You have just finished your H&E stain, but under the microscope, you notice that the nuclear stain (hematoxylin) is pale or weak. What is the corrective action?
Bring the slide back down to water (xylene, descending grades of alcohol, water).
Re-stain the slide with all steps of the H&E technique.
Note: If this is autopsy tissue, this result may be ACCEPTABLE. No action needed.
After staining, you realize you completely forgot to use eosin. There is no cytoplasmic staining at all. How do you fix this?
Rehydrate the slide (xylene, descending grades of alcohol, water).
Stain with eosin (ensuring adequate time).
Follow the protocol through dehydration, clearing, and mounting (DCM).
The slide you are looking at has a “muddy” or unclear appearance. You suspect the hematoxylin was not differentiated properly. What should you do?
Rehydrate the slide (alcohols will remove most of the eosin).
Dip the slide 1-2 times in acid alcohol.
Proceed with the rest of the staining protocol (blueing, eosin, dehydration, clearing, mounting).
The slide looks muddy, but this time you suspect it’s because the blueing agent wasn’t rinsed off adequately, or the eosin was contaminated with it. How can you correct this?
Rehydrate the slide. (The alcohols will remove most of the eosin, and the water will remove the blueing agent).
Apply eosin again (stain with eosin).
Continue with the rest of the protocol (dehydration, clearing, mounting).
You completed the blueing step, but forgot to put the slide in eosin. There is no cytoplasmic staining. What is the corrective action?
Bring the slide back down to water (xylene, descending grades of alcohol, water).
Stain with eosin.
Proceed with dehydration, clearing, and mounting (DCM).
The cytoplasmic staining on your slide is very pale. You know you used eosin, but you may not have left the slide in it long enough. How do you fix this?
Rehydrate the slide (xylene, descending grades of alcohol, water).
Stain with eosin again, ensuring you allow adequate time.
Follow the protocol until the end (dehydration, clearing, mounting).
You look at your slide and realize you must have skipped the hematoxylin step entirely, as there is no nuclear staining. What is the corrective action?
Rehydrate the slide (xylene, descending grades of alcohol, water).
Re-stain the slide with all steps of the H&E technique.
After coverslipping, you notice water droplets under the coverslip. What is the likely cause and the corrective action?
Cause: The 100% alcohol or the xylene used for clearing is contaminated with water.
Corrective Action:
Replace the 100% alcohol and xylene buckets with fresh reagents.
Take the slides back to fresh 100% alcohol twice. Dip until the slides are no longer streaky.
Clear the slides with fresh xylene, ensuring the xylene is not contaminated with water.
Your H&E slide shows white spots in the tissue section or patchy, irregular staining. What is the most likely problem, and how do you fix it?
Problem: Incomplete dewaxing. There is still wax on the tissue before the stains were applied.
Corrective Action:
Ensure the slides stay longer in the dewaxing xylene. Avoid using dewaxing xylene that is contaminated with water.
Proceed with the rehydration steps.
Re-stain the slide starting from the hematoxylin step.
The nuclear staining on your slide is not crisp; it looks poorly defined. What factors during tissue processing could cause this?
This can be due to:
Incomplete fixation.
Incomplete dehydration or incomplete clearing during processing.
Heat being introduced in reagents other than the paraffin wax (heat should only be used in the paraffin steps).
Prolonged wax infiltration.
Prolonged slide drying after sectioning.
Besides simply not leaving the slides in hematoxylin long enough, what are some other reasons for pale nuclear staining?
Other reasons include:
The hematoxylin was over-oxidized or depleted.
The section was over-differentiated (left in acid alcohol too long).
An acidic fixative was used on the tissue.
The tissue was over-decalcified or over-fixed.
Your slide shows dark, overly stained nuclei. What could be the causes, and what is the corrective action?
Causes:
The section was left in hematoxylin too long.
The differentiation step (acid alcohol) was too short.
The tissue section is too thick.
Corrective Action:
Rehydrate the slide.
Increase the time the slide spends in the acid alcohol (re-differentiate).
Proceed with the next steps (blueing, etc.).
If the section is too thick, the section may need to be recut.
Instead of the usual blue-black, the nuclei on your stained slide appear red or brown. What are the likely causes and the corrective action?
Causes:
The hematoxylin is breaking down (oxidation issue).
The blueing step was not done properly.
Corrective Action:
First, check the oxidation status of the hematoxylin.
Remember that sections must be blued properly—it is not possible to over-blue them.
To fix the current slide, rehydrate it and re-stain it starting from the hematoxylin step.
The cytoplasmic (eosin) staining on your slide is too pale. What are the potential causes, and how can you prevent this in the future?
Causes:
The pH of the eosin solution is greater than 5.
The eosin was contaminated with blueing agent.
The slides were left in the low-grade alcohols during dehydration (DCM) for too long.
The tissue sections are too thin.
Prevention: Adjust the pH of the eosin with acetic acid, completely remove blueing agent before going into eosin, and monitor timing in alcohols. Ensure correct tissue thickness.
You have a slide where the cytoplasm is stained too darkly. What is the corrective action to fix the current slide?
Bring the slide back down to 95% alcohol. (This will help differentiate, or remove some of, the excess eosin).
Proceed with the rest of the dehydration, clearing, and mounting (DCM) protocol.
What are the potential causes of dark cytoplasmic staining that a pathologist might see on a slide?
The eosin solution is too highly concentrated.
The slide was left in the eosin for too long.
The slide was not left in the low-grade alcohols long enough during dehydration (DCM).
The tissue section itself is too thick.
You notice a blue-black precipitate on top of the tissue, and in some areas, it has a metallic sheen. What is the problem, and how do you correct it?
Problem: Stain precipitation, likely from the hematoxylin.
Corrective Action:
To fix the current slide: Rehydrate it, remove the hematoxylin staining with acid alcohol, rinse in water, and then re-stain the slide starting from the hematoxylin step.
Prevention: Filter the staining solutions regularly to prevent this from happening. (Alternatively, you may need to recut and re-stain the tissue).
“The eosin was not properly differentiated.” What does this mean, and what factors during tissue processing or staining can cause this problem?
This means the eosin was not adequately removed from the tissue during the dehydration steps, leading to a lack of contrast. Causes include:
Incorrect or inadequate fixation of the tissue.
Incorrect dehydration and clearing during the initial tissue processing.
Inadequate time spent in the low-grade alcohols during the staining/dehydration (DCM) process.
An incorrect pH of the eosin solution.
Your pathologist comments that the eosin on a slide was not properly differentiated. What is the corrective action for that specific slide?
Bring the slide back down to 95% alcohol. This will help remove (differentiate) the excess eosin.
Proceed with the rest of the dehydration, clearing, and mounting (DCM) protocol.
Your H&E slide shows uneven staining across the tissue. What are three potential causes related to reagent quality and equipment?
Water from the fixative may have contaminated the infiltrating paraffin during processing.
The alcohols used during processing or staining are contaminated with water.
The reagent levels in the processor or staining dishes are insufficient, meaning the slides were not fully covered.
Besides reagent contamination, what is another potential cause of uneven H&E staining, and what are the general corrective actions for these issues?
Cause: Equipment malfunction during the tissue processing cycle.
Corrective Actions:
Change reagents regularly to prevent contamination and depletion.
Check processing equipment for any malfunctions (e.g., improper vacuum, temperature, or fluidics).
A pathologist comments that there is poor contrast between the nucleus and the cytoplasm on your slide. What is the general approach to identifying and correcting this problem?
Diagnosis: First, determine which stain is inadequate. Is the nuclear stain too pale or too dark? Or is the cytoplasmic stain too pale or too dark?
Corrective Actions:
Once the weak stain is identified, adjust the timing for that step during re-staining.
Check the pH of the staining solutions (especially eosin) and adjust if necessary.
Monitor the quality of all solvents, including water and alcohols, to ensure they are not contaminated.
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Accessioning Quiz46
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Grossing Quiz45
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Processing Quiz58
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Cryostat Quiz16
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Decalcification Quiz10
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Embedding Quiz10
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Fixation Quiz20
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Fixation, Accessioning, and Grossing54
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IHC Quiz35
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Decalcification33
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Microtomy Quiz73
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Tissue Processing46
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Pigment Quiz40
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IHC33
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Embedding Troubleshooting42
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Microtomy Troubleshooting89
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GSM Troubleshooting25
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H&E Troubleshooting22
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PAS21
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Alcian Blue20
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Congo Red13
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Masson Trichrome19
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GSR13
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Oil Red O18
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VVG21
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Cryostat18
