HISTOLOGICAL TECHNIQUES

Question 1

Paraffin sectioning basic process:

Answer 1

Tissues are first fixed to avoid degradation, embedded in paraffin, cut into serial sections with a rotary microtome, and stained to contrast cellular elements.

Question 2

Fixatives function

Answer 2

preserve proteins and cellular structure

Question 3

_______shrinks tissue and causes excessive hardening

Answer 3

alcohol

Question 4

swells tissue and prevents overhardening.

Answer 4

acid such as acetic acid

Question 5

Not an ideal fixative; a mixture of alcohol and acetic acid.

Answer 5

Carnoy’s fixative

Question 6

Carnoy’s fixative*=
glacial acetic acid 100 ml +
100% ethanol 300 ml

Question 7

A commonly used fixative that is better at preserving structure than Carnoy’s is _____

Answer 7

Dietrich’s FAA** =
95% ethyl alcohol 30.0 ml +
formalin 10.0 ml +
glacial acetic acid 2.0 ml +
distilled water 60.0 ml

Question 8

Formaldehyde is a gas, which is sold in solution as formalin, which is about 40% formaldehyde in water.

Question 9

A fixative that is widely used for embryological material, since it will not overharden yolky material, is _______

Answer 9

Bouin’s Fluid** = picric acid, saturated 75.0 ml +
aqueous*** formalin 25.0 ml +
glacial acetic acid 5.0 ml

Question 10

though an excellent fixative for lipid-rich tissues that doesn’t harden the tissues, also stains the tissue yellow

Answer 10

picric acid

Question 11

For fixation and all steps that follow that require a fluid, the rule of thumb is that the amount of fluid used should be ____× the volume of the tissue.

Answer 11

10

Question 12

Specimens should not be left in Carnoy’s fixative ________ days.

Answer 12

more than 2–3

Question 13

Tissues should be fixed and stored in the refrigerator at _____

Answer 13

4ºC

Question 14

If you have chosen Carnoy’s as your fixative, then washing in ____ instead of water is both adequate and faster.

Answer 14

70% alcohol

Question 15

By going through a graded series of alcohols, the ______that are set up by each transfer are minimized, thereby minimizing harm to the specimen.

Answer 15

convection currents

Question 16

After the water has been removed, a _______, such as ________, which is miscible with both 100% alcohol and paraffin, makes a bridge between the alcohol and paraffin.

Answer 16

clearing agent; xylene or toluene

Question 17

(Toluene, Xylene) is less harsh on tissue than (toluene, xylene), causing less shrinkage and hardening

Answer 17

Toluene; xylene

Question 18

Microwaving the specimen during fixation and dehydration cuts down considerably on the time needed in each solution and improves infiltration of the solutions into the tissue. The specimen should not be heated over _____—this causes overdenaturation of regions that are relatively impermeable and stain nonuniformly.

Answer 18

70ºC

Question 19

Higher melting points indicate harder paraffin that can be cut into thinner sections.

Question 20

The paraffin block is to be mounted onto a piece of metal, synthetic fiber, or wood, all of which can be referred to as “_______,” which fit into the chuck holder of the microtome

Answer 20

chucks

Question 21

Paraffin scraps from trimming can be remelted, filtered, and reused. Paraffin improves with reheating, since impurities are volatilized off with each heating.

Question 22

Paraffin sections are typically cut at between _______ in thickness. For routine sectioning, set the thickness to between _______

Answer 22

4 and 12 µm; 8 and 10 µm

Question 23

Once a ribbon of sections begins to form, the end of the ribbon can be lifted with a ___________.

Answer 23

moistened camel’s-hair paintbrush

Question 24

Remember, the ribbon will not be straight unless the top and bottom sides of the block are precisely parallel to one another. When retrimming the block, the knife must first be removed from the knife carriage.

Question 25

Hygroscopic substance used as drying agent

Answer 25

Desiccant

Question 26

Type of staining dish that can handle up to 9 slides at a time

Answer 26

Coplin jars

Question 27

______________are the most commonly used general nuclear and cytoplasmic stains.

Answer 27

Hematoxylin and eosin (H&E)

Question 28

_______, (a basic dye) stains acidic components, primarily nucleic acids, a dark blue and is therefore used as a nuclear stain. ______ (an acidic dye) has an affinity for cytoplasmic elements.

Answer 28

Hematoxylin (Tap water is slightly basic, and a basic solution is needed to change the color of the bound hematoxylin from a rusty brownish color to a dark, almost black, blue.); Eosin (yellowish-red)

Question 29

______stains glycosaminoglycans (e.g., mucus, chondroitin sulfate of cartilage) and is a simple addition to the H&E series that adds useful information. Because ------- is used at a very acidic pH, it must be used prior to hematoxylin in the staining series to avoid removing any bound hematoxylin.

Answer 29

Alcian blue

Question 30

Why do you think you need to fix a tissue before processing it for paraffin sectioning?

Answer 30

Fixation preserves tissue structure by preventing autolysis, microbial growth, and degradation while hardening the sample for accurate paraffin sectioning and staining.

Question 31

How can you counterbalance the shrinking effects of alcohol in a fixative?

Answer 31

by adding acetic acid, which swells the tissue and prevents excessive hardening.

Question 32

After fixing a tissue, what must you do to it prior to embedding it in paraffin?

Answer 32

wash it to remove excess fixative, dehydrate it through a graded series of alcohols, and clear it with a solvent like xylene or toluene to prepare it for paraffin infiltration.

Question 33

What type of embedding molds can be used for paraffin embedding, and why is it important that they be watertight?

Answer 33

metal, plastic, or paper boxes. It is important that they be watertight to prevent molten paraffin from leaking, ensuring proper infiltration and solidification around the tissue for stable sectioning.

Question 34

When trimming a paraffin block for cutting, what shape should the block face be?

Answer 34

like a trapezoid, with the top and bottom edges parallel and the sides slanted.

Question 35

What common tool do you use to handle paraffin sections? How do you store paraffin sections before mounting them on slides?

Answer 35

A moistened camel’s-hair brush is commonly used. Before mounting, the sections are stored in a ribbon box to protect them from air currents and dust.

Question 36

Before mounting sections on a slide, what must you do to ensure that the sections won't come off the slide during processing?

Answer 36

expand them on warm water (5–10°C below the paraffin melting point) and allow them to fully dry for at least 6–8 hours (or overnight) to ensure proper adhesion during processing.

Question 37

Why is toluene or xylene the first step in a staining series when processing paraffin sections?

Answer 37

it removes paraffin from the tissue sections, allowing them to rehydrate and properly absorb stains.

Question 38

Why do you think a graded series of alcohols is used in a staining series? Why not take the slides directly from 100% alcohol to water?

Answer 38

to gradually transition the tissue from a non-polar solvent (like xylene) to water, preventing sudden osmotic shock and distortion of the tissue. Directly transferring slides from 100% alcohol to water would cause convection currents, leading to uneven rehydration and potential tissue damage.

Question 39

What is a routine nuclear stain? What is a routine cytoplasmic stain? What other stains do you know about, and what components do they stain?

Answer 39

Hematoxylin – stains nuclei blue to purple by binding to acidic components like DNA. Eosin – stains cytoplasm pink to red by binding to basic proteins. Alcian Blue – stains acidic mucopolysaccharides (mucus, cartilage) blue. Oil Red O – stains lipids red.