Transmission Electron Microscopy

Robert C. Burghardt1, Robert Droleskey2

1 Texas A&M University, College Station, Texas, 2 USDA/ARS/SPARC/FFSRU, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2B.1
DOI:  10.1002/9780471729259.mc02b01s03
Online Posting Date:  December, 2006
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Abstract

Transmission electron microscopy has long been an important analytical tool in the field of microbiology. This unit describes preparation techniques for examining particulate samples as well as samples presenting more complex ultrastructural considerations that require analysis in thin sections. Negative staining is a useful technique for routine examination of particulate samples in suspension ranging from bacteria to purified macromolecules. In order to investigate the relationships between microbes and the environments with which they interface, fixed samples can be prepared for imaging in sections of 60‐ to 90‐nm thickness. Due to the many steps in sample preparation for ultrastructural analysis of thin‐sectioned samples, the major steps in the process are divided into fixation and initial processing of samples for thin sectioning, the embedment of samples into a plastic resin for sectioning, ultramicrotomy, and staining of samples. Procedures for immunolocalization of antigens in negatively stained and thin‐sectioned preparations are also considered.

Keywords: transmission electron microscopy; negative staining; thin sectioning; embedding; colloidal gold immunostaining

     
 
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Table of Contents

  • Safety
  • Basic Protocol 1: Negative Staining for TEM Using the Drop Application Method
  • Alternate Protocol 1: Negative Staining for TEM Using Direct Deposition of Sample and Stain onto Grids
  • Alternate Protocol 2: Negative Staining for TEM Using Spray Droplet Application
  • Support Protocol 1: Preparation of Grids for Negative Staining
  • Basic Protocol 2: Protein A–Gold Staining and Negative Staining for Immunoelectron Microscopy
  • Alternate Protocol 3: Staining for Immunoelectron Microscopy Using Gold‐conjugated Secondary Antibodies
  • Fixation and Initial Processing of Samples for Thin Sectioning
  • Basic Protocol 3: Fixation and Initial Processing of Tissue Samples
  • Alternate Protocol 4: Fixation and Initial Processing of Cell Pellets
  • Alternate Protocol 5: Direct Fixation and Initial Processing of Bacteria in Suspension
  • Alternate Protocol 6: Fixation and Initial Processing of Monolayer Cells in Culture
  • Alternate Protocol 7: Fixation and Initial Processing of Samples for Immunogold Labeling of Thin Sections
  • Basic Protocol 4: Embedding of Tissues and Cell Pellets for Thin Sectioning
  • Alternate Protocol 8: Resin Embedding of Monolayer Cells on Glass Chambered Slides
  • Alternate Protocol 9: Resin Embedding for Thin Section Immunogold Labeling
  • Basic Protocol 5: Overview of Ultramicrotomy
  • Basic Protocol 6: Heavy Metal (and Immunogold) Staining of Thin Sections
  • Alternate Protocol 10: Immunogold Post‐Embedding Staining of Thin Sections
  • Support Protocol 2: Etching of Epoxy Embedding Resin to Increase Immunogold Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Negative Staining for TEM Using the Drop Application Method

  Materials
  • Samples: bacterial or viral suspensions
  • Negative staining solution: phosphotungstic acid staining solution (see recipe), uranyl acetate staining solution (see recipe), or ammonium molybdate staining solution (see recipe)
  • 37% formaldehyde (optional)
  • Desiccant
  • Several sets of self‐locking fine‐point forceps
  • Formvar‐ or carbon‐coated Formvar hexagonal 300‐ or 400‐ mesh copper grids (see protocol 4)
  • Whatman no. 4 filter paper
  • E‐Series Germicidal Ultraviolet lamp (Spectroline) or equivalent, with UV monitor and UV goggles (optional)
  • 35‐mm petri dish (optional)
  • Grid box (Electron Microscopy Sciences or Ted Pella)

Alternate Protocol 1: Negative Staining for TEM Using Direct Deposition of Sample and Stain onto Grids

  • Hand‐held glass nebulizer (Electron Microscopy Sciences or Ted Pella, Inc.)
  • Stand that can support self‐locking forceps holding grid while sample spray is applied

Alternate Protocol 2: Negative Staining for TEM Using Spray Droplet Application

  Materials
  • Samples: bacterial or viral suspensions
  • Primary antibody against antigen of interest and irrelevant primary antibody of same species/class as control
  • Immunogold antibody dilution buffer (see recipe)
  • Protein A–gold or protein A plus protein G–gold solution: e.g., Structure Probe/SPI Supplies or Nanoprobes (see suppliers appendix) or Aurion (http://www.aurion.nl)
  • 96‐well microtiter plate
  • Humidified chamber: e.g., Tupperware or Rubbermaid plastic container with sealable lid, containing water‐saturated paper towels
  • Several sets of self‐locking fine‐point forceps
  • Formvar‐coated 300‐ or 400‐mesh nickel grids.
  • Additional reagents and equipment for negative staining (see protocol 1)

Support Protocol 1: Preparation of Grids for Negative Staining

  • Colloidal gold‐conjugated secondary antibody directed against primary antibody

Basic Protocol 2: Protein A–Gold Staining and Negative Staining for Immunoelectron Microscopy

  Materials
  • Tissue sample
  • TEM primary fixative 1 (see recipe)
  • Phosphate/sucrose rinse buffer (see recipe), 4°C
  • Osmium tetroxide post‐fixative (see recipe)
  • Uranyl acetate staining solution (see recipe)
  • Graded ethanol series: 50% and 75% ethanol (4°C) and 95% and 100% ethanol (room temperature)
  • Acetone or propylene oxide
  • Polypropylene cutting board
  • Single‐edged razor blades
  • Fixation vials: dram vials with caps
  • Platform rocker or orbital shaker (optional)
  • Additional reagents and equipment for embedment ( protocol 12)

Alternate Protocol 3: Staining for Immunoelectron Microscopy Using Gold‐conjugated Secondary Antibodies

  • Cell suspension in buffered saline or serum‐free culture medium
  • 2.5% molten agar, between 40°C and 60°C

Basic Protocol 3: Fixation and Initial Processing of Tissue Samples

  • TEM primary fixative 2 (see recipe)
  • Bacteria in suspension
  • 2.5% molten agar, between 40° and 60°C
  • 15‐ml conical centrifuge tubes
  • Horizontal mixer (e.g., Adams Nutator Single Speed Orbital Mixer)
  • Tabletop centrifuge
  • Polyethylene microcentrifuge tubes

Alternate Protocol 4: Fixation and Initial Processing of Cell Pellets

  • Cultured cells grown in Nunc Lab‐Tek chamber slides
  • TEM primary fixative 2 (see recipe)

Alternate Protocol 5: Direct Fixation and Initial Processing of Bacteria in Suspension

  • Tissue or cell sample
  • TEM primary fixative 3 (see recipe)
  • Aldehyde quenching solution (see recipe)

Alternate Protocol 6: Fixation and Initial Processing of Monolayer Cells in Culture

  Materials
  • Fixed and processed tissue ( protocol 7), cell pellet ( protocol 8), or bacterial sample ( protocol 9)
  • Luft's epoxy mixture (see recipe)
  • Acetone or propylene oxide
  • Orbital shaker
  • Transfer forceps or applicator stick
  • Embedding molds or capsules (available from Ted Pella; BEEM flat embedding molds, cat. no. 111‐2, PTFE flat embedding molds, cat. no. 10509, or BEEM embedding capsules, size 00, cat. no. 13)
  • 60°C oven

Alternate Protocol 7: Fixation and Initial Processing of Samples for Immunogold Labeling of Thin Sections

  Materials
  • Fixed and processed monolayer cells in chamber slides ( protocol 10)
  • 50%, 75%, 95%, and 100% ethanol
  • Mollenhauer's no. 2 resin mixture (see recipe)
  • Single‐edged razor blade

Basic Protocol 4: Embedding of Tissues and Cell Pellets for Thin Sectioning

  Materials
  • Fixed and processed tissue or cell sample ( protocol 11)
  • LR White resin (Polysciences)
  • LR White accelerator (Polysciences)
  • Embedding molds (available from Ted Pella; BEEM flat embedding molds, cat. no. 111‐2, PTFE flat embedding molds, cat. no. 10509, or BEEM embedding capsules, size 00, cat. no. 13)
  • Cooled water bath or cooling block (optional)
  • Jeweler's saw
  • Plexiglas pegs (Ladd Research, cat. no. 21830; http://www.laddresearch.com/)
  • Cyanoacrylate glue (e.g., Superglue, Krazy Glue)

Alternate Protocol 8: Resin Embedding of Monolayer Cells on Glass Chambered Slides

  Materials
  • Thin sections collected on copper grids ( protocol 15)
  • Uranyl acetate staining solution (see recipe)
  • Lead citrate staining solution (see recipe)
  • Whatman no. 4 filter paper
  • Spray bottle with distilled H 2O
  • Grid box

Alternate Protocol 9: Resin Embedding for Thin Section Immunogold Labeling

  Materials
  • Thin sections on 300‐mesh Formvar‐coated nickel grids ( protocol 15)
  • Immunogold blocking buffer (see recipe)
  • Primary antibody against antigen of interest and control (irrelevant) primary antibody of same Ig class
  • TBS‐Tween (see recipe)
  • Reagents for colloidal gold labeling—one of the following:
    • Colloidal gold‐labeled secondary antibody against species from which primary antibody was obtained
    • Biotinylated secondary antibody against species from which primary antibody was raised, and streptavidin‐conjugated colloidal gold
    • Colloidal gold‐labeled protein A and/or protein G
  • TEM primary fixative 2 (optional; see recipe)
  • Whatman no. 4 filter paper
  • Spray bottle
  • Grid box
  • Additional reagents and equipment for etching of epoxy embedding resin (optional; see protocol 18) and uranyl acetate/lead citrate staining of thin sections (see protocol 16)

Basic Protocol 5: Overview of Ultramicrotomy

  Materials
  • Plastic etching solution: 5% (w/v) sodium metaperiodate (prepare fresh)
  • Thin sections on 300‐mesh Formvar‐coated nickel grids ( protocol 15)
  • TBS‐Tween (see recipe)
  • Spray bottle
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Figures

  •   FigureFigure 2.B0.1 Aspects of the negative staining procedure. (A) Grids can be conveniently handled using either self‐closing forceps (top) or conventional fine‐tipped forceps with a rubber O‐ring to keep the jaws closed (bottom). Specimen to be examined can be applied either by (B) floating grid on a drop of the specimen or by (C) direct application to the grid. Stain can be applied as illustrated in B or C. Washing (D) and fluid removal (E) are performed in subsequent steps of the procedure.
  •   FigureFigure 2.B0.2 Thin sections (∼70 to 80 nm) prepared on a diamond knife as seen through a stereo microscope. Trapezoid‐shaped sections are cut sequentially, adhering to the knife edge and forming ribbons during sectioning (black arrow). They are visible as the result of reflected light from above, which generates an interference pattern that can be used to estimate section thickness. As sections accumulate, they can be separated into smaller ribbons, which float on the water surface and can be aligned for collection onto a grid. The width of the trough of the diamond knife shown is ∼10 mm.
  •   FigureFigure 2.B0.3 Transmission electron micrograph of cultured chicken spleen cells infected with avian reticuloendotheliosis virus, embedded using . (A) Clusters of virus particles (arrows) are seen adjacent to the plasma membrane of an individual spleen cell. (B) Higher magnification of an area of plasma membrane with shedding virus (arrows) similar to that shown in A. Bars = 1.0 µm (A) and 0.1 µm (B).
  •   FigureFigure 2.B0.4 Monolayer processing in chambered slides. (A) The 4‐well chambered slide consists of plastic wells mounted onto a glass slide (1) with a silicone gasket. When the plastic chamber is removed (2), a thin layer of unpolymerized epoxy resin is added (3) at the thickness of the silicone gasket (4). Embedded wells containing cells are removed from the glass slide (5) and pieces of the resin embedded wells are mounted onto plastic pegs for sectioning (6). (B) Transmission electron micrograph of a HeLa cell monolayer grown and processed for electron microscopy on a 4‐well chambered slide. In this preparation, a cross‐section of the monolayer was prepared following removal of the embedded monolayer from the glass slide, and an additional layer of fresh resin (5 in panel A) was added to the substrate side. A piece of the monolayer was then cut with a jeweler's saw and mounded on plastic pegs (6 in panel A) for sectioning. Bar = 0.5 µm.
  •   FigureFigure 2.B0.5 Transmission electron micrographs of Salmonella typhimurium negatively stained using the direct deposition of sample and stain procedure (). (A) An example of an even distribution of bacteria (arrowheads) over the surface of a 300‐mesh Formvar‐coated hexagonal grid. Grid stained with 0.25% ammonium molybdate. (B) Higher magnification of a bacterium from the same grid. Peritrichous flagella are visible radiating from the cell (arrows) while individual fimbrae are barely evident around the bacterium (arrowhead) at this magnification. Bars = 10 µm (A) and 0.5 µm (B).
  •   FigureFigure 2.B0.6 Higher‐magnification transmission micrographs of negatively stained organisms showing structural detail obtainable with negative stain. (A) Salmonella typhimurium stained with 0.5% ammonium molybdate by direct deposition of sample and stain showing type 1 fimbrae (arrowheads) and a single flagellum in the field of view. Stain is evenly distributed around the cell, which allows these structures to be easily discernible. (B) Bacteriophage (T‐even type) isolated from the intestinal contents of a sheep stained with 0.2% phosphotungstic acid. Stain is distributed to allow for the recognition of tail fibers (arrowheads). Phage with intact (black arrows) and empty heads (white arrows) are present in the preparation. In the background of the preparation are globular aggregates of protein from the growth medium used to culture the phage and its host bacterium. Bars = 250 nm (A), 50 nm (B).
  •   FigureFigure 2.B0.7 Immunogold‐labeled and negatively stained preparation of Salmonella typhimurium prepared using . Bacteria were deposited on the grid and incubated with an anti‐fimbrial primary antibody; this was followed by incubation with protein G–gold and negative staining with 1% ammonium molybdate. The 10‐nm gold particles (arrows) decorate the fimbrial structures as seen in Figure A.
  •   FigureFigure 2.B0.8 Propionibacterium fixed and initially processed for immunogold labeling and embedded in LR White resin. In panels (A) and (B) shown at the same magnification, the LR White–embedded sections were labeled first with a primary monoclonal antibody specific for this particular isolate of Propionibacterium; this was followed by secondary labeling with 10‐nm protein G–gold (white arrows). Panel A shows inherent specimen contrast without staining uranyl acetate and lead staining of the section, whereas panel B shows the added contrast obtained by employing those stains. Note that while the ultrastructural detail of cells is improved with heavy metal staining, the visualization of gold particles can be more difficult and may require examination of sections at higher magnification.

Videos

Literature Cited

   Aldrich, H.C. and Mollenhauer, H.H. 1986. Secrets of successful embedding, sectioning, and imaging. In Ultrastructure Techniques for Microorganisms (H.C. Aldrich and W.J. Todd, eds.) pp. 101‐132. Plenum, New York.
   Anderson, N. and Doane, F.W. 1973. Specific identification of enteroviruses by immuno‐electron microscopy using a serum‐in‐agar diffusion method. Can. J. Microbiol. 19:585‐589.
   Bremer, A., Henn, C., Engel, A., Baumeister, W., and Aebi, U. 1992. Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy 46:85‐111.
   de Bruijn, W.C. 1973. Glycogen, its chemistry and morphologic appearance in the electron microscope. I. A modified OsO4 fixative which selectively contrasts glycogen. J. Ultrastruct. Res. 42:29‐50.
   Fassel, T.A., Mozdziak, P.E., Sanger, J.R., and Edminston, C.E. 1997. Paraformaldehyde effect on ruthenium red and lysine preservation and staining of the staphylococcal glycocalyx. Microsc. Res. Tech. 36:422‐427.
   Hayat, M.A. and Miller, S.E. 1990. Negative Staining. McGraw‐Hill Publishing, N.Y.
   Hazelton, P.R. and Gelderblom, H.R. 2003. Electron microscopy for rapid diagnosis of infectious agents in emergent situations. Emerg. Infect. Dis. 9:294‐303.
   Karlyshev, A.V., McCrossan, M.V., and Wren, B.W. 2001. Demonstration of polysaccharide capsule in Campylobacter jejuni using electron microscopy. Microsc. Res. Tech. 69:5921‐5924.
   Luft, J.H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409‐414.
   Mollenhauer, H.H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39:111‐114.
   Mollenhauer, H.H. and Droleskey, R.E. 1997. Image contrast in sections of epoxy resin‐embedded biological material: Maintenance of a proper anhydride‐epoxy ratio during tissue impregnation. Microsc. Res. Tech. 36:417‐421.
   Ohi, M., Li, Y., Cheng, Y., and Walz, T. 2004. Negative staining and image classification: Powerful tools in modern electron microscopy. Biol. Proced. Online 6:23‐34.
   Pegg‐Feige, K. and Doane, F.W. 1983. Effect of specimen support film in solid phase immunoelectron microscopy. J. Virol. Methods 5‐6:315‐319.
Key References
   Doane, F.W. and Anderson, N. 1987. Electron Microscopy in Diagnostic Virology. Cambridge University Press, New York.
  Includes a number of protocols dealing with the use of electron microscopy in diagnostic virology.
   Hayat, M.A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications. Cambridge University Press, New York.
  An excellent general reference covering all aspects of biological electron microscopy.
   Hoppert, M. and Holzenburg, A. 1998. Electron Microscopy in Microbiology. Springer‐Verlag., New York.
  A good general reference with some protocols.
Internet Resources
  http://www.ncbi.nlm.nih.gov/ICTVdb/#ICTVdB
  The Universal Virus Database, ICTVdB, is a resource that provides an Index of Viruses and includes searchable of virus isolates, species, genera, families, orders, and images of many viruses, as well as links to genomic and protein databanks.
  http://www.polysciences.com/shop/assets/datasheets/305A.pdf
  Polysciences, Inc. Technical Data Sheet no. 305A for LR White resin.
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