186
5.
SECTIONING
mastered the variety of skills necessary to achieve this. It is fairly easy to
obtain a good micrograph at 50,000 diameters, but it is quite another
matter to achieve real resolution at 100,000 diameters or more when the
investigator is approaching a whole host of technical limitations which
conspire to defeat him.
5.25. Summary of Sectioning Technique
Woven into this and the previous chapter have been a number of
precautions that must be taken for successful sectioning, as well as a
discussion of difficulties that may be encountered. It seems wise to bring
these together in the form of a summary or a check list that can be consulted in time of frustration. The beginner should be encouraged by the
knowledge that ultrathin sectioning is a proven possibility, and that if
he is having difficulties or is unsuccessful, it is probably because of some
seemingly small flaw in his technique. He should take stock of every key
step and not attempt shortcuts.
(1) Knife contamination. This is the most common difficulty, and the
fingerprints which produce oily films are the worst offenders. Dirty towels,
impure solvents, oily tools, contaminated containers, trough walls—all
must be considered.
(2) Block face too large. The cutting forces deflect the knife edge with
consequent digging and skipping, and often wide variation in section
thickness along with the skipping. Even thin sections are apt to be useless
because of fine-order chatter. This is second only to contamination as the
principal cause of failure.
(3) Knife tilted too far forward. This favors digging and subsequent
skipping. It is also apt to produce fine chatter which may not be apparent
while sectioning is in progress, thus giving a false sense of security.
(4) Knife tilted too far backwards. There will be much skipping and
occasional thick sections.
(5) Cutting speed too fast. This favors fine chatter which may not be
apparent during the sectioning. Be sure you can follow the block with
your eyes as it descends towards and across the knife if the microtome is
being cycled by hand.
(6) Unsteady cutting. The microtome must be cycled rhythmically;
any interruption or break in this rhythm will require a period of readjustment before good uniform sectioning can begin again.
5.
SECTIONING
mastered the variety of skills necessary to achieve this. It is fairly easy to
obtain a good micrograph at 50,000 diameters, but it is quite another
matter to achieve real resolution at 100,000 diameters or more when the
investigator is approaching a whole host of technical limitations which
conspire to defeat him.
5.25. Summary of Sectioning Technique
Woven into this and the previous chapter have been a number of
precautions that must be taken for successful sectioning, as well as a
discussion of difficulties that may be encountered. It seems wise to bring
these together in the form of a summary or a check list that can be consulted in time of frustration. The beginner should be encouraged by the
knowledge that ultrathin sectioning is a proven possibility, and that if
he is having difficulties or is unsuccessful, it is probably because of some
seemingly small flaw in his technique. He should take stock of every key
step and not attempt shortcuts.
(1) Knife contamination. This is the most common difficulty, and the
fingerprints which produce oily films are the worst offenders. Dirty towels,
impure solvents, oily tools, contaminated containers, trough walls—all
must be considered.
(2) Block face too large. The cutting forces deflect the knife edge with
consequent digging and skipping, and often wide variation in section
thickness along with the skipping. Even thin sections are apt to be useless
because of fine-order chatter. This is second only to contamination as the
principal cause of failure.
(3) Knife tilted too far forward. This favors digging and subsequent
skipping. It is also apt to produce fine chatter which may not be apparent
while sectioning is in progress, thus giving a false sense of security.
(4) Knife tilted too far backwards. There will be much skipping and
occasional thick sections.
(5) Cutting speed too fast. This favors fine chatter which may not be
apparent during the sectioning. Be sure you can follow the block with
your eyes as it descends towards and across the knife if the microtome is
being cycled by hand.
(6) Unsteady cutting. The microtome must be cycled rhythmically;
any interruption or break in this rhythm will require a period of readjustment before good uniform sectioning can begin again.