Techniques in Histology

In preparing tissue for routine light microscopic study, which procedure immediately precedes clearing the specimen with an organic solvent?

• Fixation

• Dehydration

• Staining

• Embedding

Answer explanation

After fixation, all the water is extracted from the tissue by dehydration with ethanol, after which the ethanol is replaced by an organic solvent (paraffin) that gives the tissue a translucent appearance (clearing).

Cellular storage deposits of glycogen, a free polysaccharide, could be detected using .... .

• PAS reaction or stain

• Toluidine Blue stain

• Trichome stain

• Immunohistochemistry

Answer explanation

Glycogen is rich in carbohydrates, and stains well using the Periodic acid-Schiff (PAS) reaction which makes it appear purple/magenta.

What will most likely happen during staining if the slices are too thick? 

• The slices will absorb more stain than needed.
• The staining may be uneven or ineffective.
• The staining will enhance the thickness of the slices.
• The slices will become transparent during staining.

In histology, what is the primary purpose of the dehydration step in tissue processing?

• To remove excess ink markings from the specimen

• To remove water from the tissue

• To increase the tissue's water content

• To remove fat element from the tissue

Hematoxylin is, indeed, repsonsible for the darker coloring of, in this case, the lower cellular layer in this tissue. 

What does this tell you about its chemical makeup? What part of the cell typically stains this color?

• Typically the nucleus stains darker. As an acidophilic structure, it attracts acid dyes, which often appear dark purple or blue.

• Typically the cytoplasm stains darker. As an acidophilic structure, it attracts basic dyes, which often appear dark purple or blue.

• Typically the cytoplasm stains darker. As a basophilic structure, it attracts basic dyes which often appear dark purple or blue.

• Typically the nucleus stains darker. As a basophilic structure, it attracts basic dyes, which often appear dark purple or blue.

Answer explanation

Basophilic structures attract basic particles (positively charged) because they are acidic themselves (negatively charged). The nucleus is one region of the cell that typically stains strongly with hematoxylin and other basic dyes because of its acidic properties (the cell nucleus contains a lot of nucleic acid (i.e., DNA)). Basic dyes, such as hematoxylin, often appear blue or dark purple.

What happens when your tissue is excessively dehydrated? Or not dehydrated properly? 

• Excessive dehydration leads to increased cellular growth.
• Improper dehydration causes cells to multiply rapidly.
• Dehydration has no effect on tissue health.
• Excessive dehydration causes cellular shrinkage and dysfunction; improper dehydration leads to edema and swelling.

In a light microscope used for histology, resolution and magnification of cells are largely dependent on which component?

• Condenser

• Objective lens

• Eyepieces or ocular lenses

• Specimen slide

• The control for illumination intensity

Why is embedding medium heated during the embedding process in histology?

• To make the tissue specimens more colorful

• To soften the medium and facilitate infiltration into the tissues

• To preserve tissue structures with heating

In histology, the primary purposes of the clearing step in tissue processing are __________ and ___________

• To remove excess ink markings from the specimen

• To replace water-soluble dehydrating agents with a medium compatible with embedding media

• To increase the tissue's water content

• To remove fat element from the tissue

During the clearing step, what is commonly used to replace the dehydrating agent in tissue specimens?

• mineral oil

• wax

• formalin

• xylene