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HISTOTECHNOLOGY D2L QUESTIONS & ANSWERS
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HISTOTECHNOLOGY D2L QUESTIONS & ANSWERS
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HISTOTECHNOLOGY D2L QUESTIONS & ANSWERSWhat thickness are paraffin sections routinely cut? - answer- 4-6 um
Why should the temperature of the flotation bath be set specifically at 5-10 °C below the
melting point of the paraffin wax? - answer- water bath that is too hot may distort tissue
morphology
Warm enough to relieve compression
Cool enough so the wax does not melt
What thickness are paraffin sections cut for the Luxol Fast Blue procedure? - answer-
10 - 15 um
Why are paraffin blocks put on ice prior to cutting? - answer- to soften the tissue;
harden the wax
What thickness are paraffin sections cut for the Congo Red procedure? - answer- 8-10
um
State the difficulties and / or problems that may be encountered in / or following paraffin
sectioning if the tissues were improperly fixed or processed in the following ways:
Incomplete fixation
Incomplete dehydration
Over dehydration
Incomplete clearing
Excessive time in the clearing agent
Incomplete filtration
Excessive infiltration time and / or wax too hot - answer- Incomplete fixation
Tissue components can separate easily on the flotation bath during cutting. Tissue
morphology is not well maintained and nuclei can appear smeared.
Improper fixation is irreversible.
b. Incomplete dehydration
Tissue will be mushy and softer than usual. It may show properly processed edges, but
a lighter or whiter colour in the centre and thicker areas.
Will likely smell like formalin. Water may be visible inside the tissue cassettes and on
the bottom of the wax bath.
c. Over dehydration
Tissue will be very brittle. Cutting will be difficult if even possible.
d. Incomplete clearing
, Tissues will appear opaque or cloudy due to two immiscible substances present
together in the tissue - alcohol and paraffin. Tissues will have a soft consistency. Tissue
will eventually dry up and shrink.
e. Excessive time in the clearing agent
Too much time in the xylene can produce brittle tissue, making it difficult or even
impossible to cut.
f. Incomplete filtration
This means that the wax will not completely replace the xylene from the clearing step.
Wax mixed with xylene will not properly harden to support the tissue. Cutting will be
very difficult. The block will still smell like xylene. Over time during storage, the clearing
agent will evaporate from the face of the block and the tissue will contract.
g. Excessive infiltration time and / or wax too hot
Prolonged time in the paraffin wax causes shrinkage and hardening of the tissue. Wax
that has been overheated breaks down, making sectioning and pick-up onto slides very
difficult.
List two techniques or procedures that make use of cryostat sections. - answer- Quick
section for rapid diagnosis (frozen section), Oil Red O, Enzymes for
immunohistochemistry, Immunofluorescence, Muscle biopsy
What is the antiroll plate made of? - answer- Plastic or plastic coated glass
At what thickness are frozen sections cut for lipid demonstration? - answer- 10 - 18 um
At what thickness are frozen sections cut for rapid H&E staining? - answer- 6 - 8 um
State (3) advantages and (2) disadvantages of frozen sectioning. - answer- a.
Advantages:
No lipid or enzyme loss
No processing artifact
Rapid diagnosis
b. Disadvantages:
Ribbons are impossible
Storage of frozen tissue is difficult, if not impossible (need -70° C freezer or liquid
nitrogen tank; can thaw and store in NBF)
Thin sections, such as 3-4um, are difficult
Cutting artifacts more likely and detail distorted
Name (2) chemicals that can be used to disinfect a cryostat. - answer- Formaldehyde
fumes, Glutaraldehyde, Germicidal spray, alcohol
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