Histology quiz 6

incomplete paraffin removal can be caused by

insufficient time in xylene, water contamination in xylene, paraffin contamination in xylene

How to fix insufficient time in xylene during rehydration step

Treat with xylene x3, then 100% alcohol x3 and then decolorize with acid alcohol

How to fix slide after water has been left on the slide during the drying step resulting in contaminated xylene

Treat with absolute alcohol to dehydrate, then put into xylene to complete deparaffinization and then continue with rehydration step

What can cause uneven staining

Insufficient reagent levels, thick and thin sections when tissue was cut, inadequate processing due to water contamination

How to fix a slide that has been processed with insufficient reagent levels

Decolorize and restain

How to fix a slide that has thick and thin sections in the tissue from being cut

Troubleshoot microtome, recut and stain slide

How to fix a slide that has inadequate processing due to water contamination

Can attempt reprocessing but it is hard on the tissue

What causes water and slides to be milky after the deparaffinization step

Presence of xylene/formula 83 on the slides

How to fix slides that are milky after deparaffinization step

Reverse process the slide

What causes smudgy nuclear staining

Incomplete fixation, overheating of tissues

How do you fix a slide where the tissue has been incompletely fixed

Unable to fix

How do you fix tissue that has been over hardened

If it is a slide issue, recut and restain. If it is a problem with embedding, see if there is more sample left over to reprocess properly

What causes pale nuclear staining

Insufficient time in hematoxylin, hematoxylin is depleted, over differentiation with acid alcohol, section is too thin

How to fix a slide that has insufficient time in hematoxylin

Restain for the correct amount of time

How to fix a slide that has been over differentiated

Restain

What causes dark/overstrained nuclei

Too long in hematoxylin, under differentiation with acid alcohol, section is cut too thick

What causes nuclei to be red

Overoxidized hematoxylin, improper bluing,

What causes nuclear bubbling

Improper fixation, rushed oven drying of cut slides before staining

What causes pale cytoplasmic staining

pH of eosin is too high, over exposure to dehydrating alcohols or section is cut too thin

How to restore the pH of eosin

Adjust pH to 4.1-4.5 with acetic acid

What causes no eosin differentiation

Slide is not in dilute alcohols long enough after eosin, improper fixation, eosin has the improper pH

What causes cytoplasmic staining to be too dark

Eosin is too concentrated, section was left in eosin for too long, section was not in dilute alcohols long enough after eosin, section is cut too thick

What causes precipitate on top of sections

Precipitate in hematoxylin, possible formalin pigment

How to fix precipitate in hematoxylin

Filter stains before use

How to fix possible formalin pigment

Remove with alkaline alcohol or alcoholic picnic acid, decolorize and restain the slide

What causes water bubbles under the coverslip

Slides not completely dehydrated before clearing in xylene

What causes areas of the slide to be unfocused

Mounting media on top of coverslip, coverslip was not applied with good technique

What causes cloudiness under the coverslip

Water contamination in xylene

What causes “cornflaking” on the slide

Tissue partially dries before the coverslip is applied

What causes retracting mounting media

Warped coverslip, mounting media is too thin, air is trapped under the section