Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 17

Table 4
Troubleshooting cryotome sectioning

Problem Cause Remedy

Sections smear or
crumple on the
blade edge

Temperature of specimen
is too high
The space under anti-roll
plate is too low

Select a lower temperature
Wait to equilibrate the object temperature
and try sectioning again

Sections shatter
at the tip of the
blade

Specimen is too cold
The anti-roll plate is not
correctly adjusted
The blade might be dull
or its clearance angle is
too steep
The specimen surface is
large

Select a higher temperature and let the
object equilibrate; if from the cryobar, let
it to adopt temperature of the clamp
Knife clearance angle of 2-5° is
recommended for disposable blades
Adjust the anti-roll plate correctly
Use another area of the blade or a new
blade
Trim the specimen parallel and increase the
section thickness

Sections curl up
when the anti-roll
plate is raised up

The anti-roll plate is too
warm

Lay down the anti-roll plate on knife and let
its temperature stabilize
Minimize air exchange within the chamber

Sections do not
run fl at under
the anti-roll plate

Dirty anti-roll plate and/
or knife
Dull blade

Clean with dry cloth or brush; it is
convenient to have some frozen in the
chamber
If necessary, use ethanol to clean blade and
anti-roll plate
Change blade or use another area of the edge

Sections curl in front of
the anti-roll plate and
do not go underneath

The anti-roll plate is too
far below the edge of
blade

Readjust the anti-roll plate

Section smear on the
top of the
anti-roll plate

Anti-roll plate goes
beyond the blade edge
and crushes the object

Readjust the anti-roll plate

Chatter on sections The chuck or blade is not

secured correctly
Specimen is too hard, too
cold or too big
The clearance angle is
incorrect
Cutting speed is too high

Check and fi x the stabilization of block
holder and blade
Modify temperature; let the object
equilibrate with specimen head
Trim the object to decrease its size
Reset clearance angle of the blade
Decrease the speed of cutting

Variable thickness
of sections

The chuck or blade is not
secured correctly
The clearance angle is
incorrect

Recheck and fasten the microtome head and
blade holder
Reset the blade angle

Sections are torn
perpendicularly
to the blade edge

Dust or nick on the blade
Leading edge of the
anti-roll plate is dirty or
damaged

Clean front and back side of the blade
Replace the blade or move to another part
of the blade
Clean or replace anti-roll plate