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46 Cards in this Set
- Front
- Back
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Xanthene Dyes what kind of dye and what color? |
synthetic dyes with 3 rings (2 benzene rings) yellow, orange, red
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What kind of dye is eosin and
what percent solution is it? |
Acidic anionic dye (contains carboxylic acid and additional halogens)
0.5%-2% eosin (dissolved in 70% EtOH/aqueous) |
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pH of eosin |
cytoplasmic counterstain if pH is btw 4 and 5 (4.5 best) |
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how eosin works with IEP |
has a pH of 4.5 proteins have an IEP around 6 it is more acidic creates more positive charge on protein makes tissue more acidiophilic |
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Eosin accentuator Which chemical and what is it |
Acetic Acid is used to decrease the pH it increases the affinity of eosin in tissue components |
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how formalin fixation changes tissue
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combines with basic
leaves carboxylic acid radicals of proteins free eosin staining not as intense |
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how mercury zinc and copper fixation changes tissue |
combines with COOH radicals leaves H=NH2 side free staining of eosin more intense |
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how to reduce eosin intensity when you have fixed with something that has given the tissue a more positive charge |
dilute eosin (not practical) reduce time in eosin increase time in differentiating alcohols start in lower % of alcohol |
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order of eosin uptake |
1st: RBC, eosinophil granules 2nd: Muscle, epithelium cytoplasm 3rd: Collagen |
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regressive staining |
Use water in alcohols to pull out the dye Start in 70 (pulls out more water)-95% |
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how to remove more eosin from tissue |
start in a higher % of water, lower % of alcohol leave in 70% and 95% longer |
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order that eosin is removed from tissue |
1-collagen, lowest affinity 2-muscle 3-eosinophils and RBC if only rbc/eos either under stained or over differentiated |
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What is eosin sensitive to
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an alkaline pH
it is on the other side of the cytoplasmic IEP |
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what happens if ammonia is not removed from the slides |
it is alkaline you may come back years later and find eosin has faded |
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how could you remove accidental eosin stain |
weak alkali solution could use the bluing agent on the H&E setup
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what are two weak alkali solutions that you could use to remove eosin staining |
dilute ammonia Dilute lithium carbonate |
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what are the types of eosin |
Eosin Y Eosin B Erythroysin Erythrosin B Phloxine Phloxine B Rose Bengal |
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what are the characteristics of Eosin Y |
orange/yellow most commonly used cheapest 2nd strongest of xanthene dyes |
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what are the characteristics of eosin B |
red/orange <5% of labs use, not as cheap strongest of the Xanthene dyes |
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what are the characteristics of erythrosyin |
reddish color 3rd strongest dye aka eosin J available in Y more yellow available in B more blue |
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About Phloxine |
Rosey Magenta Commonly used with Eosin as a double cytoplasmic stain-RBCs darker red 4th strongest erytrosin BB 2 diff types Phloxine and Phloxine B |
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Rose Bengal |
Deep pink, not used very often Least strong of the xanthene dyes Can be used with bacterial staining and as a counterstain for hematoxlyin so increase time |
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List the natural cytoplasmic dyes |
Saffron Hematoxlyin-Phloxine -Saffron (HPS) |
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About Saffron |
Natural made from the dried stigma of flower its an acidic dye dissolved into an alcohol solution, keeps covered don't want it to evap, stains collagen yellow |
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Hematoxlyin-Phloxine-Saffron (HPS) |
Very expensive Stains-nuclei blue, RBC, muscle and cell cyto pink/red, collagen and bone yellow |
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List the red synthetic cytoplasmic dyes |
Biebrich scarlet Acid Fuchsin Ponceau de xlyidone, ponceau s Chromotrope |
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List the synthetic yellow dyes |
Picric Acid Tartazine (Yellow # 3) Mentanil Yellow Martius yellow |
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List the Green cytoplasmic dyes |
Fast green Light green SF Yellowish Malachite green |
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List the blue cytoplasmic dyes |
Aniline blue Methylene blue |
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What is used to deparaffinize |
hen pxylene toluene limonene aliphatic hydrocarbons 3x can do longer |
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Purpose of hydration |
Removes xylene from slides to hydrate slides, most staining solutions are aqueous Start in absolute alcohol, introduce water Different blocks will have different protocols best around a minute, could to 15-20s with agitation |
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How to remove formalin pigment |
saturated alcoholic picric acid alcoholic ammonia solution |
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How to remove Mercury pigment |
Lugolize/dezenkerize use weigert or lugol iodine solution 5m Place in 5% sodium thiosulfate to remove brown color from iodine Rinse in running tap for 1-10m |
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How to remove picric acid pigment |
Need to remove yellow color Wash in running tap for 1-10m |
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Why may you need to go into distilled water before hematoxylin |
Sometimes pH of running water is very alkaline or acidic, also tap water may contain impurities. Will interfere with hematoxylin staining May need to do everything in distilled water if endemic bacteria |
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What affects the staining time of hematoxylin |
Age of solution Strength of solution Pathologist preference Fixative used Decal tissue Tissue thickness Progressive vs regressive |
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Warm water rinses do best to remove what |
Aluminum salts from the slide |
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When is differentiation preformed? |
When doing a regressive stain (removes non-specific collagen mucin and nucleoplasm stain)
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What can be used to speed up differentiation |
HCl rather than acetic acid Aqueous is faster that alcoholic acid |
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What can be used to blue? |
Dilute ammonia Dilute lithium carbonate Scotts tap regular tap |
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Why do you have to remove all of the bluing agent |
it will carry over and could alter the pH of eosin and cause poor staining |
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Why do we go into 70% alcohol before eosin |
bc eosin is made up with 70% and don't want to dilute the alcohol in eosin. 10 s with agitation or 1m without also HOLD |
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What factors does dehydration depend upon? |
Vary based on time spent in eosin Longer time in alcohol, the more eosin removed If agitating would scale back time |
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What steps effect staining |
Patient treatment, Obtain tissue, grossing, fixation, decal, processing, embedding, microtomy, slide drying, medical condition, clean glassware, deparaffinze, alcohols, water, pigment removal, staining, counterstaining, dehydrate, clear, mounting media, coverslip, slide storage |
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IEP of nucleic acids and hematoxylin |
nucleic acids=1.5-2.0 hematoxylin=2.2-2.9 |
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IEP of cytoplasmic proteins and eosin |
cytoplasmic=6 eosin= 4-6 |
