Routine Staining Theory Flashcards
What dye pH is a basophilic tissue component attracted to?
Basic
True or False: Eosinophilic and Acidophilic are interchangeable terms when dealing with cell structures.
True
Describe the difference between euchromatin and heterochromatin.
Chromatin is the genetic material in the nucleus. Heterochromatin is stainable. Euchromatin is nonstainable. Heterochromatin is stainable because it is condensed, but it is that way because it is gene-poor. Euchromatin is gene-rich, unwound, and trancriptionally active.
Name 3 types of hematoxylins.
Ehrlich, Delafield, Harris, Mayer, Gill, Weigert, and Phosphotungtic Acid Hematoxylin.
Can eosin be differentiated?
Yes. Alcoholic eosin is usually differentiated in 95% alcohol.
What is the purpose of a mordant?
To help attach the dye to the tissue.
What are the 6 natural dyes? Which of these can be made synthetically?
Carmine, Indigo, Brazilin, Orcein, Saffron, and Hematoxylin. Indigo, Brazilin, and Orcein can be made synthetically.
Can limonene be used to deparaffinize slides as well as clear tissue on the processor?
Yes. Deparaffinization reagents include xylene, toluene, limonene, and aliphatic hydrocarbons. Limonene will take a little longer to deparaffinize.
How often should you change/rotate deparaffinizing xylene?
Depends on how many slide racks are being stained. Deparaffinzation reagents won’t work if they become saturated with paraffin.
Why can frozen sections be stained so much faster than permanent sections?
Frozen sections are often stained by hand with techs adding agitation. Most automatic stainers are unable to move slides faster than 30 seconds per reagent. Also, carryover is less concerning when doing fewer slides for rapid diagnosis.
At what point during slide staining is fixation pigment removal performed. What pigments can be removed?
After deparaffinization and rehydration but before staining. Formalin and Mercury pigments can be removed.
How do you remove the yellow color from tissue fixed with picric acid?
By washing in water or 50% alcohol.
Can you leave slides in water steps during staining if you are interrupted?
Yes, it is a holding step.
Can you leave slides in xylene if you are interrupted during manual staining?
Yes, no harm will come to the slides. It is a holding step.
Can you leave slides in differentiating reagents if you are interrupted during staining?
No! If you do this, some or all of the dyes will be removed.
It is not possible to “overblue” slides after hematoxylin staining. True or False?
True.
Can you leave slides in the bluing solution if you are interrupted during manual staining?
If you do, you have a higher risk of tissue sections “washing” or falling off the slides.
Can slides be left in the alcohol steps if staining is interrupted?
Depends on which alcohol steps. If slides are in 100% alcohol, then yes. If slides are in differentiating alcohol after eosin staining, then, of course, no.
What are some reasons that tissue may loosen or “wash” from slides during staining?
Poor fixation, too long in bluing solutions, tissue type, improper drying step (for FFPE).
How would the hematoxylin on stained slides look if they passed through depleted bluing solution?
Reddish purple. Slides will not be blued. This can be partially or totally.
What happens during tissue staining if the staining reagents are below the level of the tissue section?
The tissue will only be partially stained.
What can happen if you skip the water steps during tissue staining?
Understaining of hematoxylin, overstaining of hematoxylin, understaining of eosin, slides may not be blued.
What is a touch prep?
Touch preps are made by pressing a glass slide to fresh tissue to imprint some surface cells onto the slide.
Name 2 methods of staining touch preps.
Rapid H & E and Toluidine Blue.
What is a polychromatic dye?
A compilation of multiple dyes resulting in a range of stained colors.
Name a polychromatic stain.
Giemsa. Giemsa is a mixture of Methylene Blue, Azure, and Eosin. It is used to detect h. Pylori.
Explain orthochromasia, polychromasia, and metachromasia.
Orthochromasia: Tissue is electrostatically attracted to one dye or another, as in the H & E stain. Polychromasia: tissue is stained by a combination of dyes in one solution giving a range of “mixed” colors. Metachromasia:
One dye reacts markedly differently with various tissue components. Pictured is the polychromatic Wright’s Giemsa and the metachromatic Toluidine Blue.
What are the two main uses of Toluidine Blue in the histology laboratory.
Frozen section staining and staining for mast cells. Mast cells stained with Toluidine Blue are pictured.
Name 3 stains that use small bowel as control tissue.
Colloidal iron, mucicarmine, alcian blue, AB-PAS.
Hematoxylin is basic and stains acidic tissue components. Would those components be negatively or positively charged?
Acidic tissue components have a negative charge. (Remember DNA has an acidic, negative anal personality and gets the blues)
True or False: An eosinophilic/acidiphilic tissue component has a positive charge.
True
Is a negative charge cationic or anionic?
Anionic.
Does a positively charged tissue component bind to Eosin or Hematoxylin?
Eosin (Remember proteins are basically positive cats who are in the pink)
What is the word that refers to everything inside the cell membrane in both eucaryotic and procaryotic cells?
Protoplasm
What is a redox reaction?
An oxidation/reduction reaction. A chemical reaction involving the transfer of electrons. Oxidation adds oxygen and loses hydrogen. Reduction adds hydrogen and loses oxygen.
Explain the PAS reaction.
Certain carbohydrates are oxidized by periodic acid to aldehydes. These react with the Schiff reagent to reveal a magenta color upon washing in water.
Why is Schiff reagent clear?
Schiff reagent (also known as leucofuchsin) loses color when sulfurous acid is added to basic fuchsin and chromophores are masked. Washing in water breaks this bond and color is revealed.
Name 3 things that can be demonstrated with a PAS reaction.
Fungi, basement membrane, glycogen, senile plaques, amyloid bodies in prostate epithelium, and various polysaccharides and glycoproteins.
What fixative has the disadvantage od interfering with the PAS stain?
Gluteraldehyde.
What is the only natural dye that comes from an insect?
Carmine comes from the shell of the Cochineal Bug. Aluminum or iron is added to help bind with tissue, resulting in mucicarmine, which stains mucin in goblet cells.
What color is the stamen of the Saffron flower?
Yellow. It is a natural connective tissue dye.
What is the name of the reddish-brown dye made from lichens?
Orcein. Lichens are boiled to make orcinol, which is then oxidized to make orcein. It stains elastin and can be synthetically made.
Hematoxylin comes from the bark of the logwood tree and stains blue-purple. What other natural dye comes from tree bark, and what color does it stain?
Brazilin comes from Brazilwood bark, it can be synthetically made and it stains red.
Where do we get the deep blue color indigo?
From the Indigo Plant, though, it can be made synthetically.
Divide the 6 natural dyes into nuclear and cytoplasmic stains.
Carmine, Brazilin, and Hematoxylin are nuclear. Indigo, Orcein, and Saffron are cytoplasmic. All nukes are red.
True or False: If the water in your lab has a high pH, then you don’t need a bluing reagent.
True.
True or False: The basic chemical structure of all synthetic dyes is a quinone ring.
True.
Chromophores and auxochromes are added to quinone rings to form synthetic dyes. Which one gives it color?
Chromophore.
True or False: Adding more of the same chromophore will not change the shade of a dye.
False. If you add more of the same chromophore, you can alter the shade of a dye. To change the color entirely, you would choose a different chromophore.
What do auxochromes do when added to synthetic dyes?
Auxochromes help in dye binding, giving the dye its affinity to tissue components. That is a negative (anionic) or positive (cationic) charge.
True or False: ONLY chromophores can alter the shade of a dye.
False. Although auxochromes don’t provide color to a dye, when attached to a chromophore, it can affect its absorption of visible light and may alter the shade.
What is the difference between a dye and a chromogen?
Dyes have quinone rings with chromophores and attached auxochromes. Chromogens do not have the auxochromes and, therefore, do not have a charge.
True or False: Eosin can be either alcoholic or aqueous.
True.
What is the difference between Eosin Y and Eosin B?
The Y stands for yellow, and the B stands for blue. The alcoholic version of Eosin Y is the most popular version in histology labs.
Dyes that use oxidation/reduction reactions to switch between being colorless and colored are called what?
Leuko dyes. Benzene rings are colorless, so when a reaction changes a quinone ring back to a benzene ring, it will become colorless. The color will return when a benzene ring changes to a quinone ring.
What is a common leuko stain?
Periodic Acid Schiff.
Digestive enzymes breakdown nutrients into molecules that can be easily transported, stored and used by cells. Which of the enzymes listed is also used in the PASD reaction?
Amylase. The “D” in PASD is for digestion. You may have thought it was for diastase, which is more commonly used in this digestion step.
Auxochromes tend to be anionic. So, how do we make them attractive to anionic tissue components?
By adding a cationic (positively charged) chemical group-a mordant.
True or False: Technically all mordants are metals.
True. The term mordant is used loosely in histology to describe substances that bind dyes to tissue components.
Is iodine a mordant?
Not technically. It is not a metal, so it should not be called a mordant. It is technically a trapping agent, when used in the Gram stain, but histo techs just say mordant.
What is the term for a mordant bonding to a dye?
Lake. The etymology comes from the same “lac” as shellac.
What are the ways mordants can help the attraction between tissue and dyes?
3 ways: Onchrome-mordant first followed by dye. Metachrome-mordant and dye are mixed and then applied to tissue. Afterchrome-the dye is applied first, followed by the mordant.
What are methods, other than mordanting, to bind dye to tissue.
Substantive, Adjective, Impregnation, and Absorption.
Name some methods of differentiation.
Ionic charges, redox reactions, excess mordant.
True or False: Substantive staining methods bind dyes directly to the tissue, and Adjective staining methods work indirectly by treating the tissue first and then adding the dye.
True.
What is impregnation?
A method used with metallic dyes. The dye is deposited onto and not into the structure. With the dye on top of the structure, it can appear bigger.
True or False: Absorption is a method for attaching a dye to tissue.
True. It happens when the dye is more soluble in the tissue than the solvent.
Why do many pathologists prefer a silver staining method for h. pylori?
The organism is much easier to see, both because it is stained black and because the silver impregnation method makes the organisms appear larger.
Explain progressive vs regressive staining.
Progressive staining occurs when slides are left in a dye for a specific amount of time and then staining proceeds to the next dye. Regressive is deliberately overstaining and then pulling some dye back out by means of a differentiating solution.
What do Methylene Blue and Light Green have in common with alcoholic Eosin?
They are all counterstains, and they are all alcohol based.
What would happen if, after counterstaining your AFB in Methylene Blue, you began running it down to Xylene, but you got interrupted and left it in 95% alcohol?
The Methylene Blue counterstain would leach out. Methylene Blue is an alcoholic dye.
True or False: The isoelectric point of a protein is the point at which it is electrically neutral.
True
True or False: You can optimize a stain for a particular protein by changing the pH of the dye.
True. Most of the special stains in the histology laboratory are dependent on pH.
The pH of a dye can be adjusted relative to the isoelectric point of a protein to increase the intensity of a stain. How is this acheived?
By adding acetic acid to acidify the dye or by adding borax or sodium carbonate to make it more alkaline.
Name some staining protocols that are good examples of changing the pH of a dye to alter staining.
Trichrome, Congo Red, Alcian Blue
Name 3 blue staining nuclear dyes.
Hematoxylin, Crystal Violet, Toluidine Blue, Methylene Blue
Name 3 red staining nuclear dyes.
Carmine, Brazilin, Nuclear Fast Red, Neutral Red
True or False: Gill’s Hematoxylin will stain mucin in goblet cells.
True. Gill’s is the only hematoxylin that represents goblet cells. Weaker staining versions of Gill’s are used in cytology.
True or False: You can skip oxidation of hematoxylin to speed preparation.
False. Hematoxylin itself has little affinity for tissue. It MUST be oxidized to hematein.
True or False: 3 progressive steps in hematoxylin production are oxidizing, ripening, and aging.
False. Oxidizing, ripening, and aging are all the same thing, so 1 absolutely necessary step. But just 1 step.
True or False: Hematoxylin can be aged naturally.
True. Aging or oxidizing naturally is done with Erlich, Delafield, and PTAH. It takes 3 to 6 months.
Which chemical oxidizer is used in Mayer, Gill, and Harris hematoxylin?
Sodium Iodate.
True or False: Once hematoxylin is oxidized to hematein, it can be used to stain tissue.
Nope. Unfortunately, we still have to add a mordant to the dye to make a lake. This will influence the color and what tissue components are stained.
Name the 2 metals that are the main mordants for hematoxylin.
Aluminum and Iron. Aluminum gives a purplish-blue color, and iron gives a blackish-blue color.
Why do we filter hematoxylin?
Initially, to remove the green sheen, which is overoxidized hematoxylin. It can cause a precipitate on the tissue slides. It’s also a good idea to filter hematoxylin daily in order to remove debris from slides and/or carryover from other solutions.
True or False: Ferric Chloride is both the mordant and oxidizer for iron hematoxylins.
True. Both Weigert and Verhoeff hematoxylins use ferric chloride.
True or False: Iron hematoxylins last longer than aluminum hematoxylins.
Absolutely False. This is why iron hematoxylins are only used in special stains and usually discarded after each use. They last for a couple of days at best.
What is the mordant for Phosphotungstic Acid Hematoxylin?
Phosphotungstic Acid. Bonus Question: What is the oxidizer?
True or False: Eosin is a synthetic Xanthene dye.
True
True or False: Since proteins in the cytoplasm have an ISP (isoelectric point) of around 6.0, Eosin works best at a pH around 7.0.
False. Eosin must be acidic relative to the protein’s ISP to make it acidophilic. It works best at a pH between 4 and 5.
True or False: The optimal pH of Eosin is 4.5.
Absolutely true.
True or False: Formalin fixed tissue stains more intensely with Eosin than metallic fixatives.
False. Mercury, zinc, and copper fixatives stain more intensely and require less time in eosin.
What are the easiest tissue components to stain with eosin?
RBCs and Eosinophils will stain 1st and most intensely.
True or False: You should see 3 shades of eosin in well stained H and E slides.
True
Name a yellow cytoplasmic dye.
Saffronin, Metanil Yellow, Tartrazine
Name a green cytoplasmic dye.
Fast green, light green, malachite green.
True or False: When staining, tissue should be clear going through xylene, then become opaque as you enter the alcohols.
True.
When staining, what does it mean when the 95% alcohol gets cloudy?
There is xylene carryover.
True or False: Decalcified tissue needs a longer staining time in hematoxylin.
True
If a pathologist says there is a staining problem on all of his/her slides, what should you do?
Ask for particulars. Check to see when the slides were stained. Recheck the control slide for that day. Stop staining until the problem is solved. Change out necessary staining steps. Run another control slide. Restain his/her slides and check them before turning them in again. Check with any other pathologist to see whether restaining their slides is necessary.
Let’s say your working alone on a busy day, and a pathologist comes to the lab and tells you the hematoxylin staining is too light on the slides he just received. He says he wants them twice as dark. Can you just restain them by hand or run the slides through the autostainer again?
Yes, you can. You already know what the problem is. You need to stop staining any other slides on the stainer, remove the coverslips, and run the slides down to water. Begin staining at hematoxylin, run straight through the staining lineup, coverslip, hand to pathologist. What did you do next, and why didn’t you need to destain the slides first?
Explain what slides would look like if the person changing the stainer switched the positions of hematoxylin and eosin.
Hematoxylin would be lighter, smudgy, and reddish due not just to reduced timing and no bluing, but wrong pH going into the dye after alcohol steps and no differentiating. Eosin would be practically nonexistant, coming from water, then going back into water and, if that didn’t remove all the eosin, going into bluing solution would.
Which special stain is regularly used by hematopathologists?
A hematopathologist interprets work from the histology and hematology labs. He/she specializes in the lymphatic system and bone marrow. He/she looks at a lot of peripheral blood smears stained with Wright Giemsa.
What is a Wright Giemsa?
A stain used by both hematology and histology labs for peripheral blood smears.
What is a peripheral blood smear?
A slide made so that a hematopathologist can interpret blood cells and platelets.
What is the Giemsa stain used in the hematology lab?
Wright Giemsa
What type of stain is the Geimsa stain?
Romanowsky, which means it is polychromatic. It is used for blood, bone marrow, bacteria, malaria, and sometimes DNA. Some versions are Jenner, May-Grunwald, and Wright.
What fixatives are recommended for the Giemsa stain?
Zinc formalin or fixatives with Mercuric Chloride to get the best nuclear fixation.
Can you fix tissue to be stained with Giemsa in 10% NBF?
Yes. Most labs do.
What control tissue is used for the Giemsa?
Bone marrow, lymph node, or spleen. Spleen is shown.
What is a good control for mast cells?
Loose connective tissue anywhere in the body, especially the lamina propria of the GI tract and skin.
Name 1 example each of an orthochromatic, metachromatic, and polychromatic stain.
H&E, Toluidine Blue, and Giemsa.
What is the special stain that has all three primary colors?
The Gram stain.
Is Gomori’s One Step Trichrome metachromatic or polychromatic?
Polychromatic.
How many steps is Gomori’s One Step Trichrome?
Several. Tissue is mordanted in Bouin’s and stained with Weigert’s before the collagen stain, then it is differentiated after the stain.
How is the word argyrophil used in histotechnology?
To denote tissue components that can be impregnated with silver salts but need a reducing agent to give a visible metallic silver. Such as the GMS, Von Kossa, Warthin Starry, and Bielschowsky.
Liver, kidney, intestine, or fungus. What one stain might use any of these for a control?
PAS. Liver for glycogen, kidney for basement membrane, Intestine for mucin, and a control with fungus for fungus.
What is autofluorescense?
Autofluorescence is tissue that fluoresces on its own due to an endogenous element or a processing technique. (examples are red blood cells, collagen, and aldehyde fixatives)
What does endogenous mean?
Intrinsic, something originating internally. Exogenous would come from an outside source.
