking.
  7. Inoculum is then removed from each well gently, so as not to scratch away the newly forming biofilm.
  8. 1 ml of fresh RPMI + MOPS media is added gently to each well.
  9. 6 well plates are wrapped in parafilm and aluminum foil and incubated for 24 h at 37 °C and 50 rpm.
    Note: It is very important that the biofilms not be shaken above 50 rpm, as this will dislodge the growing biofilm from the plate bottom.
  10. After 24 h, media is gently removed so as not to disturb the biofilm and replaced with 1 ml of fresh RPMI + MOPS.
    Note: Tilting the 6 well plates and gently placing your pipet tip in the corner of the well helps to prevent too much disruption of the fragile biofilm.
  11. 6 well plates are re-wrapped as before and incubated another 24 h at 37 °C and 50 rpm.
  12. After the second 24 h incubation, the media is removed from the wells and the biofilms are gently washed 1x in 1 ml of 1x PBS. It is important to slowly pipet in the PBS so as not to disturb the delicate biofilms. The PBS wash can be removed immediately after adding it to the biofilm.
  13. Harvest each biofilm in 2 ml of 1x PBS and store in a 15 ml conical tube.
    Note: Harvesting is best done by adding 1 ml of PBS into the well and scraping away the biofilm with a plastic spatula, and adding the biofilm to the conical tube. Then repeat this process with the second 1 ml of PBS to collect any remnants.
  14. Conical tubes are gently centrifuged at 720 x g for 5 min at room temperature.
  15. Supernatant is discarded and the pellet is resuspended in 2 ml of Fixative.
  16. Tubes can be stored at 4 °C until ready for imaging by a trained transmission electron microscopist. It is recommended that the tubes not be stored more than a few days.
  17. The following steps are a summary of those performed by a trained transmission electron microscopist.
    1. Cells are postfixed in 1% osmium tetroxide.
    2. Samples are then dehydrated by soaking for 10 min each in increasing concentrations of ethanol (30%, 50%, 70%, 85%, 90%, 95%, and 100%).
    3. Next, samples are rinsed 3 x 10 min in 100% propylene oxide then embedded in Spurr’s resin.
    4. 70 nm sections are cut and placed on copper grids and poststained for 5 min with 8% uranyl acetate in 50% ethanol and for 5 min in Reynold’s lead citrate stain.
    5. Samples can now be analyzed by TEM. A sample picture of a reference strain Candida albicans biofilm is shown below (Figure 1). Most pictures will only show one or two cells at a time, and usually cross-sectioned so that you can see inside the cells. Scale bar represents 1 μm.


      Figure 1. Transmission Electron Microscopy image of a Candida albicans biofilm cell

Recipes

  1. YPD + uridine
    Bacto-yeast extract (1%)
    10 g
    Bacto-peptone (2%)
    20 g
    Dextrose (2%)
    20 g
    Uridine
    0.08 g
    Distilled water
    1,000 ml
    Combine all ingredients and aliquot into 100 ml bottles. Autoclave bottles on liquid cycle to sterilize.
  2. RPMI + MOPS
    RPMI 1640
    10.4 g
    MOPS
    34.5 g
    Distilled water
    700 ml
    Combine all ingredients in a 2 L flask. Use a 10 ml pipette to adjust pH to 7.0 using Sodium hydroxide (5 N). Bring volume up to 1 L with distilled water. Filter sterilize RPMI + MOPS into 500 ml bottles. Use at 37 °C, but can store for up to 6 months at 4 °C to prevent contaminant growth.
  3. 1x PBS                                                                                                               
    Sodium phosphate monobasic monohydrate (mw = 137.99; 0.038 M)
    2.62 g
    Sodium phosphate dibasic anhydrous
    11.5 g
    Sodium chloride
    43.84 g
    Distilled water
    500 ml
    Add above ingredients to 450 ml distilled water. Adjust pH to 7.4 by adding 1 M NaOH if necessary. Bring final volume to 500 ml distilled water. This makes 10x PBS stock. Dilute in distilled water 1:10 for final concentration of PBS.
  4. Fixative
    2.5% glutaraldehyde
    2.0% paraformaldehyde
    Add above ingredients to 0.1 M sodium cacodylate trihydrate. Use caution with this reagent, as it is very toxic.
  5. Reynold’s lead citrate stain
    Lead nitrate
    1.33 g
    Sodium citrate
    1.76 g
    Distilled water
    30 ml
    1 N Sodium hydroxide
    8 ml
    Place all ingredients in a 50 ml flask and shake forcefully for 1 min, then intermittently for 30 min until fully dissolved. Then add 8 ml of NaOH and bring volume up to 50 ml with distilled water. Filter sterilize before using.

References

  1. Bowling Green State University (2012). Reynold’s Lead Citrate Stain. Retrieved from www.bgsu.edu/departments/biology/facilities/MnM/TEM/ReynoldsLeadStain.pdf.
  2. Nett, J. E., Sanchez, H., Cain, M. T., Ross, K. M. and Andes, D. R. (2011). Interface of Candida albicans biofilm matrix-associated drug resistance and cell wall integrity regulation. Eukaryot Cell 10(12): 1660-1669.
  3. Nett, J., Lincoln, L., Marchillo, K., Massey, R., Holoyda, K., Hoff, B., VanHandel, M. and Andes, D. (2007). Putative role of beta-1,3 glucans in Candida albicans biofilm resistance. Antimicrob Agents Chemother 51(2): 510-520.
  4. Taff, H. T., Nett, J. E., Zarnowski, R., Ross, K. M., Sanchez, H., Cain, M. T., Hamaker, J., Mitchell, A. P. and Andes, D. R. (2012). A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog 8(8): e1002848.

简介

透射电子显微术是允许对单个细胞的不同部分成像的显微镜检查的形式。 对于白色念珠菌生物膜,它通常用于可视化酵母和菌丝的固定样品的细胞壁。 此协议描述如何生长,收获和修复白色念珠菌生物膜准备透射电子显微镜。

材料和试剂

  1. 含有生长的白色念珠菌菌落的平板
  2. 500ml0.22μm过滤器(Corning,目录号:431118)
  3. 铝箔
  4. 细菌蛋白胨(BD Biosciences,目录号:211677)
  5. 细菌酵母提取物(BD Biosciences,目录号:212750)
  6. 细胞刮刀(BD Biosciences,Falcon ,目录号:353085)
  7. Difco葡萄糖(BD Biosciences,目录号:215530)
  8. 戊二醛( Sigma-Aldrich,目录号:G5882)
  9. 硝酸铅(PbNO 3)(Electron Microscopy Sciences,目录号:17900)
  10. MOPS(Thermo Fisher Scientific,目录号:BP308)
  11. 4%四氧化锇(Electron Microscopy Sciences,目录号:19140)
  12. parafilm
  13. 多聚甲醛(Electron Microscopy Sciences,目录号:15710)
  14. 环氧丙烷(Electron Microscopy Sciences,目录号:20410)
  15. RPMI 1640(Sigma-Aldrich,目录号:R6504)
  16. 二水合三甲基膦酸钠(Sigma-Aldrich,目录号:C0250)
  17. 氯化钠(Thermo Fisher Scientific,目录号:S671-3)
  18. 柠檬酸钠(Electron Microscopy Sciences,目录号:21140,500gm)
  19. 氢氧化钠(Sigma-Aldrich,目录号:59223C)
  20. 无水磷酸氢二钠(Thermo Fisher Scientific,目录号:BP332-500)
  21. 磷酸二氢钠一水合物(Thermo Fisher Scientific,目录号:S369-500)
  22. Spurr树脂试剂盒(Polyscience,目录号:01916-1)
  23. 尿苷(Sigma-Aldrich,目录号:U3750)
  24. 乙酸乙酯(Electron Microscopy Sciences,目录号:22400)
  25. YPD +尿苷(参见配方)
  26. RPMI + MOPS(请参阅配方)
  27. 1x PBS(请参阅配方)
  28. 固定剂(见配方)
  29. 雷诺铅柠檬酸菌(参见食谱)

设备

  1. 15ml锥形管(Corning,目录号:CLS430055)
  2. 6孔聚苯乙烯板(Grenier Bio-one,目录号:657160)
  3. 血细胞计数器
  4. 以可调温度和速度摇动培养箱
  5. 具有训练有素的显微镜的透射电子显微镜设备

程序

  1. 在15ml锥形管中5ml的YPD +尿苷培养物接种在2至10天龄的板上生长的白色念珠菌的单个菌落(菌落的年龄可以基于生长而变化 特定应变速率)。 大多数< em>念珠菌菌株和物种在该培养基中生长良好,但如果另一种培养基通常用于特定菌株,则可以代替。
  2. 将接种物在30℃和200rpm下温育过夜
  3. 过夜培养物的细胞浓度通过血细胞计数器上的细胞计数来确定
  4. 在RPMI + MOPS中将接种物稀释至1×10 6个细胞/ml
  5. 将1ml稀释的导管加入6孔板的每个孔中 注意:接种物扩散覆盖整个井底。
  6. 6孔板在30℃温育60分钟而不摇动
  7. 然后从每个孔中轻轻取出接种物,以便不刮除新形成的生物膜
  8. 向每个孔中轻轻加入1ml新鲜的RPMI + MOPS培养基
  9. 将6孔板包裹在石蜡膜和铝箔中,并在37℃和50rpm下孵育24小时 注意:非常重要的是,生物膜不会在50 rpm以上摇动,因为这会从板底移出生长的生物膜。
  10. 24小时后,轻轻地除去培养基,以不干扰生物膜,并用1ml新鲜的RPMI + MOPS替换。
    注意:倾斜6孔板并轻轻地将移液管尖端放在孔的角落,有助于防止对脆弱的生物膜造成太大的破坏。
  11. 6孔板如前所述重新包装,并在37℃和50rpm下再培养24小时
  12. 在第二个24小时温育后,从孔中除去培养基,并将生物膜在1ml的1×PBS中轻轻洗涤1次。重要的是在PBS中慢慢吸取,以免干扰精细的生物膜。 PBS洗液可以在添加到生物膜后立即除去。
  13. 收获每个生物膜在2毫升1×PBS,并存储在15毫升锥形管 注意:收获最好是通过将1ml PBS加入孔中,并用塑料刮刀刮去生物膜,并将 生物膜添加到锥形管中。然后用第二个1ml PBS重复该过程以收集任何残留物。
  14. 弃去上清液,将沉淀重悬于2ml固定剂中
  15. 管可以储存在4℃,直到准备好由训练的透射电子显微镜成像。建议试管不要储存超过几天。
  16. 以下步骤是由训练有素的透射电子显微镜执行的步骤的总结
    1. 将细胞在1%四氧化锇中后固定
    2. 然后通过以增加浓度的乙醇(30%,50%,70%,85%,90%,95%和100%)浸泡10分钟使样品脱水。
    3. 接下来,将样品在100%环氧丙烷中漂洗3×10分钟,然后包埋在Spurr树脂中
    4. 切割70nm切片并置于铜网上,并用8%乙酸双氧铀在50%乙醇中染色5分钟,并在雷诺氏柠檬酸铅染色中染色5分钟。
    5. 现在可以通过TEM分析样品。 参照菌株白色念珠菌生物膜的样品照片如下所示(图1)。 大多数图片将一次只显示一个或两个单元格,通常横截面,以便您可以看到单元格内部。 比例尺表示1μm。


      图1.白色念珠菌生物膜细胞的透射电子显微镜图像

食谱

  1. YPD +尿苷
    细菌酵母提取物(1%) 10克
    细菌蛋白胨(2%)
    20克
    葡萄糖(2%)
    20克
    尿嘧啶
    0.08克
    蒸馏水
    1000 ml
    将所有成分混合并等分至100 ml瓶中。 高压灭菌瓶上的液体循环灭菌。
  2. RPMI + MOPS
    RPMI 1640
    10.4克
    MOPS
    34.5克
    蒸馏水
    700 ml
    将所有成分混合在2L烧瓶中。 使用10 ml移液管,使用氢氧化钠(5 N)将pH调节至7.0。 用蒸馏水将体积升至1升。 过滤灭菌RPMI + MOPS到500毫升瓶。 在37°C使用,但可以在4°C下存储长达6个月,以防止污染物生长
  3. 1x PBS                                                                                                            
    磷酸二氢钠一水合物(mw = 137.99; 0.038M)
    2.62克
    磷酸氢二钠
    11.5克
    氯化钠
    43.84克
    蒸馏水
    500 ml
    将上述成分加入到450ml蒸馏水中。 如果需要,通过加入1M NaOH将pH调节至7.4。 将最终体积加入500ml蒸馏水中。 这使10x PBS股票。 在蒸馏水中稀释1:10,用于PBS的最终浓度
  4. 固定剂
    2.5%戊二醛
    2.0%多聚甲醛
    将上述成分加入0.1M二甲胂酸钠三水合物中。 使用此试剂时要小心,因为它很有毒。
  5. 雷诺铅柠檬酸盐染色
    铅硫酸盐
    1.33克
    柠檬酸钠
    1.76克
    蒸馏水
    30 ml
    1N氢氧化钠
    8 ml
    将所有成分放在50ml烧瓶中,强力摇动1分钟,然后间歇30分钟,直到完全溶解。 然后加入8ml NaOH,并用蒸馏水使体积达到50ml。 在使用前过滤灭菌。

参考文献

  1. 鲍灵格林州立大学(2012)。 雷诺铅柠檬酸盐污渍。 检索自www.bgsu.edu/departments/biology/facilities/MnM/TEM/ReynoldsLeadStain.pdf。
  2. Nett,J.E.,Sanchez,H.,Cain,M.T.,Ross,K.M.and Andes,D.R。(2011)。 白色念珠菌生物膜基质相关耐药性和细胞壁完整性的界面 Eukaryot Cell 10(12):1660-1669。
  3. Nett,J.,Lincoln,L.,Marchillo,K.,Massey,R.,Holoyda,K.,Hoff,B.,VanHandel,M.and Andes,D。(2007)。 β-1,3葡聚糖在白色假丝酵母中的推定作用生物膜抗性。 Antimicrob Agents Chemother 51(2):510-520。
  4. Taff,H.T.,Nett,J.E.,Zarnowski,R.,Ross,K.M.,Sanchez,H.,Cain,M.T.,Hamaker,J.,Mitchell,A.P.and Andes,D.R。(2012)。 假丝酵母生物膜诱导的基质葡聚糖传递途径:对药物的影响 PLoS Pathog 8(8):e1002848。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Taff, H. T. and Andes, D. R. (2013). Preparation of Candida albicans Biofilms for Transmission Electron Microscopy. Bio-protocol 3(14): e822. DOI: 10.21769/BioProtoc.822.
  2. Taff, H. T., Nett, J. E., Zarnowski, R., Ross, K. M., Sanchez, H., Cain, M. T., Hamaker, J., Mitchell, A. P. and Andes, D. R. (2012). A Candida biofilm-induced pathway for matrix glucan delivery: implications for drug resistance. PLoS Pathog 8(8): e1002848.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。