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 DYE

S
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
pg. 1 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023
Molybdenum Solution
+ molybdic acid; demo of collagen and course
reticulin
 ALUM HEMATOXYLIN
Natural ripening (2 months); chemical ripening w/
Ehrlich’s Sodium Iodate; 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,
Harris
exfoliative 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
Gill’s
formation of 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
+ ferric ammonium sulfate; demonstrate myelin &
Loyez 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);
Hematoxylin (PTAH)
demonstrate structures in paraffin, celloidin, & frozen
sections; staining time (12-24 hours)

COCHINEAL DYE
S

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

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 SYNTHETIC DYE
S

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

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 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

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 DYE

S

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

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 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

 STAINS FOR BIOGENIC AMINES 

 Cells produced by polypeptide hormones,  MELANIN STAINS 
active amines, or amine precursors can be  Melanin – normally seen in eyes, skin,
found individually or as a group. and substantia nigra; may also be found in
Traditional classification of staining melanomas
patterns based upon the ability of cells to  Fontana-Masson – commonly used
reduce ammoniacal silver nitrate to metallic method which relies upon melanin granules
silver (black deposit in a tissue section): to reduce ammoniacal silver nitrate; stain
1. Chromaffin – have cytoplasmic granules melanin pigment in cells of malignant
that appear brown when fixed with melanoma
dichromate solution; reaction is associated  Schmorl’s – method used in reducing
with adrenal medulla or extra-adrenal properties of melanin to stain granules blue-
paraganglion tissues green
2. Argentaffin – reduce silver solution to  DOPA-oxidase – an enzyme
metallic silver after formalin fixation; histochemical method known to be the most
reaction is associated with carcinoid tumors specific method of all; requires frozen
of the gut sections for best results; DOPA substrate
3. Argyrophil - pre-reduction step acted upon by DOPA-oxidase in melanin-
necessary producing cells to produce a brownish black
deposit
 TYPES OF STAINS   Bleaching – remove melanin to get a good
look at cellular morphology (Ocular takes
ARGENTA CHROMAF ARGYROP hours to bleach & skin takes minutes)
FFIN FIN HIL  Formaldehyde-induced fluorescence –
Diazo Modified Grimelius used to highlight biogenic amines and
Giemsa melanin in tissues; formalin contribute a
Fontana- Schmorl’s Pascual’s strong yellow autofluorescence to unstained
Masson tissues with these substances
Schmorl’s Wiesel’s  Pseudomelanin of melanosis coli – PAS
Autofluoresc positive
ence  Pseudomelanin pigment – usually found
 Grimelius – Bouin’s preferred fixative in macrophages
 True Melanin – PAS negative

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IRON (HEMOSIDERIN)
- present in areas of old hemorrhage; can be
deposited in tissues with iron overload
 Perl’s iron stain – classic method of
demonstrating iron in tissues

FAT STAINS
 Oil Red O stain – rapid & simple stain;
can identify neutral lipids and fatty acids in
smear and tissues; useful in identifying fat
emboli in lung tissue or clot sections of
peripheral blood

CONNECTIVE TISSUE STAINS

 Trichome stain – helps to highlight
supporting collagenous stroma in sections
from a variety stroma; helps determine the
pattern of tissue injury; aid in identifying
normal structures
 Chronic active hepatitis with
collapse in liver / Cerebral abscess in
brain / Scleroderma with fibrosis of
submucosa in stomach

GIEMSA STAIN
 Giemsa Stain - identify components in a
variety of tissues (e.g Skin with mast cells in
the dermis, & Esophagus with eosinophils)
 Wright’s Stain - stain peripheral blood
smears

GOMORI METHENAMINE
SILVER STAIN “GMS”
- stain for fungi and Pneumocystis carinii;
organisms are outlined by brown to black
stain; also a stain for Cryptococcus
neoformans & Coccidioides immitis

PAS (PERIODIC ACID-SCHIFF)

- Very sensitive, but specificity depends
upon interpretation; all-around useful stain
for many things; stains glycogen, mucin,
mucoprotein, glycoprotein, & fungi; pre-
digestion step with amylase remove staining
for glycogen; useful for outlining tissue
structures
 Candida in lung / Glycogen in Ewing’s
sarcoma / Nodular glomerulosclerosis in
kidney

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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 Fontana Mason (Silver Nitrate)
elements
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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pg. 8 STAINING & CYTOPATHOLOGY | Arriola, Stephanie Kate G. | MLS 4C | 2023
 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 & Brown & Brenn (Crystal violet); Nc
Gram Negative Callum & Good Pasture (Gentian violet)
Bacteria
Acid Fast Bacilli Kinyoun’s (Cold); Zeihl Neelsen (Hot)
(AFB)
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 (alcohol

CELLS FOR ANALYSIS  acetone or formalin – substitute fixative)
1.) EXFOLIATIVE CYTOLOGY
- Microscopic study of cells that have been  METHODS OF SMEAR
desquamated from epithelial surfaces PREPARATION for CYTOLOGY 
- Father of Exfoliative cytology: Dr. George 1. Streaking
N. Papanicolaou 2. Spreading
3. Pull-apart
- Usually recommended for: 4. Touch preparation
→ For assessing malignant or cancerous
conditions  SMEAR FOR BODY FLUIDS
→ For detection of asymptomatic cancer in REQUIRES:
women a. Sufficient alcohol fixative in different
→ For assessment of female hormonal concentration
activity in case of b. 50% Alcohol: pleural and peritoneal fluids;
sterility and endocrine disorders applicable for all types of specimen
→ Determination of genetic sex 50% alcohol – pleural and peritoneal
→ Determination of the presence of possible fluid; also applicable to all types of specimen
infection  Saccomanno’s fixative – highly
preferable (50% alcohol + carbowax);
2.) FINE NEEDLE ASPIRATION can replace 50% alcohol
CYTOLOGY or FINE NEEDLE  Equal parts of 95% alcohol and
ASPIRATION BIOPSY (FNAB) ether: best fixative
- collects specimens through a syringe  95% alcohol: most lab use for
- 18 to 27 gauge (most coomon: 22-23) routine prep
attached to  slide should be kept at a distance of 1
10 cc syringe FOOT for the spray (Too near = may
disperse; Too far = insufficient fixing)
 Specimen: representative tissues from
deeply seated When fluid spx is more than few drops:
lesions: lungs, mediastinum, abdominal o Centrifugation: 2000 rpm for 2 minutes;
organs supernatant
(liver, pancreas), retroperitoneal organs ⇒ decanted ; sediment, smeared (directly to
(kidney, glass)
adrenal, LN) – taken by
clinicians/pathologists When there’s excess sediment:
with the aid of laparoscopy, CT scan, or USD o Run as cell block or cytocentrifuge/cytospin
on
 Slide fixation: most crucial for directly adhesive coated slide
prepared
smear  ADHESIVE AGENTS FOR
 95% alcohol / spray – recommended CYTOLOGY 

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2023
1.) pooled human serum or plasma endocervical
2.) celloidin ether alcohol canal
3.) leuconostoc culture 2. Vaginal scrape – for px with
hysterectomy
 GYNECOLOGIC SAMPLING FOR 3. Lateral vaginal scrape – for
PAP SMEAR  hormonal evaluation
 Sampling for T-zone for the detection of 4. Four quadrant vaginal scrape - for
dysplasia and localizing of vaginal adenosis
carcinomas of cervix 5. Vulvar scrape – for detection of
1. Endocervical brush – samples of herpetic lesion or carcinoma

 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 Cytoplasm is pale blu-gray & finely vacuolated; may
Cells 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

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 BLUE-GREEN / BLUE-GRAY
Ferning Phenomenon Mucus upon drying = high persistence of estrogen
(Palm-Leaf Pattern) effect diagnostic 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

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