CHAPTER 10

FROZEN SECTIONS
The Frozen Section Method is normally utilized when a rapid
diagnosis of the tissue in question is required, and is especially
recommended when lipids and the nervous tissue elements are to be
demonstrated. Frozen sections, both fixed and unfixed, have many
applications in histotechnology, and are commonly used for:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances
such as lipids and carbohydrates
4. Immunofluorescent and immunocytochemical staining
5. Some specialized silver stains, particularly in neuropathology
Fresh, completely unfixed tissues, or tissues that have been briefly
treated with formalin may not require embedding anymore; instead
they may be frozen and cut in a freezing microtome or cryostat. Two
methods of preparing frozen sections may be resorted to:
1. Cold Knife procedure
2. Cryostat procedure (Cold Microtome)
Cold Knife Procedure
Tissue blocks can be frozen by adapting a conventional freezing
microtome gas supply of carbon dioxide gas from a CO2 cylinder, or by
using a specially made piece of equipment known as cryostat. Almost
any microtome can be utilized for the purpose, provided means are
made available for freezing and maintaining the specimen and the
knife at low temperatures, usually by utilizing the carbon dioxide
technique.
A piece of filter paper soaked in gum syrup is placed on the
microtome stage, and short bursts of CO2 are applied, freezing the
filter paper to the stage. The selected block of tissue approximately 3-5
mm. thick, is then oriented on the stage, applied with a few drops of
gum syrup and frozen solid with several intermittent bursts of CO 2,

each for l-2 seconds duration, at intervals of around 5 seconds. It

should be frozen just to the point where it will be firm enough to
section. The tissue is then lifted up to the knife manually and trimmed
until the surface is flat. The surface is then warmed with until the
finger until hard frozen tissue starts to thaw and becomes visible to the
naked eye. This is the Dew Line, the point at which sections may then
be cut at 10 micra thickness.
Sections do not form ribbons but rather stick to the knife blade
and should therefore, be removed with a camel hairbrush or finger
moistened with water. They are then transferred to a dish of distilled
water to separate, and picked up individually for mounting and
staining. The water dish is usually placed on a dark or black
background, in order to see the sections which are usually colorless or
very light in color.
Tissues that have been frozen too hard will usually chip into