Name: Jenra D.
Eran BSMT 3 D
ACTIVITY 8: Study Questions
1. What are the reagents to be used in staining? Enumerate them chronologically.
1. Deparaffinization (if using paraffin-embedded tissues):
- Xylene: Used to remove paraffin wax from tissue sections.
- Alcohols (usually ethanol): Used to remove xylene and rehydrate the tissue. Common
concentrations are 100%, 95%, and 70% ethanol.
2. Hydration:
- Water: The tissue sections are rehydrated using distilled water to remove any remaining
alcohol.
3. Staining:
- Hematoxylin: A basic dye that stains nuclei blue/purple.
- Eosin: An acidic dye that stains cytoplasm and extracellular matrix components pink to red.
- Safranin O, Giemsa, or Azan for additional tissue differentiation.
4. Differentiation (if necessary):
- Acid Alcohol (usually 1% hydrochloric acid in alcohol): Used to differentiate the hematoxylin
stain by removing excess stain.
- Ethanol or Acid Alcohol: Used for differentiation to remove excess eosin or adjust the color
intensity.
5. Dehydration:
- Ethanol (95%, 100%): Used to remove water from tissue sections.
6. Clearing:
- Xylene: Used again for clearing the tissue after dehydration, preparing it for mounting.
7. Mounting:
- Mounting Medium: Typically Canada Balsam or Permount to mount the tissue on the
microscope slide for viewing.
- Cover slip: Placed over the tissue after mounting to protect the sample and facilitate
microscopy.
2. What are the possible causes of
a. Overstaining
Overstaining occurs when the tissue is exposed to the staining reagent for too long or in excess,
leading to overly dark or saturated coloration. Possible causes include:
- Excessive staining time: Leaving the tissue in the stain for longer than recommended can
result in an overly intense color.
- Too concentrated stain solution: Using a higher concentration of stain than recommended can
lead to overstaining.
- Insufficient washing or differentiation: If the tissue is not properly rinsed or differentiated after
staining, excess stain may remain, resulting in overstaining.
b. understaining
Understaining occurs when the tissue does not absorb enough of the stain, resulting in pale or
faint staining. Possible causes include:
- Insufficient staining time: Not allowing enough time for the stain to adequately bind to the
tissue.
- Diluted stain solution: Using too weak a concentration of the staining reagent can lead to
under-coloring of the tissue.
- Improper pH or temperature of stain: Some stains require specific pH or temperature
conditions to work effectively; deviations can cause poor staining.
- Insufficient tissue fixation: Poor fixation can cause the tissue to be less receptive to staining.
3. What are the precautionary measures to observe in staining?
- Use of Personal Protective Equipment (PPE): Always wear gloves, a lab coat, and safety
goggles to protect from harmful chemicals.
- Proper Ventilation: Work in a well-ventilated area or under a fume hood, especially when
using solvents like xylene and alcohol, which release toxic fumes.
- Avoiding Contamination: Use clean containers and tools for each reagent to avoid
cross-contamination between steps.
- Correct Reagent Handling: Follow the specific instructions for each reagent to ensure correct
concentration and exposure times.
- Proper Disposal: Dispose of used chemicals (e.g., xylene, alcohol) and biological waste
according to safety guidelines to avoid environmental contamination or personal injury.
- Monitoring Staining Time: Ensure that staining times are strictly adhered to, as prolonged
exposure to reagents can cause overstaining or other issues.
- Maintain Temperature and pH: Ensure reagents are stored and used at the correct
temperature and pH, as these factors affect the staining process.
4. Can you identify the specific purpose of the listed procedures in Section 8.4?
1. Deparaffinization: Removal of paraffin wax from embedded tissue to make the tissue
accessible for staining and sectioning.
2. Hydration and Rehydration: Transitioning from a hydrophobic (alcohol and paraffin) to a
hydrophilic (water) environment to facilitate the staining process.
3. Staining: Applying specific reagents (e.g., hematoxylin, eosin) to the tissue to highlight
different cellular structures for observation under a microscope.
4. Differentiation: The process of removing excess stain or adjusting the intensity of the color to
achieve the desired contrast.
5. Dehydration and Clearing: Removing water and reintroducing a substance (such as xylene)
that makes the tissue transparent, preparing it for mounting.
6. Mounting: Securing the stained tissue sample onto a slide and adding a cover slip, which
prepares the sample for long-term preservation and examination.
