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H&E Trouble-Shooting
Lab
| Question | Answer |
|---|---|
| White spots in section after deparaffinization | Section not dried enough or not in xylene long enough; place in absolute alcohol then xylene or xylene longer |
| Nuclei too pale (hematoxylin light) | Not in hematoxylin long enough, hematoxylin overoxidized, differentiation too long; restain |
| Nuclei overstained (hematoxylin dark) | Too long in hematoxylin, sections too thick, differentiation too short, Decolorize, restain, check pH, rinse bluing cup, dehydrate quickly |
| Red, reddish-brown nuclei | Hematoxylin old, hematoxylin not ripened, insufficient bluing (can't overblue) |
| Pale staining w/ eosin | Eosin pH >5 (maybe bluing carryover), sections too thin, over-dehydrated |
| Cytoplasm overstained, differentiation poor | Eosin too conc'd (eosin-phloxine), section stained too long, insufficient dehydration, eosin in alcohols |
| Blue-black precipitate on top of sections | Overoxidzed hematoxylin; filter hematoxylin |
| Microscopic water bubbles in section | Incomplete dehydration; alcohol, xylene, recoverslip |
| Difficulty bringing some ares or tissue in focus w/ light microscope | Mounting medium on coverslip; recoverslip |
| Mounting medium retracted from edge of coverslip | Coverglass warped or mounting medium thinned w/ too much xylene; recoverslip, cap mounting medium |
| Water & slides turn milky following rehydrating alcohols | Xylene incompletely removed; change alcohols, dehydrate slides |
| Slides hazy & milky in last xylene | Water in xylene, change alcohol, redehydrate, change xylene |
| Mounted, stained sections don't show usual transparency & crispness under light microscope | Mounting medium too thick; recoverslip |
| Hazy blue nuclei | Too much heat on processor, too hot paraffin, under-fixed; begin dehydration w/ 65% to 70% |
| Uneven H&E staining, nuclei show poor chromatin detail | Water in clearant, water or fixative in infiltrating paraffin, solution levels insufficient; if open processor in high humidity use toluene instead of xylene, if closed processor check for malfunction |
| Dark basophilic staining of nuclei & cytoplasm, esp. around edges of tissue | Laser & electrocautery heat artifact, can't fix |
| Brown stippling resembling pigment & section shows glossy black nuclei | Section air-dried before coverslipping; remove coverslip, set in water several min, dehydrate, clear, remount |
| "Cornflaking" artifact caused by? | Section drying before it is coverslipped |
| Poor contrast between nuclei & cytoplasm | Understained nuclei, overstained cytoplasm; run control, check/change solutions |
| Less than three shades of eosin | Increase time in low dehydrating alcohols, best eosin differentiation occurs in 70% |
Created by:
CCF
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