1. Alum Hematoxylin – progressive staining.

TYPES/METHODS OF STAINING Counterstained with Congo red and Safranin.

 Direct – aqueous or alcohol soln. Nucleus is blue-stained. Red-stained with Acid.
 Indirect – requires mordant (insoluble tissue- Blueing – neutralize acid. Cold water slows, Warm
mordant complex); accentuator (fast reaction) water accelerates, Very Cold water produces pink
 Progressive – definite sequence, correct timing, artifacts
relies on selective affinity of dyes. NOT WASHED  Ehrlich’s – regressive stain. For mucopolysacch,
OR DECOLORIZED (DA. Diffused and obscure
cartilage, cement lines of bones (15-40 mins.)
effect)
Deep red.
 Regressive – first overstained then excess stain
 Harris – routine nuclear, EC, sex chromosomes
is removed (Differentiation/Decolorization –
selective removal) (5-20 min.) Dark purple.
 Metachromatic – stained color is different from  Cole’s – routine purposes, sequence with
stain itself (Thiazine and Triphenylmethane Celestin blue. Mixed with iodine. (10 mins.)
groups)  Mayer’s – in Celestin Blue Hemalum method of
Methyl violet ~ cresyl blue ~ safranin ~ bismarck nuclear stain. (boil 5 mins. and cool)
brown ~ basic fuchsin ~ methylene blue ~ 2. Iron Hematoxylin – for tissues stored in alcohol for
thionine ~ toluidine blue ~ azure A, B, C years with tendency to fail to stain. Black/Gray
 Counterstain – contrast the staining of stain. Permanent stain. For photomicrography.
components  Weigert’s – standard. For muscle fibers, CT,
Cytoplasmic previously acid-stained tissues. (active for 1-2
Eosin Y, Eosin B, Phloxine B, Rose Bengal
days)
Picric Aid, Orange G
 Heidenhain’s – regressive. For cytoplasmic and
Light Green SF, Lissamine Green
Nuclear nuclear. (mordant is used separately)
Neutral Red, Sfranin Red, Carmine  Phosphotungstic Acid (PTAH) – progressive. For
Methylene Blue, Toluidine blue, Celestine blue, paraffin, celloidin, frozen sections. (12-24 hrs.)
hematoxylin Nuclei, fibrin, muscle striations, fibroglia - (blue)
 Microanatomical – differentiate nucleus & Collagen, bone and cartilage - (orange-red to
cytoplasm brownish-red)
Cytoplasmic – cytoplasm and nucleus B. COCHINEAL DYES – female cocchineal bug
Negative – bacterial morphology (req. black extract. Carmine dye is formed from alum
background) treatment. For nuclear stain of fresh materials
 Metallic impregnation – not absorbed but and smear preps.
precipitates. Opaque black deposits (gold + picric acid = neuropathological studies
chloride & silver nitrate)
+ aluminum chloride (Best’s Carmine) = glycogen
 Vital – cytoplasmic phagocytosis. Only
C. ORCEIN – vegetable extract. Blue/Violet. For
demonstrates nuclear if cell is dead due to
membrane permeability elastic fibers or as an indicator.
Intravital – dye injection II. SYNTHETIC DYES – Coal Tar derivative. Aka Aniline
Supravital – biopsy tissues placed in stain dish dyes.
(neutral red, janus green, tryphan blue, nile 1. Chromophores – definite atomic group. Visible
blue, thoinine, toluidine blue) colors
CLASSIFICATION OF DYES 2. Chromogens – benzene compounds. Not
According to composition: permanent stain.
I. NATURAL – from plants or animals 3. Auxochrome – forms salts from electrolytic
A. HEMATOXYLIN – tree ether extract. Hematin is dissociation
the coloring agent. Ripening is oxidizing 4. Dye – chromophore + auxochrome attached to a
hematoxylin to form hematin. (air exposure, HC benzene ring.
H2O2, HgCl2, KMNO4, Na perborate, Na iodate)  Acid dyes – for acid-, chromic-fixed tissues
*Has low affinity, thus needs mordants (alum,  Basic dyes – HgCl2-, formaldehyde- fixed
iron, chromium, copper salts) tissues
  Neutral dyes – for alcohol. (water-insoluble) 16. CARMINE – chromatin stain. For fresh materials
and smear preps. +aluminium chloride (Best
OTHER STAINS USED IN HISTOPATHOLOGY
Carmine) for glycogen and mucin (Mucicarmine)
1. EOSIN – for CT and cytoplasm. Routine
counterstain after Hematoxylin and before
Methylene blue. Background stain. For
chromate and picric-fixed tissues, and acid-
decalcified materials. Naturally Red dye.
 Eosin B – bluish -red
 Eosin Y – yellowish. More common for its water
solubility
2. METHYLENE BLUE – basic nuclear. For plasma
cells. Cytological exams of fresh sputum,
bacterial stain, grading of milk, diagnosis of
diptheria, vital staining for nervous tissue,
indicator of colon and Aerogenes group in
bacteriology.
3. METHYLENE VIOLET – metachromatic. Reddish-
purple of leokocyte nuclei in methylne blue
4. TOLUIDINE BLUE – nuclear stain, substitue for
thionine in fresh frozen sections. For Nissl
granules or Chromophilic bodies.
5. CRYSTAL VIOLET – nuclear/chromatin. For
amyloid in frozen sections and platelets in blood
6. ANILINE BLUE – cytoplasmic. Counterstain
epithelial sections
7. BASIC FUCHSIN – plasma stain. For Acid-fast,
Smooth muscles. Aldehyde detection
(Fuelgen’s, Schiff’s). CT, mucin, elastic tissue
(Van Gieson’s)
8. GIEMSA STAIN – stain blood. Differentiate
leokocytes.
9. CELESTINE BLUE – routine stain for fixed
sections. Resistant to strong acid dyes. Good
nuclear stain with alum hematoxylin.
10. MALACHITE GREEN – contrast to Ascaris eggs,
erythrocytes, bacteria spore. Decolorizer,
counterstain.
11. METHYL GREEN – green in acid. Gives false
positive to mucin
12. BISMARCK BROWN – contrast to Gram’s tech.,
Acid-Fast, Pap method, diphtheria.
13. PRUSSIAN BLUE – intravital. Microanatomical
contrast. Circulatory system.
14. ORCEIN – for elastic fibers and dermatological
studies
15. PICRIC ACID – contrast to Acid fuchsin. For CT
(Van Gieson’s), cytoplasmic, contrast to basic
dyes, counterstain to crystal violet.