Pathology Special Stains for FFPE Tissue StainingBioGenex
Special stains refer to alternative staining techniques that are used when H&E stains do not provide all the cellular information required. These techniques use a variety of dyes and methods so that pathologists can visualize tissue morphology and detect the presence of particular cell types, structures or pathogens (e.g. bacteria).BioGenex offers broadest special stain menu anywhere (over 30 special stains), including:
Grocott’s Methenamine Silver (GMS) Stain
Reticulin Stains
Trichrome Stains
Giemsa Stain
Periodic Acid-Schiff (PAS) Stains
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journey
Special stain in histopathology
1. Special stain in HIstopathology
Presented by Dr. Mahesh
Guide: Dr. S. P. Hiryur
- Associate Professor
2. • Special stains are used to identify certain normal and
abnormal substance present in the cells and tissue,
which can not be identified on routine
Haematoxylene & Eosin staining or are better
appreciated on special stain.
3. Periodic Acid-Schiff (PAS) stain
• Principle:
Substance containing vicinal glycol groups
or their amino or alkylamino derivatives are
oxidized by periodic acid into dialdehydes which on
reaction with schiff’s reagent give insoluble purple-
magenta compound.
• Dyes : 1%periodic acid
Schiff’s reagent
• Control : Appendix
• Result :
PAS +ve substance : Purple –magenta color
Nuclei : Blue
5. • PAS Diastase reaction : Glycogen is diastase
sensitive, hence section containing glycogen
when pretreated with diastase, the enzyme
will digest the glycogen and will give
negative PAS reaction.
• The purpose of using PAS with diastase
staining procedure is to differentiate
glycogen from other PAS positive elements
such as mucin that may be present in the
tissue sample.
6. Pocedure :
•Dewax the section and bring to water level
•Dip the slide in copplin jar containing Per iodic
acid for 10 min
•Wash well in tap water for 15-20 min
• Dip the slide in coplin jar containing Schiff
reagent for 20 min
•Rinse in distilled water
•Counter stain with hematoxylene for 5-10
seconds
•Wash in tap water
• Clear in xylene and mount in DPX.
7. • Uses of PAS:
• PAS is used to demonstrate glycogen and neutral
mucoprotein.
• In diagnosis of poorly differentiated adenocarcinoma of
various tissue like stomach, pancreas, lung.
• In diagnosis of hepatocellular carcinoma.
• Seminoma, Dysgerminoma
• RCC- Renal cell carcinoma: Clear cell type
• To demostrate – intracytoplasmic crystal in alveolar soft
part sarcoma
• To demostrate basement membrane e.g. adenoid cystic
ca.
• To demostrate neutral mucin in gastrointestinal tract
8. • To demostrate fungi in tissue sample which are positive
due to high carbohydrate content in their cell wall
like. Candidiasis
Actinomycosis
Histoplasmosis
Blastomycosis
• It can be used to confirm the metastatic malignancy.
Finding mucin positive tumor cells in an area that does not
contain mucin producing cells would indicate the tumor
did not arise from that area.
• In diagnosis of ALL- block positivity and AML- diffuse
cytoplasmic positivity in M5, M3 and cytoplasmic
granular/block positivity in M6 and cytoplasmic granular
and blebs positivity of blast in M7.
11. • Presence of glycogen will be evidenced by loss
of staining after enzyme treatment when
compared to the untreated sections.
PAS StainPAS with Diastase
12. Mucin
• Mucins are high moleqular weight substance containing
acidic groups and divided into two major categories-
epithelial and stromal.
• Epithelial mucin (membrane bound or secreted) are
composed of central protein core and sialic acid
containing chains of carbohydrate(polysaccharides)-
that may be sulfated or nonsufated.
• Stromal mucin known as glycosaminoglycans contain
hyaluronic acid may be sulfated or non sulfated. They
are better known as myxoid substance.
• Their functions are lubrication, chemical barrier and cell
signaling.
13. The types of mucin are as follows:
Neutral - Found in glands of stomach and in prostate. They
stain with PAS but not with Alcian blue, colloidal iron and
mucicarmine.
Acid (simple, or non-sulfated) - Are the typical mucins of
epithelial cells containing sialic acid. They stain with PAS,
Alcian blue at pH 2.5, colloidal iron. They resist
hyaluronidase digestion. E.g. salivary glands.
Acid (simple, sulfated -mesenchymal) - These contain
hyaluronic acid and are found in tissue stroma. They do
not stain with PAS, but do stain with Alcian blue at pH 2.5,
colloidal iron, and metachromatic dyes. They digest with
hyaluronic acid. They can be found in sarcomas.
14. • Acid (complex, epithelial) - These are found in
adenocarcinomas of colon, breast,ovary, mucoepidermoid
carcinoma . PAS is usually positive. Alcian blue is positive at
pH 1, and colloidal iron, mucicarmine, and metachromatic
stains are also positive. They resist digestion with
hyaluronidase.
• Acid (complex, connective tissue) - Found in tissue stroma,
cartilage, and bone and include substances such as
chondroitin sulfate or keratan sulfate. They are PAS
negative but do stain selectively with Alcian blue at pH 0.5.
15. Stains for mucin
• Alcian blue
• PAS
• Mayer mucicarmine
• Hale colloidal iron stain
16. Alcian blue
• PRINCIPLE
• Alcian blue is a copper phthalcyanin dye and contains
positively charged groups capable of salt linkage with
certain polyanions. These polyanions consist of the
sulphate and carboxyl radicals of the acid mucins
and the phosphate radicals of the nucleic acids that
do not react. Consequently, only the acid mucins are
stained. By varying the pH of the solution of Alcian
blue more information can be gained concerning the
types of acid mucin present.
17. Alcian blue
• At pH 2.5 it stains – Acidic mucin that include acid-simple or
non-sulfated and acid –simple mesenchymal mucin.
• At pH 1 it stains acid-complex or sulfated mucin and at pH
0.5 it stains acid-complex connective tissue mucin.
• It does not stain neutral mucin.
• Dyes : 1%Alcian blue in 3%acetic acid
Results : Acid MPS – deep blue
Nuclei – faint blue
• PAS-Alcian blue is best ‘pan-mucin’ stain combination, since it
demostrates mucin both neutral, slightly acidic and highly
acidic.
18. REAGENTS:
3% Glacial Acetic Acid
Alcian Blue Solution:
3% glacial acetic acid-
100.0ml
Alcian blue -1.0 gm
Mix, adjust pH to 2.5, using
acetic acid. Filter, add a
crystal of thymol, label with
initial and date.
Nuclear Fast Red
(Kernechtrot):
Aluminum sulfate 25.0 gm
Distilled water 500.0 ml
Nuclear fast red 0.5 gm
Dissolve the aluminum
sulfate in the water. Add
the nuclear fast red,
dissolve
with aid of heat. Filter,
add a crystal of thymol
SPECIMEN
Standard paraffin section fixed in neutral buffered formalin
19. PROCEDURE: ( Alcian Blue)
1. Hydrate slides to distilled water.
2. Stain with3% acetic acid, 3 minutes.
3. Stain with alcian blue solution, for 30 seconds.
4. Wash in running water for 2 minutes, rinse in
distilled.
5. Nuclear-fast red, 5 minutes, wash in tap water.
6. Dehydrate, clear, and mount.
20. USES
• It is useful to differentiate gastric metaplasia into
complete and incomplete. Complete metaplasia is
small intestinal type, shows well defined brush border
and well formed goblet cells. Whereas incomlete
metaplasia is colonic type and shows multiple
irregular mucin droplets of varying size in the
cytoplam and absence of brush border.
• It is useful in diagnosis of salivary gland pleomorphic
adenoma, mucoepidermoid carcinoma, in stroma of
chordoma at sacrococcygeal region.
• Excessive amounts of non-sulfated acidic
mucosubstances are seen in mesotheliomas, certain
amounts occur normally in blood vessel walls but
increase in early lesions of atherosclerosis.
21. The goblet cells of the gastrointestinal tract are filled with
abundant acid mucin and stain pale blue with this Alcian blue stain.
22. Colonic mucosa showing sialomucin that have acid and
neutral mucopolysaccharide stain purple on alcian blue-PAS.
23. Mixed salivary gland. Acid mucopolysaccharides in mucous
cells are stained blue (Alcian blue at pH 2.5).
25. MUCICARMINE STAIN –SOUTHGATE’S – MUCIN
PRINCIPLE :-
Positively charged carmine-mucicarmine complex
bonds with the negatively charged acid mucin. The
carmine solution is highly complex. Strongly
sulphated mucins are variable in their reaction,
neutral mucin do not stain at all, while other acidic
mucin stain strongly.
27. PROCEDURE: ( Mucicarmine stain)
1. Deparaffinize and hydrate to distilled water.
2. stain with mayer's hematoxylin for 2 minutes.
3. Wash in running tap water.
4. Differentiate in acid alcohol.
5. Rinse in tap water.
6. Blue in scott’s tap water substitute.
7. Wash in running tap water.
8. Stain with mucicarmine solution for 20
minutes.
9. Wash in running tap water.
10. Dehydrate, clear and mount.
28. Uses:
• Mucicarmine is useful to identify acidic mucin.
• It is used to identify adenocarcinoma particularly
of gastrointestinal tract.
• It also stains the capsule of fungus- cryptococcus
neoformans found in lung and nervous tissue of
immuno-compromised patient.
29. Colonic Mucosa showing sialomsucin --
- magenta
RESULTS:
Mucin - magenta
Nuclei - black
Other tissue elements- yellow
31. HALE’S COLLOIDAL IRON STAIN
• Positive staining with hale’s colloidal iron
stain is considered diagnostic feature of
chromophobe renal cell carcionoma and has
been used as discriminatory feature to
differentiate it from other renal tumour.
Result
• Acid mucopolysaccharides: blue
• Nuclei: red
33. Procedure: (Hale’s colloidal iron stain)
• Bring the section to water level.
• Place into working colloidal iron for 15-20 minutes.
• Wash with distilled water.
• Wash with running tap water for 5 minutes to remove all
traces of colloidal iron.
• Wash with distilled water.
• Place into freshly made perls’ solution for 10 minutes.
• Wash with distilled water.
• Counter stain with neutral red for 1 minute.
• Dehydrate, clear ad mount.
34. a.Classic chromophobe RCC
b. classic chromophobe RCC
on hale’s colloidal iron stain
c. Eosinophilic chromophobe
d. Eosinophilic chromophobe
on hale’s colloidal iron stain
e.oncocytoma
f. oncocytoma diffusely
negative cytoplasmic
reaction with hale‘s colloidal
iron stain
35. RETICULIN STAIN-GOMORI’S METHOD
• Reticulin stain demonstrate both reticular fibers
and basement membrane material.
• Reticular fibers consist of very thin fiber of type
III collagen which are widespread in connective
tissue throughout the body.
• Basement membrane are largely composed of
type IV collagen and laminin.
36. Principle:
Reticulin fibers have little natural affinity for
silver solutions. On treatment with potassium
permangenate it produce sensitised sites on fibers
where silver deposition can be initiated. The silver is
in the form readily able to precipitate as metallic
silver. The optimal ph for maximum uptake of silver
ions is 9.0. A reducing agent formalin causes
deposition of silver in the form of metal.
37. • Dyes used: Silver nitrate 10%
NaOH 10%
KMnO4 1% aqu.
oxalic acid 5% aqu
Iron alum 2.5%
Formalin 10%
• Control: Cirrhosis of liver
• Result: Reticular fiber – Black
Nuclei- Gray
Other elements- According to counter
stain used
38. Pocedure :
•Dewax the section and bring to water
•Treat with 1% potassium permanganate solution for 3 min
•Rinse in tap water
•Bleach in 5% oxalic acid solution for 2 minute
•Wash well in tap water
•Treat with 2.5% iron alum for 1 min
•Rinse in distilled water
•Impregnation with ammonical silver solution for 3 min
•Distilled water wash
•Reduce in 10% aqueous formalin with agitation for 3 minute
•Wash in tap water
•Dehydrate ,clean and mount.
39. Uses of reticulin stain
1. Diagnosis of liver cirrhosis.
2. To distinguish epithelial neoplasms from non-
epithelial neoplasms.
Foci of carcinoma have reticulin around tumour
nest but not in between tumour cell, whereas in
most sarcomas and large cell lymphoma reticulin
separates single cells.
3. To differentiate between in-situ and invasive
carcinoma
41. Trichrome stain
• This stain is mainly used to evaluate the type
and amount of extracellular material like-
collagen, fibrin, muscle and elastic fiber.
Various technique includes:
• Masson trichrome stain
• Van gieson stain
• Mallory, Phosphotungstic or phosphomolybdic
acid stain
• Verhoeff-Van Gieson(VVG) stain
42. The general rule in trichrome staining is that
the less porous tissues are colored by the
smallest dye molecule; whenever a dye of large
molecular size is able to penetrate, it will
always do so at the expense of the smaller
molecule.
43. Masson trichome stain
• This method is used for detection of collagen
fibers in the tissues such as skin, stomach,
intestine and lung.
• The collagen fibers will be stained blue and their
nuclei will be stained black.
44. Masson trichrome stain
Principle:
As per the name 3 dyes are used which
selectively stain muscle, collagen fibers, fibrin
and erythrocytes. As general rule in trichrome
stain less porous tissues are stained by small
dye molecule. Acid fuchsine stain all the
connective tissue, PMA( phosphomolybdic
acid) competes with fuchsine and gain access
to collagen displacing fuchsine. If reaction
stopped at appropriate time, collagen will be
free to be stained by Methyl Blue.
45. • Reaent used – Weigerts iron hematoxylin solution
- Phosphomolybdic- phosphotungstic
acid solution
- Biebrich scarlet acid fucshin solution
- Aniline blue solution
- 1% acetic acid solution
• Result - Nuclei : Black
Collagen : Blue
Cytoplasm, Muscle, RBC : Red
• Positive control : skin, lung, stomach, intestine
46. Pocedure: Masson trichrome stain
•Dewax the section and bring to water
•Wash in tap water
•Stain with weigerts iron hematoxylene for 10 minute
•Rinse well in running tap water for 10 minute
•Wash in distilled water
•Stain in biebrich scarlet acid fuchsin for 10-15 minute
•Wash in distilled water.
•Differentiate in Phosphomolybdic-phosphotungstic acid
solution for 10-15 minute
•Transfer section directly(without rinse) to aniline blue
solution and stain for 5-10 minute
47. •Rinse briefly in distilled water and differentiate in1%
acetic acid solution for 2-5 minute
•Wash in distilled water
•Dehydrate through alcohol, clear in xylene and mount in
DPX.
49. Uses of Masson trichrome stain
• It is used to differentiate between collagen and smooth
muscle in tumour.
• To identify increased collagen deposition in condition
like cirrhosis, keloid, benign prostatic hyperplasia,
membranoproliferative glomerulonephritis etc.
50. VAN GIESON STAIN
Principle
When using combined solution of picric acid and
acid fuchsin, the small molecules of picric acid penetrate all
the tissue rapidly, but are only firmly retained in the close
textured red blood cells and muscle. The larger molecules
of ponceau S displaces picric acid molecule from collagen
fibres, which has larger pores and allow larger molecules to
enter.
It is used for detection of collagen.
Result -- Nuclei : Blue / Black
Collagen : Red
Cytoplasm, muscle, fibrin, RBCs : Yellow
51. • Results:
• Collagen – deep red
• Muscle, – yellow
cornified epithelium
• Nuclei – blue to black
52. Phosphotungstic acid- Hematoxylene
test (PTAH) test:
• Principle
There is much more phosphotungstic acid in the
solution then hematein. The phosphotungstic acid binds all
the available hematein to form blue lake pigment. This lake
stains muscle cross striations, fibrin, nuclei, and other
tissue elements blue. The rest of phosphotungstic acid
stains the red-brown components, such as collagen.
USES
• Traditionally used for demonstration of muscle striations,
glial cells and fibrin.
• This technique has been largely replaced by
immunohistochemistry techniques
53. RESULTS
• Muscle, cross striations : Blue- black
• Fibrin and neuroglia – Deep blue
• Connective tissue: Pale orange-pink to brownish red
• Bone and cartilage- yellowish to brownish red
56. Verhoeff-Van Gieson(VVG) stain :
• This method is used for identifying elastic fiber in tissue
such as skin, aorta etc.
• Result – Elastic fiber: Blue-black to black
- Nuclei: Blue to black
- Collagen: Red
- other tissue elements: Yellow
58. This section shows elastic cartilage and elastic fibers (arrow),
which are dark-stained linear structures embedded
in the cartilage matrix.
59. Fibrin
• Fibrin is formed by polymerization of smaller
soluble fibrinogen.
• Found in tissue damage and acute inflammation,
fibrinoid necrosis in vessel wall.
• Stained by : Mallory PTAH
MSB Stain
60. MSB (Maritus, Scarlet, Blue)Stain for
fibrin:
• This is trichrome method for selective demostration of
fibrin.
• Dyes Used :
Maritus Yellow
Crystal Scarlet
Methyl Blue, PTA.
• Results :
Fibrin : Red
RBC’S : Yellow
Muscle : Red
Collagen : Blue
63. Stain for Amyloid
Amyloids are insoluble fibrous protein. They arise
from inappropriately folded protein and polypeptides
present naturally in the body. These misfolded structure
alter their configuration and interact with each other or
other cell components forming insoluble fibrils. Abnormal
accumulation of amyloid fibrils leads to amyloidosis and
play role in various pathological disease.
Stains used to demostrate amyloid:
• Congo red
• Crystal/methyl violet
64. Congo red stain
Principle :
Amyloids are homogeneous and eosinophillic,
proteinaceous deposits, that are extracellular and
may become sufficiently large enough to cause
damage to surrounding tissues. When stained with
the congo red stain the amyloid will show
birefringence an apple green color, under the
polarizing microscope.
65. Reagent:
• Solution a - 0.5% Congo red in 50% alcohol
• Solution b - 0.2% potassium hydroxide in 80%
alcohol
• RESULTS:
Amyloid, elastic fibers, --- red to pink
eosinophilic granules,
Nuclei – blue
66. PROCEDURE: (Congo red stain)
1. Deparaffinize and hydrate to water.
2. Stain in Congo red solution for5 minutes.
3. Differentiate with alcoholic potassium hydroxide
solution for 3-10 seconds.
4. Wash in water, stain nuclei in alum hematoxyllin,
differentiate and blue.
7. Dehydrate, clear in xylene & mount.
67. It is used to demostrate amyloid deposits in
• renal amyloidosis- in patients on dialysis for
long time
• Medulary carcinoma of thyroid,
• Vessel wall in case of Alzheimer’s disease
• Cardiac arrhythmias, etc.
68. Positive red staining is present around the large central artery and
a smaller vessel to its upper right. The right panel shows the green
birefringence that is diagnostic of amyloid when the Congo red
stain is viewed with polarized light.
All amyloids have a fibrillar ultrastructure that gives this reaction.
69. Crystal/Methyl violet for amyloid
• Crystal violet or methyl violet stain are used for
metachromatic amyloid staining.
• They stain amyloid as purple-red in blue background.
Reagents used
• Crystal/methyl violet
• 95% alcohol
• 1% aqueous ammonium oxalate
• 0.2% acetic acid
70. Preparation of solutions
• Dissolve 2 gm crystal/methyl violet in 20ml 95%
alcohol and 80ml of 1% aqueous ammonium oxalate
dissolve using minimum of heat. Cool and filter
71. Procedure
• Bring the section to water level.
• Stain in crystal/methyl violet solution for 5 minute.
• Wash and differentiate in 0.2% acetic acid controlling
differentiation microscopically arresting differentiation
in water and repeating untill good contrast is obtained
between amyloid and background.
• Wash and mount in DPX.
RESULT
Amyloid - Purple
Backgrond - Blue
75. Oil red o stain
PRINCIPLE :
Staining with oil-soluble dyes is based on
the greater solubility of the dye in the lipid
substances than in the usual hydroalcoholic
dye solvents.
Result
Lipid – Red
Nuclei- Blue
76. • REAGENTS :
• 60% isopropanol
• Alum hematoxylin:
• Glycerin Jelly mounting medium
• Oil Red O working solution:
o To make oil red O stock solution
• Oil red O - 0.5gm
• Isopropanol 100 ml
Dissolve the dye in isopropanol, using the very
gentle heat of water bath. This is the stock solution.
o To make working oil red O solution
• Dilute 30ml of stock stain with 20ml of distilled water
and allow it to stand for 10 minutes, filter into coplin jar
and cover immedeiately.
• Working solution should be made fresh each time.
77. PROCEDURE : (Oil red O stain)
• Fix slides in 10% formalin if fresh.
• Wash well in tap water for 1-10 minutes to drain off
excess water.
• Rinse with 60% isopropanol.
• Stain with freshly prepared oil red O working solution
for 15 minutes.
• Rinse with 60% isopropanol.
• Lightly stain nuclei with alum hematoxylin 5 dips.
• Rinse with distilled water.
• Mount with aqueous mounting media, Glycerin Jelly.
78. Fat emboli seen as red dot within capillaries of lung on Oil red
O stain
80. Uses
• Oil red O stain is used for staining neutral
triglycerides, lipids and lipoprotein.
• Tumors arising from fat cells (liposarcomas) can be
differentiated from other types of tumors.
• Fat occurring in an abnormal place, such as fatty
emboli that may develop after either a bone fracture
or an injury that crushes a fatty body area.
• Lipid storage disorder like nieman-pick disease
• To demonstrate fat or lipids in conidition like fatty
liver.
• In hematological condition like burkitt’s lymphoma
and sea-blue histiocytic syndrome
81. FAT - SUDAN BLACK B - PROPYLENE GLYCOL
PRINCIPLE :
Sudan Black B is a dye that is insoluble in water but
dissolves in fat. Therefore this dye will accumulate in fat
globules within cells.
• It is slightly basic dye and will combine with acidic
groups in compound lipids, thus staining phospholipids
also.
Result
Fat- Blue/ Black
Nuclei- Red
82. REAGENTS :
• 85% Propylene Glycol:
Propylene glycol -85.0 ml
Distilled water -15.0 ml
• Hematoxylin:
• Sudan Black B/Propylene:
Sudan Black B -0.7 gm
Propylene glycol -100.0 ml
83. PROCEDURE : ( Sudan black stain)
1. Fix slides in 10% formalin if fresh.
2. Wash well in tap water, rinse in distilled water, drain
off excess water.
3. Propylene glycol, two changes, 5 minutes each.
4. Stain with Sudan Black, 7 minutes, agitate.
5. Dip in 85% Propylene glycol, 3 minutes.
6 Rinse in distilled water.
7. Stain with Nuclear Fast Red, 3 minutes.
8. Wash in water.
9. Wash in tap water, rinse in distilled.
10. Mount with aqueous mounting media, Glycerin Jelly.
84. Uses
• Sudan black is used for staining neutral
triglycerides, lipids, lipoprotein and phospholipid.
• In hematological disorder it can be used to
differentiate blast as it stains the myeloblast, but
does not stain lymphoblast.
86. MASSON FONTANA METHOD- FOR MELANIN
Melanin is a nonlipid, non hematogenous
pigment. It is a brown-black pigment present
normally in the hair, skin, retina, iris and certain
parts of the CNS.
PRINCIPLE:
Melanin is insoluble in organic solvents but soluble in 1M
sodium hydrooxide.
It is slowly bleached by strong oxidising agents.
The solutions of ammoniacal silver nitrate are reduced by
melanin to black metallic silver this is the basis of Masson
Fontana method for demostrating melanin.
87. USES:
• To identify melanin and argentaffin
granules.
• In diagnosis of malignant melanoma
• Argentaffin granules are found in
carcinoid tumors.
88. Melanin pigment of skin showing
RESULTS:
Melanin, argentaffin
granules - Black
Nuclei -red
90. VON KOSSA METHOD FOR CALCIUM
PRINCIPLE:
Tissue sections are treated with silver nitrate
solution, silver is deposited by replacing the the
calcium and then it is reduced by the strong light
and visualized as metallic silver.
USE:
Abnormal deposits of calcium may be found in any
area of the body. With the H&E stain, calcium
appear deep blue-purple. On von kossa method it
appear black.
92. PERL’S- PRUSSIAN BLUE REACTION FOR
IRON
PRINCIPLE :
Dilute mineral acid hydrolysis releases ferric
iron from protein bound tissue deposits, which in
the presence of ferrocyanide ions, is precipitated as
highly coloured and highly water soluble complex,
potassium ferric ferrocyanide- prussian blue.
• Ferrous ion do not produce a coloured reaction.
• Tissue deposits containing ferric iron are invariably
hemosiderin
93. USE:
• To demonstrate ferric iron in tissue sections.
• Small amounts of iron are found normally in
spleen and bone marrow.
• Excessive amount are present in
hemochromatosis- with deposits found in the
liver and pancreas and hemosiderosis- with
deposits in the liver, spleen, and lymph nodes.
• To access the bone marrow iron content
94. REAGENTS:
• 2% Potassium Ferrocyanide:
Potassium ferrocyanide - 2.0 gm
Distilled water - 100.0 ml
• Neutral-fast Red:
Neutral red - 1gm
Distilled water - 100ml
Glacial acetic acid - 1ml
• 2% Hydrochloric Acid:
Conc. Hydrochloric acid, - 2.0 ml
Distilled water - 100.0 ml
CONTROL: Postmortem lung – high number of heart
failure cells that contain hemosiderin
95. PROCEDURE: ( Perl’s prussian blue)
1. Deparaffinize and hydrate to distilled water.
2. Stain with mixture of equal volume of aqueous 2%
potassium ferrocyanide and 2% HCl solution for 30
minutes.
3. Thorough wash with distilled water.
4. Counter stain with 0.5% Neutral red lightly.
5. Wash in tap water.
6. Dehydrate, clear, and mount.
98. Ziehl-Neelsen (ZN) Stain for Mycobacterium
Bacillus
Mycobacteria are difficult to demonstrate by the
Gram technique because they possess a capsule
containing a long chain fatty acids that make
them hydrophobic.
Phenolic acid or heat may be used to reduce the
surface tension and increase the porosity.
99. • PRINCIPLE
Mycobacterias (tubercle bacilli) have a lipid-rich cell
wall which is capable of taking up strong phenol dye
solutions (eg. carbol fuchsin solution) such that the
dye is retained upon subsequent differentiation in
acid or alcohol. They are said to be acid and alcohol
fast (AAFB = acid and alcohol fast bacilli).
Results
• Mycobacteria - Red
• Background - pale Blue
100. Method
• Bring the section to water level.
• Flood sections with carbol-fuchsin and heat to steaming
by passing the flame of spirit swab underneath the slides
on metal rack till, vapour just being formed.
• Wash the slides in distilled water. Shake off excess liquid.
• Decolourise the slides with 20% H2SO4
• Wash well in tap water, till no more red colour runs off
the surface when the slides is dipped in water
• Wash thoroughly with water.
101. • Counter stain in methylene blue solution, 30
seconds.
• Blot and differentiate by alternate dehydration and
rehydration until the background is a delicate pale
blue.
• Examine microscopically screening at high power
and confirming all suspicious organism with an oil
immersion lens.
104. Wade fite faraco stain
• This stain is used for staining leprocy bacilli in
tissue sample.
• Leprocy bacilli are much less acid and alcohol fast
than tuberculous bacilli. Therefore alcohol is
removed from hydrating and dehydrating steps
and 10% sulphuric acid is used as decolouriser in
place of acid-alcohol solution.
• The section are also deparaffinised using ground
nut oil and xylene mixture to protect more
delicate waxy coat of lepra bacilli.
105. PROCEDURE
• Warm section and deparaffinised using mixture of two part
xylene and one part of ground nut oil for 10 minutes.
• Blot dry and wash in water untill section is uniformly
wetted.
• Stain with carbol fuschin for 20-30 minutes.
• Wash in tap water and blot dry.
• Decolorize in 10% sulphuric acid till no more red colour
appear while dipping in water.
• Wash in tap water and counter stain with 0.2% methylene
blue for 5-10 seconds.
• Blot dry and do not mount.
• Smears should be seen in oil immersion lens.
107. Warthin-Starry method for spirochetes
(Warthin & Starry 1920)
PRINCIPLE
Organisms are demonstrated by silver
impregnation technique.
Solutions
Acetate buffer. pH 3.6
Sodium acetate 4.1 g
Acetic acid 6.25 ml
Distilled water 500 ml
1 % silver nitrate in pH 3.6 acetate buffer.
108. • It can also be used to demostrate
- Helicobacter pylori in gastric mucosa
- Leptospira organism in renal biosy
- Cat scratch disease associated bacilli on
lymph node biopsy
109. H. Pylori on gastric mucosa on Warthin starry stain
112. Gomori methenamine silver stain for
fungi
• GMS staining is a silver staining technique for
demonstrating fungi in tissue sections.
• It is primarily based on staining the polysaccharides
in fungal cell walls, and can be used to demostrate
the basement membrane.
PRINCIPLE
This method depends upon the reduction of the
silver by the aldehyde groups produced after
oxidation of fungal wall components with chromic
acid.
116. Giemsa stain
• PRINCIPLE
This method is a modified version of the original
Giemsa technique used for haematological smears
and gives good results for sections. Giemsa is a
Romanowsky stain which contains azure B and eosin
Y and is capable of making subtle distinction in
shades of staining. The acidic groupings of the
nucleic acids and proteins of the cell nuclei
determine their uptake of the basic dye azure B and
the presence of basic groupings result in an affinity
for acidic dyes and their staining by eosin.
117. • It can be used for histopathological diagnosis of malaria
and some other spirochete protozoan, and blood
parasites.
Results
• Micro-organism, fungi - purplish-blue
parasites
• Nuclei- blue to violet
• Erythrocytes- salmon pink
• cytoplasm - light blue
• Collagen, muscle and bone - pale pink
120. Gram method for Bacteria
• Gram staining differentiate bacteria into 2 classes
depending on their cell wall structure and composition
• Gram positive and Gram negative.
• PRINCIPLE
Crystal Violet stains the nucleic acids of the bacteria (and
background tissue) and after treatment with iodine, the
sections are differentiated in acetone and counterstained
with basic fuchsin. The tissue background and Gram-
negative bacteria lose their blue staining and are
subsequently stained with counter stain basic fuchsin.
Gram-positive bacteria resist the decolourisation and retain
the crystal violet/iodine blue staining.