Basic Techniques For Transmission Electron Microscopy

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.

Preface

1 Chemical Fixation

Introduction

Hazards, Precautions, and Safe Handling of Reagents

Fixatives

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

Fixation

Vascular Perfusion

Vapor Fixation and Staining

Microwave Fixation

Artifactual Electron-Dense Granules Occurring during Double Fixation

2 Rinsing, Dehydration, and Embedding

Introduction

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

Methacrylates

Styrenes

Water-Miscible Embedding Media

Acrylamide-Gelatin-Jung Resin

Durcupan

Gelatin

Glutaraldehyde-Carbohydrazide (GACH)

Glycol Methacrylate (GMA)

Hydroxypropyl Methacrylate (HPMA)

LEMIX

Lowicryl K4M

Melamine Mixture

Polyacrylamide

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

Methacrylate-Styrene

Rigolac-Styrene

Silicone (Rhodorsil 6349)-Araldite CY 212

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

Embedding

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

Labeling

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

Introduction

Examination of Glass Knives

Diamond Knives

Grids

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

Introduction

Ralph Knife

Sectioning

Serial Sectioning

Cryofixation and Cryoultramicrotomy

Specimen Collection

Encapsulation

Fixation and Cryoprotection

Coolants

Freezing

Freezing Instrument

Sectioning

4 Positive Staining

Introduction

Stains

Acridine Orange

Alcian Blue

Bismuth

Cobalt

Concanavalin A

Diaminobenzidine-Osmium Tetroxide

Gold

Indium

Iodide-Osmium Tetroxide

Iodine

Iron

Lanthanum

Lead

Molybdenum Blues

Oxalate-Glutaraldehyde

Phosphotungstic Acid (PTA)

Platinum

Potassium PermanganateCitrate

Potassium Pyroantimonate-Osmium Tetroxide

Ruthenium Red

Silicotungstic Acid

Silver

Sodium Tungstate

Tannic Acid-Ferric Chloride

Tannic Acid-Uranyl Acetate

Tetraphenylporphine Sulfonate

Thallium Ethylate

Thiosemicarbazide and Thiocarbohydrazine

Uranyl Acetate

Vanadium

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

Giemsa

Hematoxylin-Phloxine Β

Hematoxylin-Malachite Green-Basic Fuchsin

Sudan III

Chromotrope 2R-Methylene Blue

5 Negative Staining

Introduction

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

Viruses

General Methods for Animal Viruses

General Methods for Plant Viruses

Specific Methods

Apoferritin

Bacteria

Fibrinogen

Microsomes

Protein Macromolecules

6 Support Films

Introduction

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

Amoeba

Anthers

Attachment of Cells to Substratum

Bacteria

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

Cartilage

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

Chromosomes

Method 1 (Polytene Chromosome Squashes)

Method 2 (Chromosomes from Human Lymphocytes)

Ciliates

Method 1

Method 2

Cream Cheese

Cysts

Diatoms

Method 1

Method 2

Echinoderms

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

Eyes

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)

Flagellates

Free Cells

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

Method 2 (e.g., Leucocytes)

Fungi

Method 1 (General Method)

Method 2 (Neurospora)

Method 3 (Filamentous Fungi)

Method 4 (Glass Bead Treatment)

Leaves

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)

Liverwort

Lobster

Method 1

Method 2

Lymphocytes (Peripheral, Human)

Marine Invertebrate Tissues

Microorganisms

Microtubules

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)

Nematodes

Olfactory Mucosa (Human)

Oocytes

Oyster

Paramecium

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)

Roots

Resetted Cells

Rust Fungi

Seeds

Seeds (Dry)

Seeds (Water-Impermeable Coat)

Single Cells from Suspensions

Thin-Layering and Immobilization

Specimen Supports

Selection of Cells for Sectioning

Embedding

Relocating a Selected Cell in the Embedded Thin Layer

Skin

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)

Spermatids

Spermatozoa

Method 1

Method 2

Method 3

Spindle Microtubules (Anaphase of Grasshopper Spermatocytes)

Sponges

Method 1 (General Method)

Method 2 (Freshwater or Marine Sponges)

Spores

Method 1

Method 2 (Dormant Spores of Bacillus subtilis)

Method 3

Surfactant over Alveolar Surface

Teeth

Tetrahymena

Tissue Containing Hard Mineral Fibers

Tissue Prepared Anhydrously in Organic Reagents

Tracheal Epithelium Tissue

Virosomes

Viruses (Plant Tissues)

Method 1

Method 2

Whole-Cell Preparations

Method 1

Method 2

Method 3 (Myoblasts)

Wood

Wool Follicles

Yeast

Method 1

Method 2

Method 3

Method 4 (Saccharomyces cerevisiae)

Method 5 (Candida albicans)

Method 6 (Cell Wall)

Method 7 (Mitotic Spindle)

Appendix

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

Tyrode

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

References

Index

Life Sciences

Physical Sciences & Engineering

Social Sciences & Humanities

Health

Basic Techniques For Transmission Electron Microscopy

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M.A. (Eric) Hayat

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