Basic Techniques For Transmission Electron Microscopy

Basic Techniques For Transmission Electron Microscopy

1st Edition - December 4, 1985

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  • Author: M Hayat
  • eBook ISBN: 9780323150293

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Basic Techniques for Transmission Electron Microscopy describes the basic techniques for transmission electron microscopy. Preparatory procedures for both eukaryotic and prokaryotic groups are presented in a step-by-step fashion, together with special preparatory methods for plant specimens and viruses. The processing of uncommon specimens and the solution of unusual, individual problems are included. This book is comprised of seven chapters and begins with a discussion on chemical fixation, with particular reference to fixatives and the hazards, precautions, and safe handling of reagents, as well as the preparation of buffers and tissue blocks. The reader is then introduced to the standard procedure for fixation, rinsing, dehydration, and embedding. Subsequent chapters focus on sectioning, cryofixation, and cryoultramicrotomy; positive and negative staining; and the use of support films. The final chapter presents a wide variety of specimens such as algae, amoeba, anthers, actin filaments, bacteria, and cells in culture. This monograph is essentially a laboratory handbook intended for students, technicians, teachers, and research scientists in biology and medicine.

Table of Contents

  • Preface

    1 Chemical Fixation


    Hazards, Precautions, and Safe Handling of Reagents


    Purification of Glutaraldehyde

    Storage of Glutaraldehyde

    Preparation of Formaldehyde Solution from Paraformaldehyde Powder

    Preparation of and Precautions in Handling Osmium Tetroxide Solution

    Regeneration of Used Osmium Tetroxide

    Preparation of Fixatives

    Preparation of Buffers

    2-Amino-2-Methyl-1,3-Propanediol (Ammediol) Buffer

    Acetate Buffer

    Barbital Buffer

    Cacodylate Buffer

    Carbonate-Bicarbonate Buffer

    Citrate Buffer

    Collidine Buffer

    Glycine-HCl Buffer

    Maleate Buffer

    Phosphate Buffer (Sörensen)

    Phosphate Buffer (Karlsson and Schultz, 1965)

    Phosphate Buffer (Maunsbach, 1966)

    Phosphate Buffer (Millonig, 1961)

    Phosphate Buffer (Millonig, 1964)

    Piperazine (PIPES) Buffer

    PM Buffer

    Ryter-Kellenberger Buffer

    Succinate Buffer

    Tris(hydroxymethyl)aminomethane Buffer

    Tris(hydroxymethyl)aminomethane Maleate Buffer

    Veronal Acetate Buffe

    Preparation of Tissue Blocks


    Vascular Perfusion

    Vapor Fixation and Staining

    Microwave Fixation

    Artifactual Electron-Dense Granules Occurring during Double Fixation

    2 Rinsing, Dehydration, and Embedding


    Standard Procedure for Fixation, Rinsing, Dehydration, and Embedding

    Gradual, Progressive Dehydration and Embedding

    Procedure for Minimizing Chemical Hazards during Specimen Preparation

    Low-Temperature Infiltration and Embedding

    Glycol Methacrylate

    Lowicryl K4M (Polar)

    Lowicryl HM2O (Nonpolar)

    Incomplete Dehydration for Lipid Preservation

    Water-Immiscible Embedding Media

    Epoxy Resins

    Dow Epoxy Resins

    Vinylcyclohexene Dioxide (VCD, ERL 4206, Spurr Mixture)

    Vinylcyclohexene Dioxide-n-Hexenyl Succinic Dioxide (HXSA)

    Polyester Resins



    Water-Miscible Embedding Media

    Acrylamide-Gelatin-Jung Resin



    Glutaraldehyde-Carbohydrazide (GACH)

    Glycol Methacrylate (GMA)

    Hydroxypropyl Methacrylate (HPMA)


    Lowicryl K4M

    Melamine Mixture


    Polyampholyte (Polyamph 10)

    Polyethylene Glycol (PEG; Carbowax)

    Diethylene Glycol Distereate

    Polyvinyl Alcohol

    Protein Aldehydes

    Mixed-Resin Embedding Media

    DER 332-DER 732

    Epon 812 (LX-112)-Araldite 502

    Epon 812-DER 736

    Epon 812-Thiokol LP-8

    Epon 812 (LX-112)-Maraglas

    ERL 4206-Quetol 653



    Silicone (Rhodorsil 6349)-Araldite CY 212

    Vinylcyclohexene Dioxide-n-Hexenylsuccinic Anhydride-Araldite RD-2


    Viscosity of Embedding Media

    Agar Preembedding (Encapsulation)

    Bovine Serum Albumin Preembedding

    Fibrin Clot Preembedding

    Direct Pelleting of Cells

    Pelleting of Cells in Agar

    Pelleting of Cells in Agarose

    General Embedding Methods


    Rapid Embedding

    Specimen Orientation

    Embedding of Hard Plant Tissues

    Orientation and Embedding of Small Specimens

    Orientation and Embedding of Single-Cell Organisms

    Embedding of Individual Cells

    Embedding of Ascomycetes (Cell Walls)

    Embedding of Bacteria and Tissue Culture Cells

    In Situ Embedding of Cells Grown on Millipore Filters

    In Situ Embedding of Cell Monolayers on Untreated Glass Surfaces

    In Situ Embedding of Virus for Immunoelectron Microscopy

    Open-Face Embedding for Correlative Microscopy

    Resin Slide Embedding for Correlative Microscopy

    Epoxy Resin Slides for Handling Unfixed Cryostat Sections

    Wafer Embedding

    Identification of Areas of Interest in Human Breast Tissue before Embedding

    Reembedding of Paraffin-Embedded Tissue in Resin

    Reembedding of Paraffin-Embedded Tissue Sections in Resin

    Pop-Off Method for Reembedding

    Reembedding of Tissue Culture Cells

    Reembedding of Thick Resin Sections

    Osmication and Flat Embedding of Large Tissue Sections

    Reembedding of Autoradiograms of Semithin Sections for Correlative Microscopy

    Retrieval of Poorly Resin-Embedded Tissue

    3 Sectioning


    Examination of Glass Knives

    Diamond Knives


    Carbon-Polymer Support Grids for X-Ray Microanalysis

    Specimen Block Trimming

    Preparation of Troughs

    Mounting the Specimen Block and Knife

    Sectioning Procedure

    Defects Appearing during Sectioning

    Serial Sectioning

    Specific Methods

    En Face Sectioning

    Sectioning Hard Mineral Fibers in Tissues

    Sectioning Hard Laminated Fibrous Tissues

    Vertical Sections of Cultivated Anchorage-Dependent Cells

    In Situ Thin-Section Microscopy of Cell Cultures

    Resectioning of Semithin Sections

    Semithin Sectioning


    Ralph Knife


    Serial Sectioning

    Cryofixation and Cryoultramicrotomy

    Specimen Collection


    Fixation and Cryoprotection



    Freezing Instrument


    4 Positive Staining



    Acridine Orange

    Alcian Blue



    Concanavalin A

    Diaminobenzidine-Osmium Tetroxide



    Iodide-Osmium Tetroxide





    Molybdenum Blues


    Phosphotungstic Acid (PTA)


    Potassium PermanganateCitrate

    Potassium Pyroantimonate-Osmium Tetroxide

    Ruthenium Red

    Silicotungstic Acid


    Sodium Tungstate

    Tannic Acid-Ferric Chloride

    Tannic Acid-Uranyl Acetate

    Tetraphenylporphine Sulfonate

    Thallium Ethylate

    Thiosemicarbazide and Thiocarbohydrazine

    Uranyl Acetate


    Lipid Preservation and Staining

    Multiple Staining

    Double Staining of Thin Sections with Uranyl Acetate and Lead Citrate

    Multiple-Grid Staining

    Removal of Bound Osmium from Thin Sections

    Staining for High-Voltage Electron Microscopy

    Staining Methods

    Precautions to Minimize Artifactual Staining Precipitates

    Removal of Artifactual Staining Precipitates from Thin Sections

    Removal of Epoxy Resins from Semithin Sections before Staining

    Staining Methods for Semithin Sections

    Azure Β for Plant Tissues

    Basic Fuchsin and Methylene Blue

    Methylene Blue-Azure II-Basic Fuchsin

    Methylene Blue-Azure II


    Hematoxylin-Phloxine Β

    Hematoxylin-Malachite Green-Basic Fuchsin

    Sudan III

    Chromotrope 2R-Methylene Blue

    5 Negative Staining


    Negative Stains

    Wettability of Support Films

    Choice of Support Film

    General Methods

    Basic Considerations

    One-Step (Simultaneous) Method

    Two-Step (Sequential) Method

    One-Side Negative Staining Method

    Pseudoreplica Method

    Agar Filtration Method

    Freeze-Dry Negative Staining


    General Methods for Animal Viruses

    General Methods for Plant Viruses

    Specific Methods





    Protein Macromolecules

    6 Support Films


    Plastic Support Films

    Formvar Films Cast on Glass

    Formvar Films Cast on Water

    Formvar Films Cast on Mica

    Collodion Films Cast on Water

    Collodion Films Cast on Glass

    Carbon Support Films

    Substrates for Carbon Evaporation

    Carbon Films Deposited Directly on Grids

    Carbon Films Prepared on Glass

    Carbon Films Prepared on Mica

    Carbon Films Using Plastic Substrates

    Carbonized Plastic Films

    Perforated Films

    Perforated Formvar Films

    Support Films with Large Holes (Micronets)

    7 Specific Preparation Methods

    Actin Filaments

    Algae (Unicellular): General Method

    Algae (Green)

    Method 1

    Method 2 (Unicellular Marine Algae)

    Method 3 (Chlamydomonas and Volvox)

    Algae (Red)

    Method 1

    Method 2



    Attachment of Cells to Substratum


    Method 1 (General Method)

    Method 2 (Glutaraldehyde and OsO4)

    Method 3 (for Extracellular Matrix)

    Method 4 (for Phage-Infected Bacteria)

    Bacterial Colonies

    Bacterial Microcolonies Grown on Solid Surfaces

    Bone (Undecalcified)

    Method 1

    Method 2

    Buffy Coat


    Cell Colonies Grown on Soft Agar

    Cells Attached to Glass Surfaces

    Cells in Culture

    Method 1 (e.g., Chick Embryo Spinal Cord Cells)

    Method 2 (e.g., Rat Liver Parenchyma and Human Glia Cells)

    Method 3 (e.g., Limpet Blood Cells)

    Cell Layers

    Cell Monolayers

    Method 1

    Method 2

    Method 3

    Method 4 (Pig Aorta Endothelial Cells)

    Method 5

    Method 6 (Human Brain Tumor Cells)

    Method 7

    Method 8 (Neuronal Cultures)

    Cell Suspensions

    Cerebrospinal Fluid Cells


    Method 1 (Polytene Chromosome Squashes)

    Method 2 (Chromosomes from Human Lymphocytes)


    Method 1

    Method 2

    Cream Cheese



    Method 1

    Method 2


    Method 1

    Method 2

    Eggs and Zygotes

    Effusion Fluid

    Embryos (e.g., Chick)

    Epidermal Hair of Seeds

    Epithelium (e.g., Cheek Pouch)

    Erythrocytes (e.g., Avian)

    Examination of Specific Parts of Identified Neurons (Somatosensory Cortex)

    Extracellular Protoplasts in Suspension


    Fetal Tissue (e.g., Pig Liver and Tooth Germs)

    Fine-Needle Aspiration Biopsy

    Method 1 (Single Glomerulus)

    Method 2

    Method 3 (Soft Tissues and Bone Tumors)


    Free Cells

    Method 1 (e.g., Cerebrospinal Fluid, Urine, Ascites, and Pleural Effusion)

    Method 2 (e.g., Leucocytes)


    Method 1 (General Method)

    Method 2 (Neurospora)

    Method 3 (Filamentous Fungi)

    Method 4 (Glass Bead Treatment)


    HeLa Cells

    Method 1

    Method 2

    Liposome Preparation

    Method 1

    Method 2

    Liquid Specimens (e.g., Milk and Fruit Juices)

    Method 1 (Milk)

    Method 2 (Orange Juice)

    Method 3 (Colostrum)



    Method 1

    Method 2

    Lymphocytes (Peripheral, Human)

    Marine Invertebrate Tissues



    Microtubules and Microfilaments

    Method 1 (Plant and Animal Tissues)

    Method 2 (Pituitary Gland)

    Mitotic Apparatus (Mammalian Cells)

    Mosquito (Freshwater)

    Muscle (Skeletal)

    Method 1

    Method 2

    Muscle (Smooth)

    Method 1

    Method 2

    Muscle (Insect)


    Olfactory Mucosa (Human)




    Parotid Gland

    Peritoneal Exudate Cells (Macrophage)

    Phage Particles

    Plant Tissues (Woody)

    Plant Tissues (Soft)

    Platelets (Human Blood)

    Method 1

    Method 2

    Method 3

    Pollen Grains (Fossil Material)

    Pollen Walls

    Potato Tuber

    Preparation and Removal of Selected Cells from Cytologic Smear Preparations

    Preservation of Larva-Substrate Relationship

    Protoplasts (Lettuce Leaf)


    Resetted Cells

    Rust Fungi


    Seeds (Dry)

    Seeds (Water-Impermeable Coat)

    Single Cells from Suspensions

    Thin-Layering and Immobilization

    Specimen Supports

    Selection of Cells for Sectioning


    Relocating a Selected Cell in the Embedded Thin Layer


    Method 1 (Fish)

    Method 2 (Frog)

    Slime Mold

    Small Specimens

    Method 1 (Small Invertebrates and Biopsy Specimens)

    Method 2 (Eggs, Embryos, and Small Isolated Organs)



    Method 1

    Method 2

    Method 3

    Spindle Microtubules (Anaphase of Grasshopper Spermatocytes)


    Method 1 (General Method)

    Method 2 (Freshwater or Marine Sponges)


    Method 1

    Method 2 (Dormant Spores of Bacillus subtilis)

    Method 3

    Surfactant over Alveolar Surface



    Tissue Containing Hard Mineral Fibers

    Tissue Prepared Anhydrously in Organic Reagents

    Tracheal Epithelium Tissue


    Viruses (Plant Tissues)

    Method 1

    Method 2

    Whole-Cell Preparations

    Method 1

    Method 2

    Method 3 (Myoblasts)


    Wool Follicles


    Method 1

    Method 2

    Method 3

    Method 4 (Saccharomyces cerevisiae)

    Method 5 (Candida albicans)

    Method 6 (Cell Wall)

    Method 7 (Mitotic Spindle)


    Commonly Used Chemicals for Stock Solutions of Specified Molarities

    Commonly Used Salts and Their Physicochemical Properties

    Earle's Balanced Salt Solution

    Hanks' Balanced Salt Solution

    Phosphate-Buffered Saline

    Balanced Salt Solutions


    Amphibian Ringer

    Mammalian Ringer

    Phosphate-Buffered Physiologic Saline

    Standard Cacodylate Washing Solution

    Krebs-Henseleit Saline (KHS)

    Locke Solution

    Physiologic Salt Solutions

    Molarities of Solutions Isotonic with Mammalian Blood

    Artificial Seawater

    Instant Seawater



Product details

  • No. of pages: 440
  • Language: English
  • Copyright: © Academic Press 1985
  • Published: December 4, 1985
  • Imprint: Academic Press
  • eBook ISBN: 9780323150293

About the Author

M Hayat

Dr. Hayat serves as Distinguished Professor in the Department of Biological Sciences at Kean University. He is an internationally renowned scholar in the fields of electron microscopy and cancer research, has authored and edited more than 31 books, several of these recently with Elsevier (dealing with immunohistochemistry of carcinomas, cancer imaging, and autophagy).

Dr. Hayat has published chapters in 54 books, and authored 10 of them. Seven major publishers have published his books: Cambridge University Press, Elsevier, Academic Press, McGraw-Hill, Plenum Press, CRC Press, and Van Nostrand Reinhold Company. Dr. Hayat’s books have been published in Japanese. He has also published a large number of articles in scientific journals and established the Electron Microscopy Center at Kean University.

Degree Information: Ph.D., Biology, Indiana University

Courses Taught: Electron Microscopy; Cell Biology; Seminar

Primary Area of Expertise: Electron microscopy, cell biology, cancer, health and disease. His research interests include immunohistochemistry and in situ hybridization of human carcinomas, including brain metastases and various primary cancers (lung, breast, prostate, colorectal, pancreatic, ovarian, gastrointestinal, and liver).

Advice For Students Preparing for Your Class: "Read the books and work very hard."

Affiliations and Expertise

Department of Biological Science, Kean University, USA

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