M E T H O D S OF S T A I N I N G

• Direct Staining - use Aqueous / Alcoholic Dye Solutions

• Indirect Staining - requires Mordant-Dye Complex
• Progressive Staining - w/o Decolorizer
• Regressive Staining - w/ Decolorizer
• Metachromatic - use of specific dyes; give tissue different
Staining color from original dye
• Intravital Staining - inject/introduce dye to any part of the body
• Supravital Staining - enter and stain living cells

NATURAL DYES SYNTHETIC DYES

Hematoxylin Acid Dyes

Cochineal Basic Dyes
Orcein Neutral Dyes
Saffron
Natural Dyes Origin
Hematoxylin Logwood / Heartwood “Hematoxylin campechianum”
Cochineal Female Cochineal Bug
Orcein Lichens
Saffron Crucos sativus

HEMATOXYLIN

Hematein Active coloring agent formed upon oxidation of hematoxylin

Natural – (2-Ehrlich) 3-4 months (longer storage)
Oxidation (Ripening) Chemical – hematoxylin to hematein instant oxidation (shorter
storage)
Mordants
Combine with tissue and hematein to form a bridge and allow staining
reaction to occur
Alum Hematoxylin Regressive or Progressive staining; Potash alum or Ammonium alum
Iron Hematoxylin + Iron Salts; Demo a much wider range of tissue structures
Cook’s Hematoxylin
+ Cupric Sulfate; Nuclei; (Blue-black); Cytoplasm (unstained /
according to counterstain)
Tungsten Solution
Phosphotungstic acid hematoxylin (PTAH); stains general tissue
structure
Lead Hematoxylin Diagnostic application in ID of endocrine cells in tumors
Molybdenum Solution + molybdic acid; demo of collagen and course reticulin

pg. 1 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023

 ALUM HEMATOXYLIN
Natural ripening (2 months); chemical ripening w/ Sodium Iodate;
Ehrlich’s used for regressive staining; suitable for acidic tissues; bring out
blue detail
Delafield’s
Natural ripening (4-6months); similar longevity with Ehrlich’s;
Longer ripening
+ mercuric chloride; for routine nuclear staining, exfoliative
Harris cytology, & stain for sex chromosomes; Oxidizing agent (very toxic);
Corrosive to automated staining machines
Mayer’s + sodium iodate; short staining time; short storage
Cole’s + alcoholic iodine; used after celestine blue
Carazzi’s
+ potassium iodate; short staining time (progressive staining);
DOES NOT stain cytoplasmic organelles
+ potassium iodate; will stain mucins; prevent the formation of
Gill’s surface ppt w/ ethylene glycol (ethandiol); sensitive to acids; may
stain GEL ADHESIVE and GLASS SLIDE

IRON HEMATOXYLIN

Weigert’s
+ ferric chloride; used when acidic solutions are to be applied
(nucleus resist decolorization w/ acids)
Heidenhain’s
+ ferric ammonium sulfate; for regressive staining of thin
sections, stain cytoplasmic inclusion
Loyez
+ ferric ammonium sulfate; demonstrate myelin & can be applied
to paraffin, frozen, or nitrocellulose sections
Verhoeff’s + ferric chloride; used for photomicrography

Regaud’s Most permanent and simplest; mordanted in dichromate; do not

dve uniform result; for mitochondria demo using the light microscope
Phosphotungstic Acid Mordant (1% aqueous phosphotungstic acid); demonstrate
Hematoxylin (PTAH)
structures in paraffin, celloidin, & frozen sections; staining time (12-
24 hours)

COCHINEAL DYES

Carmine
Widely used as powerful chromatin & nuclear stain for fresh
material & smear preparations
Picrocarmine (Carmine + Picric acid) extensively used in neuropathological studies
Best’s Carmine Stain Combined with aluminum chloride; used for glycogen demonstration

pg. 2 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023

 SYNTHETIC DYES

Eosin Counterstain after hematoxylin & before methylene blue; Routinely used red acid
dye
Methylene Blue Basic nuclear stain; used as an indicator or dye; used in vital staining for nervous
tissues, bacterial staining, & milk evaluation and diagnosis for Diptheria
Picric Acid Acid dye that is a fixative, decalcifying agent, tissue softenet, & differentiator
Methylene Violet Metachromatic dye formed after methylene blue is heated in fixed alkali
Toluidine Blue for staining of Nssl granules & chromophilic bodies
Crystal Violet Nuclear stain; for amyloid in frozen section & platelets in blood
Cresyl Violet Commonly used in histology to stain nervous system; stain acidic components of
neuronal cytoplasm (Nssl bodies) a violet color
Gentian Violet Crystal Violet + Methyl Violet + Dextrin
Aniline Blue Cytoplasmic stain for counter staining of epithelial sections
Basic fuchsin Plasma stain for deep staining of acid-fast organisms, mitochondria,
differentiation of smooth muscles with picric acid
Acid fuchsin (Masson stain) May be used to stain collagen, smooth muscle, or mitochondria; used as nuclear and
cytoplasmic stain in Mallory’s trichrome method; traditional stain for mitochondria
Von Gieson’s stain Picric acid + Acid fuchsin; for connective tissues demonstration
Celestine Blue Resistant to strong acid dyes as a nuclear stain
Malachite Green Weakly basic dye; counterstain for Ascaris eggs & RBC, & as a bacterial spore
stain
Methyl Green Stains chromatin green in the presence of acid
Bismarck Brown Counterstain for acid fast, Papanicolaou, & gram’s technique
Iodine Colored salt of ferric ferrocyanide normally used for manufacture of paints
Alcian Blue Oldest of all stains; originally used for microscopic study of starch granules; stains
amyloid, starch, cellulose, carotenes, & glycogen
Neutral Red Basic dye recommended for observing all granules & phagocytic cell vacuoles
Congo Red Can be used as an indicator and stain for axis cylinders in embryos
Janus Green B Used for demonstration of mitochondria during vital staining
Victoria Blue Used in neuroglia in frozen sections demonstration
Night Blue Substitute for carbol fuchsin in acid fast technique
Acridine Red 3B Demo deposits of calcium salts and possible sites of phosphatase activities
Acridine Orange Basic acridine fluorochrome which gives green fluorescence for DNA & red
fluorescence for RNA
Rhodamine B Used by Griesbach with osmic acid to fix and stain blood & glandular tissues
Benzidine Used in hemoglobin staining
Lysochromes (Oil soluble False dyes used for intracellular fats demonstration
dyes)
Gold Sublimate solution Used for metallic impregnation containing gold chloride and x mercuric chloride
Osmic Acid Fixative and stain; give fat a black color
Silver Nitrate Used in spirochete, reticulum, & other fiber stains

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 BASIC FUSCHIN

Carbol fuchsin Basic fuchsin + phenol

Coleman’s Feulgen Basic fuchsin + sodium metabiosulfate + HCL
reagent / Schiff’s
reagent
Mallory’s fuchsin Basic fuchsin + 95% ethyl alcohol
stain
Gomori’s Aldehyde Basic fuchsin + 70% alcohol + HCL
fuchsin

OIL SOLUBLE DYES

Sudan Black Greater affinity for phospholipids which colors the lipid black
Sudan IV (Scharlach R) Recommended for TAG (neutral lipids) giving intense red stain
Sudan III Fat soluble stain giving lipid a lighter orange color

Types of
Mucopolysaccharides

Neutral
Found in glands of GI tract & in prostate; stain with PAS but NOT alcian blue,
colloidal iron, mucicarmine, or metachromatic dyes
Acid (simple, or non- Typical mucins of epithelial cells containing sialic acid; stain PAS, alcian blue (pH
sulfated) 2.5), colloidal iron, & metachromatic dyes; resist hyaluronidase digestion
Contain hyaluronic acid & found in tissue stroma; DO NOT stain with PAS; stain with
Acid (simple,
mesenchymal) alcian blue (pH 2.5), colloidal iron, & metachromatic dyes; digest hyaluronic acid;
can be found in sarcomas
Acid (complex, or Found in adenocarcinomas; PAS usually positive; alcian blue (pH 1), colloidal iron,
sulfated, epithelial) mucicarmine, & metachromatic dye positive; resist hyaluronidase digestion
Found in tissue stroma, cartilage, & bone; include substances like chondroitin
Acid (complex,
connective tissue) sulfate or keratin sulfate; PAS Negative; DO NOT stain selectively with alcian blue
(pH 5.0)

MUCIN STAINS
Variety of stains for mucin
Colloidal iron (“AMP”) Iron particles are stabilized in ammonia and glycerin and are attracted to acid
mucopolysaccharides; require formalin fixation; phospholipids and free nucleic
acids may also stain; actual blue color comes from Prussian blue reaction; tissue
can be predigested with hyaluronidase to provide more specificity
Alcian Blue The pH of this stain can be adjusted to give more specificity
PAS stains glycogen, as well as mucins, but tissue, can be pre-digested with diastase
to remove glycogen
Mucicarmine Very specific for epithelial mucins

pg. 4 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023

 STAINS FOR BIOGENIC AMINES
 Cells produced by polypeptide hormones, active amines,  Pseudomelanin pigment – usually found in
or amine precursors can be found individually or as a macrophages
group.  True Melanin – PAS negative
Traditional classification of staining patterns based
upon the ability of cells to reduce ammoniacal silver nitrate IRON (HEMOSIDERIN)
to metallic silver (black deposit in a tissue section): - present in areas of old hemorrhage; can be deposited in
1. Chromaffin – have cytoplasmic granules that appear tissues with iron overload
brown when fixed with dichromate solution; reaction is  Perl’s iron stain – classic method of demonstrating
associated with adrenal medulla or extra-adrenal iron in tissues
paraganglion tissues
2. Argentaffin – reduce silver solution to metallic silver
FAT STAINS
after formalin fixation; reaction is associated with
 Oil Red O stain – rapid & simple stain; can identify
carcinoid tumors of the gut
neutral lipids and fatty acids in smear and tissues; useful in
3. Argyrophil - pre-reduction step necessary
identifying fat emboli in lung tissue or clot sections of
peripheral blood
TYPES OF STAINS
ARGENTAFFIN CHROMAFFIN ARGYROPHIL CONNECTIVE TISSUE STAINS
Diazo Modified Giemsa Grimelius  Trichome stain – helps to highlight supporting
Fontana-Masson Schmorl’s Pascual’s collagenous stroma in sections from a variety stroma;
Schmorl’s Wiesel’s helps determine the pattern of tissue injury; aid in
Autofluorescence identifying normal structures
 Grimelius – Bouin’s preferred fixative  Chronic active hepatitis with collapse in liver /
Cerebral abscess in brain / Scleroderma with
fibrosis of submucosa in stomach
MELANIN STAINS
 Melanin – normally seen in eyes, skin, and substantia
nigra; may also be found in melanomas
GIEMSA STAIN
 Fontana-Masson – commonly used method which  Giemsa Stain - identify components in a variety of
relies upon melanin granules to reduce ammoniacal silver tissues (e.g Skin with mast cells in the dermis, & Esophagus
nitrate; stain melanin pigment in cells of malignant with eosinophils)
melanoma  Wright’s Stain - stain peripheral blood smears
 Schmorl’s – method used in reducing properties of
melanin to stain granules blue-green GOMORI METHENAMINE SILVER STAIN “GMS”
 DOPA-oxidase – an enzyme histochemical method - stain for fungi and Pneumocystis carinii; organisms are
known to be the most specific method of all; requires outlined by brown to black stain; also a stain for
frozen sections for best results; DOPA substrate acted Cryptococcus neoformans & Coccidioides immitis
upon by DOPA-oxidase in melanin-producing cells to
produce a brownish black deposit PAS (PERIODIC ACID-SCHIFF)
 Bleaching – remove melanin to get a good look at - Very sensitive, but specificity depends upon
cellular morphology (Ocular takes hours to bleach & skin interpretation; all-around useful stain for many things;
takes minutes) stains glycogen, mucin, mucoprotein, glycoprotein, & fungi;
 Formaldehyde-induced fluorescence – used to pre-digestion step with amylase remove staining for
highlight biogenic amines and melanin in tissues; formalin glycogen; useful for outlining tissue structures
contribute a strong yellow autofluorescence to  Candida in lung / Glycogen in Ewing’s sarcoma /
unstained tissues with these substances Nodular glomerulosclerosis in kidney
 Pseudomelanin of melanosis coli – PAS positive

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 HISTOSTAIN SUMMARY

CONNECTIVE TISSUE
Collagen 1. Van Gieson’s / Weigerts-Van Gieson’s
2. Masson’s Trichrome stain
3. Silver Impregnation
4. Mallory’s Aniline Blue Stain
Elastic Fibers 1. Weigert’s
2. Orcein or Taenzer-Unna Orcein
Reticular Fibers 1. Weigert’s
2. Gomori’s

CELLULAR PARTS
Golgi Apparatus Da famos (cobalt)
Argentaffin cells Diazo (Brentamine Fast Red)
Melanin & Silver elements Fontana Mason (Silver Nitrate)
Spermatozoa Bergh’s (Carbolfuchsin)
DNA & RNA Taft’s (RNA= pyronine; DNA= Methylene Blue)
Barr Bodies Guard’s (Biebrich scarlet)
Hemoglobin Benzidine

ELEMENTS & SUBSTANCES

Lipids Sudan Black, Sudan IV, Scharlach R
Simple Fats Sudan Black, Scharlach R
Neutral Fats Menchik’s (Nile blue sulfate)
Cholesterol Schult’z modification of Leibermann Burchard
Bile pigments Gmelin’s / Smith’s
Minerals Calcium = Von Kossa, Carr’s, Dahl’s

CENTRAL NERVOUS SYSTEM

Astrocytes Ramon & Cajal’s (Mercury & Gold)
Glia Fibers Holzer’s Crystal Violet (Violet)
Myelin Luxol Fast Blue (Blue)
Neuron Cresyl Violet (Pink Violet)
Nerve Fibers Protargol
Negri Bodies Schleifstein

CARBOHYDRATES
Glycogen 1. PAS by McManus
2. PAS Technique with Diastase digestion
3. Best Carmine

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 AMYLOID
Kraijan’s Amyloid Method /
Modified Bennhold’s
Leib’s Method
Highman’s Method
Gram’s Iodine Method
Congo Red Method

PROTEINS
Mucin Mayer’s mucicarmine
Fibrin Fraser Lendrum

MICROORGANISMS
Gram Positive & Gram Brown & Brenn (Crystal violet); Nc Callum & Good
Negative Bacteria Pasture (Gentian violet)
Acid Fast Bacilli (AFB) Kinyoun’s (Cold); Zeihl Neelsen (Hot)
Spirochetes Levaditi

Fungus – Gomor’s methenamine silver nitrate

 Entamoeba – Gridley’s
 Rickettsia – Pinkerton’s

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Best Carmine

CYTOPATHOLOGY

Branch of pathology that studies and diagnoses diseases on the cellular level
PAP Smear – most common use to detect cervical cancer at an early treatable stage
AKA cytology - “study of cells”

2 METHODS OF COLLECTING CELLS FOR ANALYSIS b. 50% Alcohol: pleural and peritoneal fluids; applicable for
1.) EXFOLIATIVE CYTOLOGY all types of specimen
- Microscopic study of cells that have been 50% alcohol – pleural and peritoneal fluid; also
desquamated from epithelial surfaces applicable to all types of specimen
- Father of Exfoliative cytology: Dr. George N. Papanicolaou  Saccomanno’s fixative – highly preferable
(50% alcohol + carbowax); can replace 50%
- Usually recommended for: alcohol
→ For assessing malignant or cancerous conditions  Equal parts of 95% alcohol and ether: best
→ For detection of asymptomatic cancer in women fixative
→ For assessment of female hormonal activity in case of  95% alcohol: most lab use for routine prep
sterility and endocrine disorders  slide should be kept at a distance of 1 FOOT for the spray
→ Determination of genetic sex (Too near = may disperse; Too far = insufficient fixing)
→ Determination of the presence of possible infection
When fluid spx is more than few drops:
2.) FINE NEEDLE ASPIRATION CYTOLOGY or FINE NEEDLE o Centrifugation: 2000 rpm for 2 minutes; supernatant
ASPIRATION BIOPSY (FNAB) ⇒ decanted ; sediment, smeared (directly to glass)
- collects specimens through a syringe
- 18 to 27 gauge (most coomon: 22-23) attached to When there’s excess sediment:
10 cc syringe o Run as cell block or cytocentrifuge/cytospin on
adhesive coated slide
Specimen: representative tissues from deeply seated
lesions: lungs, mediastinum, abdominal organs ADHESIVE AGENTS FOR CYTOLOGY
(liver, pancreas), retroperitoneal organs (kidney, 1.) pooled human serum or plasma
adrenal, LN) – taken by clinicians/pathologists 2.) celloidin ether alcohol
with the aid of laparoscopy, CT scan, or USD 3.) leuconostoc culture

Slide fixation: most crucial for directly prepared GYNECOLOGIC SAMPLING FOR PAP SMEAR
smear  Sampling for T-zone for the detection of dysplasia and
95% alcohol / spray – recommended (alcohol carcinomas of cervix
acetone or formalin – substitute fixative) 1. Endocervical brush – samples of endocervical
canal
METHODS OF SMEAR PREPARATION for CYTOLOGY 2. Vaginal scrape – for px with hysterectomy
1. Streaking 3. Lateral vaginal scrape – for hormonal
2. Spreading evaluation
3. Pull-apart 4. Four quadrant vaginal scrape - for localizing
4. Touch preparation of vaginal adenosis
5. Vulvar scrape – for detection of herpetic lesion
SMEAR FOR BODY FLUIDS REQUIRES: or carcinoma
a. Sufficient alcohol fixative in different concentration

pg. 8 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023

 Papaniculau Method – staining method of choice for exfoliative cytology
 Endometrial curettings – use to assess hormonal status in non-pregnant women

CELLS FOUND IN VAGINAL SMEARS

Mature Superficial Cells Have dark pyknotic nuclei with true acidophilia characteristic
Intermediate Cells Medium-sized polyhedral or elongated cells with basophilic cytoplasm
showing vacuoles
Navicular cells – boat-shaped present during pregnancy and
menopause suggesting combined estrogen-progesterone effect
 Pregnancy cells – round, oval, or boat-shaped cells showing
glycogen accumulation at their center and double-walled periphery
Parabasal Cells Thick, round to oval, swollen cells showing fried egg appearance
Endometrial Cells Similar to parabasal cells but with slightly cylindrical, less basophilic
cytoplasm; present 1-10 days post menstruation
Basal Cells Small, round to oval cells with large nuclei occupying more than half
of cell volume; found before puberty and after menopause
Endocervical Glandular Cells Cytoplasm is pale blu-gray & finely vacuolated; may present a
honeycomb appearance

Doderlain bacilli Most common gram-positive rods found normally in vaginal flora;
Large number of these signify cellular destruction due to low
vaginal pH & may be suggestive of Diabetes mellitus infection
Candida albicans Causes Candidiasis; commonly seen in Diabetic patients &
immunocompromised patients
Koilocytes Cells with atypical nucleus surrounded by a perinuclear halo; the
presence is diagnosed as low as low-grade squamous
intraepithelial lesion (Bethesda)
Trichomonas Vaginalis Causes Strawberry Cervix; pear-shaped organism and stains
BLUE-GREEN / BLUE-GRAY
Ferning Phenomenon (Palm- Mucus upon drying = high persistence of estrogen effect diagnostic
Leaf Pattern) pf early pregnancy

Methods of Reporting Cytologic Smears for Cancer Diagnosis

Class I Absence of atypical or abnormal cells
Class II Atypical but no evidence of malignancy
Class III Suggestive but not conclusive of malignancy
Class IV Strongly suggestive of malignancy
Class V Conclusive of malignancy

NON-GYNECOLOGIC EXFOLIATIVE SPECIMENS

 Respiratory tract specimens (sputum, BAL.BW, BB)
 Gastrointestinal specimens (GW, GA)
 Urine – 1st voided urine discarded due to overnight cell degeneration; 2nd voided urine (at least 50 mL)
 Peritoneal, pleural and pericardial fluid

pg. 9 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023