HISTOPATHOLOGY LECTURE: STAINING
1. Staining is essential at arriving at a diagnosis
based on tissue examination
a. TRUE
b. FALSE
Most of the time, yes, that is true. Again, most cells are
not visible without being stained. But there are cases na
you don't need a stain to view cells.
 Does anyone have an idea or an example of a
case that does not need a stain to examine
cells?
o Pag sa tissue culture doc, where cells
are viewed under phase contrast
microscope.
o Metallic impregnation does not need
stains.
 Can someone tell me why there is a need to
rehydrate the section prior to staining?
o Because most of our stains are
hydrophilic.
o Most dyes are water soluble thus the
tissue need to be rehydrated for those
dyes to work
 When do we NOT rehydrate?
o if alcoholic stains are used
In either cases, whether we rehydrate or not,
remember that is necessary to first remove paraffin.
(I have a question doc bakit sa book doc need parin mag
undergo ng hydration even if alcoholic stain ang gamitin
pero until 70% alcohol lang?)
 because xylene is not miscible with low grade
alcohols. we stop at 70% because that is the
closest concentration in which our alcoholic
stains are miscible
2. Routine H&E staining.
a. Indirect, progressive
b. Indirect, regressive
c. Direct, differential
d. Direct, metachromatic
 why is routine h&e indirect and regressive?
o indirect because H&E makes us of a
mordant (aluminum salt) and regressive
because the nucleus is overstained and
later decolorized and counterstained.
o Indirect doc kasi hematoxylin needs a
mordant, like the iron in Wiegert’s
hematoxylin.
 what is the mordant in Ehrlich's hematoxylin?
o Potassium alum
 Differentiate orthochromatic and
metachromatic staining.
o the usual color of the dye is termed the
orthochromatic color; while
metachromatic is the altered color.
o Ortho - structures stained kay same
color sa dye mismo; Meta - diff color
ang structure sa color ng dye
o Orthochromatic: the color that the tse
imparts is the same with the dye
o Meta: the color that the tse imparts is
different from the dye
 Silver nitrate when applied to a tissue will
impart a brown to black colour. can we say this
is also metachromatic?
o Orthochromatic and metachromatic are
terms applied to dyes and stains. they
are absorbed by the tissue.
o Hence, metallic salts such as silver
nitrate are not absorbed and once
reduced, they are only attached to the
structures' surface as deposits and
precipitates.
o Brown to black color is due to oxidation
of the tissue due to the contact of silver
nitrate to tissue.
______________________________________________
CARBOHYDRATES
1. What is the major storage form of
carbohydrates in humans?
a. Cellulose
b. Glycogen
c. Starch
d. Mucopolysaccharides
 Give me two MAJOR organs where glycogen is
normally stored.
o Liver
o Skeletal Muscles
The other form of carbohydrate that we detect in
histopathologic specimens is mucin or mucus that are
secreted by epithelial cells and glandular cells.
HISTOPATHOLOGY LECTURE: STAINING
Both glycogen and mucin are stained by the periodic
acid Schiff (PAS) technique.
PAS is a type of HISTOCHEMICAL STAIN. Histochemical
stains take advantage of the chemical changes that
occur in the tissues once a chemical agent is applied to
them. Periodic acid OXIDIZES the 1,2 glycol group of
polysaccharides (such as glycogen) and of mucin in the
process, forming aldehydes.
the aldehydes then react with the Schiff reagent
(particularly the basic fuchsin in Schiff's reagent)
producing a magenta coloured product when rinsed in
water.
REMEMBER: NOT ALL CARBOHYDRATES WILL REACT
WITH PAS.
PAS will only be positive for those with the capability of
producing aldehydes once they are oxidized. Once we
have already stained the aldehyde-producing
substances (glycogen and mucin), we now proceed with
counterstaining to provide better contrast and
visualization.
 What counterstain do we usually use?
o Hematoxylin
Hematoxylin. With the sole goal of visualizing other
tissue elements aside from those that have stained with
PAS
“Naay nag answer ug Alcian Blue” (legwark ganern!)
coz…
 alcian blue acts as a differential stain to
distinguish acid mucopolysaccharides. PAS
muna tayo.
 What tissue substances would PAS be positive?
o Glycogen granules, mucin
o Glycolipids, glycoproteins
o Thin layers of reticular connective tissue
o Neutral mucins in GI tract
o Glycosaminoglycans and proteoglycans
o Fungal organisms
 Fungal cell walls have high
levels of carbohydrates that
stain positively with PAS (clap
clap Adriel Bacus)
Glycogen in Liver
HISTOPATHOLOGY LECTURE: STAINING

Abnormal glomeruli showing you thickenings of the

This is esophageal candidiasis
basement membranes
The structures there that are stained with magenta are
Candida albicans

A normal glomerulus with normal basement This is tubulocystic renal carcinoma.

membranes

 Polysaccharides
 Neutral mucus substances
 Tissue basement membranes
 Fungal organism
Those are the major substances that will be PAS positive

 QUESTION: what's the importance of diastase?

o Diastase hydrolyses and extracts starch,
glycogen, and breakdown products of
tissue polysaccharides po Doc
o Diastase can assist in the diagnosis of
glycogen storage diseases po :)
o it is glycogen-specific (KOREK!)
o since other substances can be PAS
positive too

For us to be sure that the stained component is

glycogen, we employ diastase.
HISTOPATHOLOGY LECTURE: STAINING
If initially it stains PAS positive, then negative after
diastase application, then the component we have
viewed is definitely glycogen.
For us to be sure that the stained component is
glycogen, we employ diastase.

If initially it stains PAS positive, then negative after

diastase application, then the component we have
viewed is definitely glycogen.

This is Pompe's Disease

Assignment: pathophysiology of Pompe's Disease and
how PAS can help us arrive at the diagnosis

Then we proceed to Best Carmine

 Is it really the best for glycogen?
o Nope. It is not as specific for glycogen
as PAS is.
It's just an alternative staining technique. PAS pa rin ang
For your comparison.
mas specific specially with diastase.
Specimen: liver
Before and after diastase application

 Where do we get diastase?

o Human saliva!
o Malt
“Pero which do we prefer? Saliva? Kay barato lang?”
 Malt doc kay para dali ra i standardiz!

“Doc, I have a question. Diba diastase (alpha-amylase)

breaks glycogen down only into shorter glucose
sequences, or perhaps into individual glucose monomers
gyud. But glucose molecules also have those 1,2-glycol
But it can also be employed in staining carbohydrates
groups diba, doc? Nganong mag-negative na siya sa
like in this photomicrograph. You have there the
PAS?”
Cyptococcus neoformans stained with carmine.
 The diastase breaks down glycogen to individual
glucose monomers and is lost in the process

Remember that after diastase application you wash the

section in running tap water. And glucose is water-
soluble. Therefore, it is removed during washing

Unlike in periodic acid reaction, they remain bound to

tissue even after washing because the carbohydrate
units have not been totally detached from the glycogen Glycogen in liver
macromolecule
HISTOPATHOLOGY LECTURE: STAINING
We now proceed to staining mucin
 So we have two general classifications of
mucopolysaccharides, what are these?
o Acid and neutral
That's where you'll find the acid mucopolysaccharides
as well.
“Unsa na tissue ang naa sa lower image?”
 Hyaline Cartilage

1. Which is NOT strongly positive with PAS?
a. Acid mucopolysaccharides
b. Neutral mucopolysaccharides
c. Glycogen
Lifted from your book:
Yup. Most likely hyaline. Medyo relatively clean ang
ECM. And in this case wala man ta nag gamit ug special
stain for the fibers sa elastic cartilage. Sige lang. Staining
on connective tissue with sir ian man mo. Review
beforehand ang normal histo ha.

Acid mucopolysaccharides are the ONLY large group of

carbohydrate compounds that are not strongly PAS
positive.

 However, we have a lot of stains for them at our

own disposal, what are these stains?
o Metachromatic staining
o Aldehyde fuchsin stain
o Colloidal iron technique
o Mucicarmine stain
o Alcian blue
A tissue stained with alcian blue and PAS-hematoxylin.
o Acridine orange

This is to differentiate between the neutral and the acid

mucopolysaccharides.

The acid mucopolysaccharides will be stained with

alcian blue. And the neutrals are stained with PAS.

The rest of the tissue is stained by hematoxylin

We'll skip Gomori's and Mucicarmine as they are not

used that much in the histopath processing but you
need to at least know them

ust enough for you to know that aldehyde fuchsin will

have greater affinity to sulfated mucins and is,
therefore, a differential stain against mucins with the
carboxylated forms

Mucicarmine is useful for fungal staining especially with

Cryptococcus neoformans
The stain used is toluidine blue.
 Therefore, this is?...
o Metachromatic stain
Correct. And look at the location sa stain.
HISTOPATHOLOGY LECTURE: STAINING
Next up: colloidal iron technique
Uses the principle of converting colloidal iron to ferric
ferrocyanide or prussian blue
Shown here is the general overview of the technique
Correction: neutral fast red
Lastly, fluorescent acridine orange technique
In this technique, mucin gives a brilliant orange
fluorescence.
This is candidiasis highlighted using acridine orange
 What's a known disadvantage for this
technique?
o it's temporary and will last for only
about 2 hrs.
o Fluorescent staining is temporary/short-
life
Last topic. Neutrals
Easy lang ni. Kung kinsa to dili magstain using our stains
for acid mucopolysaccharides, mao to atong neutrals.
And for you to be more confident that they are really
neutrals, stain them with PAS
To clarify: neutrals do not stain with alcian blue,
colloidal iron, mucicarmine or metachromatic dyes like
toluidine blue and azure A BUT THEY WILL STAIN WITH
PAS
The technique using alcian blue and PAS is actually a
means to differentiate them
For the preparation of the different stains we discussed,
I'll leave that to you and your lab instructors
Correction lang sa video:
I mentioned the "cytoplasm is acidic". That should be
basic.
I also forgot to insert potassium hydroxide as the
accentuator for Loeffler's methylene blue.
I apologize for the misinformation.
Have a good day. Enjoy the rest of the week
“END OF DISCUSSION”
Thank you Doc!