Basic Techniques For Transmission Electron Microscopy

Basic Techniques For Transmission Electron Microscopy

1st Edition - December 4, 1985

Write a review

  • Author: M Hayat
  • eBook ISBN: 9780323150293

Purchase options

Purchase options
DRM-free (PDF)
Sales tax will be calculated at check-out

Institutional Subscription

Free Global Shipping
No minimum order

Description

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

    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

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

Ratings and Reviews

Write a review

There are currently no reviews for "Basic Techniques For Transmission Electron Microscopy"