Unit 1: Special staining and specimen handling
Histotechnology-II & Cytology notes · PTU syllabus (BMLS605-18)
On this page
- Unit summary
- Cryostat sections and their applications
- Special stains for connective tissue
- Metachromatic staining and metal impregnation
- Minerals and pigments
- Carbohydrates, proteins and nucleic acids
- Lipids
- Bacteria and fungi in tissue sections
- Handling specialised tissues
- Enzyme histochemistry
- Vital staining, neuropathology and museum techniques
- Key terms
- Quick revision
- Important questions
Unit summary
Special stains and techniques show what routine H&E cannot. This unit covers cryostat sections and their uses, special stains for connective tissue, metachromasia, metal impregnation, minerals and pigments, demonstration of proteins, nucleic acids, carbohydrates, lipids, bacteria and fungi in tissue, handling special tissues, enzyme histochemistry, vital staining, neuropathology and museum techniques.
After this unit you can
- Use cryostat sections for rapid diagnosis and special studies
- Perform special stains for connective tissue, carbohydrates, lipids and pigments
- Demonstrate microorganisms and enzymes in tissue
- Handle special specimens and prepare museum specimens
PTU syllabus topics
- Cryostat sectioning and its diagnostic applications
- special staining for connective tissue elements (trichrome, elastic, reticulin, collagen fibres)
- metachromatic staining
- metal impregnation
- minerals and pigments
- demonstration of proteins
- nucleic acids
- carbohydrates
- lipids
- bacteria and fungi in tissue sections
- handling of specialised tissue (eyeball, bone marrow, muscle biopsy, uncalcified bone, whole brain/lungs)
- enzyme histochemistry (phosphatases, dehydrogenases, oxidases, peroxidases)
- vital staining
- neuropathological and museum techniques
PAS
Glycogen, mucin, fungi
Magenta
Masson's trichrome
Collagen vs muscle
Collagen blue or green, muscle red
Reticulin (silver)
Reticulin fibres
Black
Perl's Prussian blue
Iron (haemosiderin)
Blue
Ziehl-Neelsen
Acid-fast bacilli
Red rods on blue
Topic 1
Cryostat sections and their applications
- Fresh tissue is frozen (isopentane cooled in liquid nitrogen, or on the cryostat's freezing bar) and cut at 5–10 µm at about −20 °C. Applications: intraoperative diagnosis and margins, lipids (oil red O, Sudan stains — dissolved by routine processing), enzymes (histochemistry), immunofluorescence (kidney and skin biopsies), muscle biopsies.
Topic 2
Special stains for connective tissue
Van Gieson
Picric acid and acid fuchsin
Collagen red; muscle and cytoplasm yellow
Masson's trichrome
Three dyes of different molecular sizes
Collagen blue or green; muscle and cytoplasm red; nuclei black
Verhoeff's elastic (EVG)
Iron haematoxylin–iodine lake
Elastic fibres black
Reticulin (Gordon and Sweets)
Silver impregnation (argyrophilic fibres)
Reticulin fibres black
Orcein
Natural dye
Elastic fibres brown; HBsAg in liver
- Uses: liver fibrosis and cirrhosis (trichrome, reticulin), vascular disease (elastic), tumour patterns (reticulin).
Topic 3
Metachromatic staining and metal impregnation
- Metachromasia: toluidine blue or methylene blue stain mast cell granules, cartilage and mucins purple–red (from blue), due to closely spaced anionic groups. Used for mast cells and amyloid (crystal violet).
- Metal impregnation: silver salts deposited on structures and reduced to black metallic silver — argyrophilic (needs an external reducer: reticulin, nerve fibres) and argentaffin (reduces silver itself: carcinoid granules — Fontana–Masson).
Topic 4
Minerals and pigments
Haemosiderin (iron)
Perl's Prussian blue
Blue — iron overload, old haemorrhage
Calcium
von Kossa (silver nitrate) or alizarin red S
Black or orange–red
Melanin
Masson–Fontana; bleaching confirms
Black
Bile pigment
Fouchet's
Green
Lipofuscin
Schmorl's, Sudan black, PAS
Blue–green or black (wear-and-tear pigment)
Copper
Rhodanine
Red — Wilson's disease
Formalin pigment
Removed by alcoholic picric acid
Brown–black artefact
- Amyloid: Congo red — orange–red with apple-green birefringence under polarised light.
Topic 5
Carbohydrates, proteins and nucleic acids
Periodic acid–Schiff (PAS)
Periodic acid oxidises glycols to aldehydes; Schiff's reagent stains magenta
Glycogen, basement membranes, fungi, mucins
PAS with diastase
Diastase digests glycogen first
Distinguishes glycogen (removed) from mucin
Alcian blue (pH 2.5)
Acid mucins
Blue — goblet cells, intestinal metaplasia
Mucicarmine
Epithelial mucin
Red — adenocarcinoma; Cryptococcus capsule
- Nucleic acids: Feulgen reaction (acid hydrolysis then Schiff's) for DNA — magenta; methyl green–pyronin — DNA green, RNA red (plasma cells). Proteins: Millon's reaction (tyrosine), immunohistochemistry for specific proteins.
Topic 6
Lipids
- Need frozen sections (lipids dissolve in processing alcohols and xylene). Oil red O and Sudan IV stain neutral fats red; Sudan black B stains lipids black. Uses: fatty liver, fat emboli, liposarcoma.
Topic 7
Bacteria and fungi in tissue sections
Gram (Brown–Brenn, Brown–Hopps)
Gram-positive blue, Gram-negative red
Bacteria in tissue
Ziehl–Neelsen
Acid-fast bacilli red
Tuberculosis
Wade–Fite
Modified ZN for M. leprae
Leprosy
Grocott's methenamine silver (GMS)
Fungal walls black
Aspergillus, Candida, Pneumocystis
PAS
Fungi magenta
Fungi
Warthin–Starry
Silver
Spirochaetes, H. pylori
Giemsa
—
H. pylori, Leishmania
Topic 8
Handling specialised tissues
- Eyeball
- Fix whole for 48 hours; open in the horizontal plane; celloidin or careful paraffin processing
- Bone marrow trephine
- Fix, decalcify gently (EDTA or formic acid), section at 2–3 µm
- Muscle biopsy
- Fresh, unfixed; snap-freeze in isopentane for enzyme stains; part in glutaraldehyde for EM
- Undecalcified bone
- Resin (methyl methacrylate) embedding — bone mineralisation studies
- Whole brain
- Suspend by the basilar artery in 10–20% formalin for 2–3 weeks before slicing
- Whole lungs
- Inflate through the bronchus with formalin to restore shape
Topic 9
Enzyme histochemistry
Phosphatases
Substrate cleaved; product captured as coloured precipitate (Gomori, azo-dye)
Alkaline phosphatase in bone and placenta; acid phosphatase in lysosomes
Dehydrogenases
Tetrazolium salts reduced to coloured formazan
Muscle fibre typing (NADH-TR, SDH), viability
Oxidases
Cytochrome oxidase with DAB
Mitochondrial myopathies
Peroxidases
DAB substrate gives brown product
Myeloid cells; basis of immunoperoxidase IHC
- Uses fresh frozen sections because fixation and heat inactivate enzymes.
Topic 10
Vital staining, neuropathology and museum techniques
- Vital staining: dyes taken up by living cells — supravital (cells removed from the body: new methylene blue for reticulocytes, Janus green for mitochondria) and intravital (dye injected into the living body: trypan blue for macrophages).
- Neuropathology: Nissl stain (cresyl violet) for neurons, Luxol fast blue for myelin, Bielschowsky silver for axons and plaques, GFAP immunostaining for astrocytes.
- 1Fix in Kaiserling I (formalin with potassium acetate and nitrate) to preserve colour
- 2Restore colour in 80–95% alcohol (Kaiserling II)
- 3Mount in Kaiserling III (glycerine, potassium acetate, water, formalin traces)
- 4Seal in a perspex jar; label with diagnosis and history
Key terms
- Cryostat
- Refrigerated microtome for frozen sections
- Argyrophilic
- Binding silver that needs an external reducer
- PAS reaction
- Periodic acid–Schiff stain for glycogen and mucins
- Feulgen reaction
- Specific stain for DNA
- Kaiserling method
- Colour-preserving technique for museum specimens
Quick revision
- Cryostat uses; lipids and enzymes need frozen sections.
- Van Gieson, Masson, EVG, reticulin, orcein.
- Metachromasia; argyrophil vs argentaffin.
- Perl's, von Kossa, Masson–Fontana, Fouchet's, rhodanine, Congo red.
- PAS ± diastase, alcian blue, mucicarmine, Feulgen, MGP; oil red O; Gram, ZN, Wade–Fite, GMS, Warthin–Starry; special tissues; enzyme histochemistry; vital stains; neuro stains; Kaiserling.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Why are lipids demonstrated on frozen sections?
- Q2.What colour does collagen stain with Van Gieson?
- Q3.Which stain demonstrates iron?
- Q4.What does PAS with diastase show?
- Q5.Which stain demonstrates fungi in tissue as black?
- Q6.Distinguish supravital and intravital staining.
Long-answer questions
- Q1.Describe special stains for connective tissue.
- Q2.Describe the demonstration of carbohydrates and lipids in tissue.
- Q3.Describe stains for microorganisms in tissue sections.
- Q4.Describe enzyme histochemistry and museum techniques.
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