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Visualization of Cell Complexity in the Filamentous Cyanobacterium Mastigocladus laminosus by Transmission Electron Microscopy (TEM)

透射电镜（TEM）法观察分析丝状蓝细菌层理鞭枝藻细胞复杂度

Dennis J. Nürnberg Dennis J. Nürnberg
GM Giulia Mastroianni
CM Conrad W. Mullineaux
GM Graham D. McPhail

发布: 2014年12月05日第4卷第23期 DOI: 10.21769/BioProtoc.1305 浏览次数: 10410

评审: Fanglian HeClaudia Catalanotti

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Cover of Molecular Microbiology, featuring study using the protocol.

Mar 2014

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Abstract

The cyanobacterium Mastigocladus laminosus (M. laminosus) is one of the most morphologically complex prokaryotes. It forms long chains of cells that are connected via septal junction complexes; such complexes allow diffusion of metabolites and regulators between neighboring cells. Cellular division occurs in multiple planes, resulting in the formation of true branches, and cell differentiation leads to the formation of specialized cell types for nitrogen fixation (heterocysts) and culture dispersal (hormogonia and necridia). Here, we describe a detailed protocol for the preparation of M. laminosus for TEM in order to visualize the ultrastructural properties of the organism. The presented preparation method is based on adding potassium permanganate as fixative which has been shown to increases the contrast of membranes (Luft, 1956), making it suitable for studies in cyanobacteria where the visualization of the photosynthetic membranes is important.

Keywords: Ultrastructure (超微结构) search Fischerella (侧生藻属) search Heterocyst (异形胞) search Hormogonia (段殖体) search Branching cyanobacteria (分枝蓝藻) search

Materials and Reagents

  1. Liquid culture of Mastigocladus laminosus
  2. Potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: BP362 )
  3. Sodium phosphate dibasic (Na2HPO4) (Thermo Fisher Scientific, Acros Organics, catalog number: 204855000 )
  4. Glutaraldehyde (25%, EM grade) (Agar Scientific, catalog number: R1020 )
  5. Low gelling temperature agarose (Sigma-Aldrich, catalog number: A9414 )
  6. Potassium permanganate (KMnO4) (VWR International, BDH, catalog number: 296444N )
  7. 100% ethanol
  8. Propylene oxide (Agar Scientific, catalog number: R1080 )
  9. Araldite CY212 (Agar Scientific, catalog number: R1042 )
  10. Methyl nadic anhydride (MNA) (Agar Scientific, catalog number: R1083 )
  11. Dodecenylsuccinic anhydride (DDSA) (Agar Scientific, catalog number: R1052 )
  12. Benzyldimethylamine (BDMA) (Agar Scientific, catalog number: R1061 )
  13. Sodium tetraborate (borax) (Sigma-Aldrich, catalog number: 221732 )
  14. Toluidine blue (Sigma-Aldrich, catalog number: 89640 )
  15. Uranyl acetate [UO2(CH3COO)2]*2 H2O] (SPI Supplies, catalog number: 02624-AB )
  16. Lead (II) nitrate [Pb(NO3)2] (Sigma-Aldrich, catalog number: 228621 )
  17. Tri-sodium citrate (TAAB, catalog number: S011 )
  18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  19. 0.125 M Sørensen's phosphate buffer (PB) (see Recipes)
  20. 4% (v/v) glutaraldehyde in PB (see Recipes)
  21. 2% (w/v) low gelling temperature agarose (see Recipes)
  22. 2% (w/v) potassium permanganate (see Recipes)
  23. Araldite (see Recipes)
  24. 1% (w/v) toluidine blue (see Recipes)
  25. Uranyl acetate (see Recipes)
  26. Reynold's lead citrate stain (see Recipes)

Equipment

  1. Centrifuge
  2. Copper grids (300 mesh) (Agar Scientific, catalog number: G2740C )
  3. Dental wax
  4. Disposable plastic Pasteur pipettes
  5. Disposable polyethylene beaker
  6. Eppendorf tubes (1.5 ml)
  7. Glass or diamond knife
  8. Hotplate
  9. Light microscope
  10. Oven (60 °C)
  11. Razor blades
  12. Rotator
  13. Rubber embedding mould
  14. Sealable glass vials (7 ml)
  15. Shaker
  16. Spatula
  17. Sterilization filters (pore size: 0.22 µm)
  18. Syringe
  19. TEM (JOEL, model: JEM-1230 )
  20. Tweezers
  21. Ultramicrotome (Reichert Ultracut E)
  22. Vortexer
  23. SPI Slide-A-GridTM storage box (SPI Slide-A-GridTM, catalog number: 02450-AB )

Procedure

English
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文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Nürnberg, D. J., Mastroianni, G., Mullineaux, C. W. and McPhail, G. D. (2014). Visualization of Cell Complexity in the Filamentous Cyanobacterium Mastigocladus laminosus by Transmission Electron Microscopy (TEM). Bio-protocol 4(23): e1305. DOI: 10.21769/BioProtoc.1305.

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微生物学 > 微生物细胞生物学 > 细胞成像

细胞生物学 > 细胞成像 > 电子显微镜

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