Biological microtechnique

241 Pages·1994·39.368 MB·English

by  Sanderson, Jeremy B

MICROSCOPY HANDBOOKS 28

Biological Microtechnique
Royal Microscopical Society MICROSCOPY HANDBOOKS
01 An Introduction to the Optical Microscope (2nd Edn)
03 Specimen Preparation for Transmission Electron Microscopy of
Materials
04 Histochemical Protein Staining Methods
05 X-Ray Microanalysis in Electron Microscopy for Biologists
06 Lipid Histochemistry
08 Maintaining and Monitoring the Transmission Electron Microscope
09 Qualitative Polarized Light Microscopy
11 An Introduction to Immunocytochemistry (2nd Edn)
12 Introduction to Scanning Acoustic Microscopy
15 Dictionary of Light Microscopy
16 Light Element Analysis in the Transmission Electron Microscope
17 Colloidal Gold: a New Perspective for Cytochemical Marking
18 Autoradiography: a Comprehensive Overview
19 Introduction to Crystallography (2nd Edn)
20 The Operation ofTransmission and Scanning Electron Microscopes
21 Cryopreparation of Thin Biological Specimens for Electron
Microscopy
22 An Introduction to Surface Analysis by Electron Spectroscopy
23 Basic Measurement Techniques for Light Microscopy
24 The Preparation ofThin Sections of Rocks, Minerals and Ceramics
25 The Role of Microscopy in Semiconductor Failure Analysis
26 Enzyme Histochemistry
27 In Situ Hybridization: a Practical Guide
28 Biological Microtechnique
Biological Microtechnique
J.B. Sanderson
Sir William Dunn School of Pathology,
South Parks Road, Oxford OX1 3RE, UK
/lIOS
SCIENTIFIC
MUSIIERS
In association with the Royal Microscopical Society
© BIOS Scientific Publishers Limited, 1994

All rights reserved. No part ofthis book may be reproduced or transmitted,

in any form or by any means, without permission.

First published in the United Kingdom 1994 by

BIOS Scientific Publishers Limited,
St Thomas House, Becket Street, Oxford, OX1 1SJ
Tel: [phone] Fax: [phone]
Transferred to Digital Printing 2005
A CIP catalogue for this book is available from the British Library.
ISBN 1872748422
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Preface

Although there is now a general trend towards molecular biochemistry in
current biological research and clinical practice, there is nevertheless still
a requirement to produce good microscopical preparations. However good
the microscope, it cannot enhance poorly prepared specimens, which
inevitably lead to unreliable results. With this in mind, Biological Micro­
technique presents the reader with the newest and also the well-proven
classical methods of slide making. A well-prepared specimen not only
displays information effectively, but is in itself an achievement and ajoy
to make. At first sight, preparing a standard thin section appears straight­
forward, and is the technique most often used in histology laboratories;
but there are other little-used techniques which can equally well inform
microscopists and answer their questions. Furthermore, the professional
reseacher can learn much from the techniques employed by skilled ama­
teurs, who often produce far superior preparations from the more limited
means at their disposal. While much ofthe information is directed towards
the professional researcher, I have tried to bear the requirements of the
amateur microscopist in mind. There is, for example, information on older
microtomes, on hand-sharpening microtome knives, and references to the
older literature and techniques more suited to the individual working
without the facilities of a modern research institute.
This book gives a general introduction to biological microtechnique for
the light microscope, for students, postgraduate workers and technicians
who are new to microscopy. Most texts in this field are either out of print,
or else concentrate upon producing and examining thin sections. This book
has been divided chapter by chapter into the steps of preparation: fixing,
embedding, microtomy or otherwise, and mounting the final preparation.
Because of the present dearth of comprehensive texts on biological micro­
technique for the light microscope, this handboook does not cover prepara­
tion techniques for electron microscopy, but references are given to
complementary texts in this field.
Histology and microscopy are often, unfortunately, taught in isolation
to students. This book is intended to present a basic introduction to
biological microtechnique, and supplements the other titles on microscopy
in the RMS Handbook series. It is not a handbook covering every commonly
used protocol orstaining method. Those thatare selected have been chosen
because they demonstrate general principles and materials used in pre­
y
vi BIOLOGICAL MICROTECHNIQUE
paring microscope slides. For example, many empirical methods for hae­
matoxylin have been published and are readily available. A rational
formula, Haematal8, which is both simple and reliable to use is published
here for this reason. An understanding of how techniques work leads to
controlled manipulation and better interpretation and diagnosis.
A practical work on biological microtechnique has not been published
for 20 years, yet significant advances have been made in biological speci­
men preparation. The resurgence ofinterest in light microscopical (partic­
ularly confocal) techniques, which have not been superseded by the
electron microscope, and indeed complement it to a greater extent than
hitherto, and the use of newer resin media for embedding in addition to
wax, mean this book will appeal to students, undergraduates, technicians
and research fellows in whose work microscopy plays a large part, or who
wish to extend their repertoire of techniques. I have assumed that they
will have recourse to academic libraries, and have included extensive
references throughout. These are intended to introduce the reader to the
basis of each technique and point to similar work. Although micro­
technique is largely a conservative art, the demands ofmodem biological
research have brought corresponding improvements, particularly in pre­
serving the structural and functional elements ofcells and tissues during
fixation and embedding. Similar advances have been made in the auto­
mated instrumentation available. Where possible, modern equipment has
been illustrated within the space available, but readers are advised to
consult manufacturers' and suppliers' catalogues.
Notes have been included in the appendix to supplement the safety
caveat published in all the Society's handbooks, and a table of selected
refractive indices has been included. Since the refractive index of moun­
tants and immersion media can readily change, they have been given to
two decimal places only, whereas those of solvents have been given to three
places. I have tried throughout to explain the reasons why operations are
carried out in the way described, particularly in the chapter on microtomy.
My own experience, from a technical viewpoint, is that this approach gives
a better understanding of why a particular technique works and helps to
develop a critical feedback for the microtomist. It is hoped that this will
lead to better work and increased confidence. In this way, I hope that those
who read this book will come to a firmer and greater understanding of
biological microtechnique, which cannot be learnt and ultimately enjoyed
except by practice.
My own professional traininghas been as a histologist and microscopist.
As a member ofthe active amateur learned societies I have been indebted
to many friends for what they have taught me. I would welcome, therefore,
suggestions offavoured methods and protocols from both those for whom
microtechnique is a profession, as well as those who make microscopical
preparations in their spare time.
Jeremy B. Sanderson
Oxford 1994
ACKNOWLEDGEMENTS vii
Acknowledgements
I have been fortunate to have the services of my colleagues in the Royal
Microscopical Society, Dr Brian Bracegirdle and Dr Savile Bradbury. Dr
Bracegirdle very kindly reviewed, commented on, and improved the clarity
ofthe initial manuscript; many ofhis suggestions have been incorporated.
Dr Bradbury, as General Editor of this series, has not only read and
advised on the text, but also helped with the line diagrams, and instructed
and encouraged me in developing my skills in microscopy.
I also wish to thank Dr Henry Teed for the production of many of the
half-tone illustrations in this book, and whose support has been generously
given. Several companies and societies have given permission for the use
oftheir pictures; they are acknowledged in place, and I am grateful to the
individuals concerned. My thanks are due to colleagues in the Department
ofHuman Anatomy, the Nuffield Department ofSurgery, as well as in my
present Department, the Sir William Dunn School of Pathology, for their
aid in the production of this book. I am indebted to Mr Douglas T.
Richardson who has also read the manuscript. It is my pleasure to record
my thanks to BIOS Scientific Publishers for their help.
This book has been greatly facilitated by the unstinted support of my
wife, Yvonne. She not only read and commented upon the manuscript, but
in addition managed a busy young family (largely unaided) until it was
completed.
Safety
Attention to safety aspects is an integral part ofall laboratory procedures
and both the Health and Safety at Work Act and the COSHH regulations
impose legal requirements on those persons planning or carrying out such
procedures.
In this and other Handbooks every effort has been made to ensure that
the recipes, formulae, and practical procedures are accurate and safe.
However, it remains the responsibility of the reader to ensure that the
procedures which are followed are carried out in a safe manner and that
all necessary COSHH requirements have been looked up and imple­
mented. Any specific safety instructions relating to items of laboratory
equipment must also be followed.

Contents

Abbreviations xv

1. Introduction 1

Collecting material for specimen preparation 3

Choice ofpreparative technique 5

Looking at preparations 8

References 10

2. Fixation 11

Function and use offixatives 11

Methods offixation 14

Immersion fixation 14

Perfusion fixation 15

Vapour fixation 16

Phase-partition fixation 16

Mechanical methods 17

When not to use fixation 17

The penetration offixatives 17

Osmolarity and pH 18

Fixing agents 19

Formaldehyde 19

Glutaraldehyde and acrolein 20

Alcohols and acetone 21

Mercuric chloride 21

Potassium dichromate 21

Picric acid and acetic acid 22

Osmium tetroxide 22

Fixative mixtures 22

Secondary fixation 29

Preservatives 29

Washing tissues 29

Microwaves in histology 30

Microwaving formalin-fixed tissues 32

Leiden fixative 34

ix