APPENDIX A
Outline of Basic Technique
At the time of writing the first edition of this book there was in
essence one standard method of preparing ultrathin sections for electron
microscopy. The beginner presumably could proceed from chapter to
chapter and not be seriously confused or diverted by alternate techniques.
This perhaps is no longer the case for our armentarium is now complex,
and this book presently includes many divergent methods. Some of these
are frankly still in developmental stages. Others are designed for special
purposes. Still others represent efforts to solve problems that remain
difficult. The beginner who would embark upon a learning program
might well feel that he needs simple guide lines to avoid going astray.
The following outline represents an effort to suggest a reasonable training program with just enough diversity to give comparative experience.
Obviously numerous substitutions could be made to fit particular needs
or experience, but the procedures which follow have been chosen for their
reliability.
Training Program
1. Select for study a "soft" mammalian organ tissue, the cells of which
illustrate a complex morphology. Avoid tissues containing much collagen
or fat. Avoid organ capsules. Pancreatic tissue of the rat or mouse is
ideal.
2. Fix tissue in three ways, collecting 6-8 samples of each, (a) Mince
tissue (Chapter 2.2) directly in Millonig's osmium tetroxide formulation
(Chapter 3.3) for 1 hour, (b) Mince tissue and fix in either phosphate
buffered formaldehyde or glutaraldehyde for 10-20 minutes, and then
transfer small blocks to Millonig's osmium tetroxide for 1 hour (Chapter
3.9). (c) Leave some of the tissue overnight or for 24 hours in buffered
formaldehyde or glutaraldehyde. Do not secondarily fix with osmium
tetroxide (Chapter 3.10).
3. Dehydrate tissue by 10 minute exposures to 70 and 95% alcohol,
followed by two 20 minute changes of 100% alcohol, and two 20 minute
changes of propylene oxide if an epoxy embedment is to be used, or two
354
Outline of Basic Technique
At the time of writing the first edition of this book there was in
essence one standard method of preparing ultrathin sections for electron
microscopy. The beginner presumably could proceed from chapter to
chapter and not be seriously confused or diverted by alternate techniques.
This perhaps is no longer the case for our armentarium is now complex,
and this book presently includes many divergent methods. Some of these
are frankly still in developmental stages. Others are designed for special
purposes. Still others represent efforts to solve problems that remain
difficult. The beginner who would embark upon a learning program
might well feel that he needs simple guide lines to avoid going astray.
The following outline represents an effort to suggest a reasonable training program with just enough diversity to give comparative experience.
Obviously numerous substitutions could be made to fit particular needs
or experience, but the procedures which follow have been chosen for their
reliability.
Training Program
1. Select for study a "soft" mammalian organ tissue, the cells of which
illustrate a complex morphology. Avoid tissues containing much collagen
or fat. Avoid organ capsules. Pancreatic tissue of the rat or mouse is
ideal.
2. Fix tissue in three ways, collecting 6-8 samples of each, (a) Mince
tissue (Chapter 2.2) directly in Millonig's osmium tetroxide formulation
(Chapter 3.3) for 1 hour, (b) Mince tissue and fix in either phosphate
buffered formaldehyde or glutaraldehyde for 10-20 minutes, and then
transfer small blocks to Millonig's osmium tetroxide for 1 hour (Chapter
3.9). (c) Leave some of the tissue overnight or for 24 hours in buffered
formaldehyde or glutaraldehyde. Do not secondarily fix with osmium
tetroxide (Chapter 3.10).
3. Dehydrate tissue by 10 minute exposures to 70 and 95% alcohol,
followed by two 20 minute changes of 100% alcohol, and two 20 minute
changes of propylene oxide if an epoxy embedment is to be used, or two
354