STAINS AND STAINING SOLUTIONS

(Hematoxylin) (Hematoxylin campechianum) (basic dye) (basic dye)

(Hematin) (natural ripening) (artificial ripening)

(___________)

● Natural dye extracted from the core or heartwood of a Mexican

tree (_____________)).

● Most valuable staining reagent

● Mostly used to any fixative and is a permanent stain

● not a true basic dye

● (_____) → active coloring agent thru ripening

● Ripening:

○ Expose to sunlight (3-4 months) → (________)

○ Adding hydrogen peroxide, mercuric oxide, potassium

permanganate, sodium perborate or sodium iodate →

(________)

(Ripened Hematoxylin) (mordants) (MORDANTS) (bridge ) (Mordant (aluminum, iron salts)) (tissue-mordant-hematoxylin linkage).)

● (__________) rarely used alone, combined with

alum, iron, chromium, copper salts which acts as

(_______).

● (___________)

○ substances that combine with the tissue and staining

solution, forming a (______) allowing staining reaction

to occur.

○ (____________) binds to the tissue,

and then hematoxylin binds to the mordant, forming

a (____________________)

Biological Stains

1. Natural dyes Cochineal dyes Logwood dyes Vegetable extracts

1. Synthetic (Artificial) dyes Aniline dyes Coal tar dyes

(Cochineal Dyes ) (Coccus cacti) (+ alum ~ carmine) (+ icric acid - picrocarmine ) (+ aluminum chloride → Best's Carmine stain )

(_________)

Old histological dye

Female cochineal bug (_______)

(____________________)

O Powerful chromatin and nuclear stain

(_________→_________)

○ Used in neuropathological studies

O (_____________________)

O Used in the demonstration of glycogen

(Orcein) (+ ammonia and air) (+ lime, soda ammonia and air) (PH indicator)

(_________)

• Vegetable dye extracted from lichens (colorless)

• (___________) → blue or violet

• (_______________) → litmus paper

• Staining of elastic fibers

• Poor staining property of litmus is instead used as (___________).

(Chromophores) (Chromogens) (Auxochromes) (Dye) ()

SYNTHETIC DYES

• Coal Tar Dyes

• Aniline Dyes

TERMS:

(____________) - capable of producing visible colors

(__________) - compounds containing chromophores

(____________) - auxillary substance that can retain the color of the dye into the tissue

(____) - consist of chromophore auxochrome (dyeing property) (coloring property) and

(Counterstain) (Van Gieson's stain) (crystal violet) (fixative. decalicifiying agent and tissue softener) (nuclear staining) (bacterial staining) (leukocyte differentiation)

Acid Dyes	Basic Dyes	Neutral Dyes
Used as a (________) to basic stains. Component of (_________) for connective tissue. (_________) for fungal microscopy Functions as a (_____________&____).	Used for (__________) (chromatin is acidic). Commonlv used in (________) in microbiology.	Primarilv used in hematology for (___________)

(stain-mordant-tissue) (acidic or an alkaline) (Primary stain and a nuclear stain)

Hematoxylin has no staining property.

○ Ripening and oxidation must happen, convert to its active

coloring substance

● Hematin with mordant such as ammonium or potassium alum

forms lake (_____________)

○ functions as cationic dye and stains anionic tissue

components.

● Hematin in an aqueous solution can be (_______/________)

dye depending on pH.

● Hematin has affinity for several tissues with an appropriate

mordant

● (________&_________)

(H&E staining) (Hematoxylin) (Eosin)

(______) is used to examine thin sections

○ (________) → stains cell nuclei blue

○ (_________) → stains cytoplasm, connective tissue

pink or red

(Alum Hematoxylin) (Iron Hematoxylin) (Tungsten Hematoxylin)

(Copper Hematoxylin)

Types of

Hematoxylin

(Alum Hematoxylin Stains (Aluminum) (Aluminum salts) (nuclei blue-purple) (Routine H&E staining)

(______________)

● Mordant: (________) (potassium or

ammonium alum)

● Appearance: Stains (_________) after

a blueing step (alkaline treatment)

● Uses: (______________)

○ Good nuclear stain

(Mercuric oxide) (Regressive) (routine nuclear staining, exfoliative cytology, staining of sex chromosomes) (Natural/Sodium iodale ) (Progressive Regressive) (frozen) (Sodium iodole) (Progressive) (frozen sections, biopsy specimens) (Natural) (Progressive) (histopathology and botanica specimens) (Sodium iodate) (Progressive Regressive) (nuclear counterstain) (special stain.) (Alcoholic iodine) (Progressive) (Celestine blue.)

	Ripaning Agent	Types of Staining/ Remarks
Harris	(_______)	Types of Staining: Remarks: Used for (______, _____, _____)
Ehrlich's	(_______)	Types of Staining: Remarks:Not an ideal stain for (_____) sections

Gill's	(______)	Types of Staining: Remarks: Used in
Delafield's	(______)	Types of Staining: Remarks: Used for
Mayer's	(_______)	Types of Staining: Remarks: Used as a (_________) to demonstrate the presence of cytoplasmic glycogen by (_______)

Cole's	(_______)	Types of Staining: Remarks: Used in sequence with (_________)

(BLUEING) (soluble red color) (insoluble blue color) (Ammonia water) (Dilute lithium carbonate) (Scott’s tap water)

● After the alum hematoxylin, (________) step converts the

initial (_________) of the hematoxylin within the

nucleus to an (_____________).

● The alkaline pH of the bluing solution causes the mordant

dye-lake to reform in the tissue and become more

permanent

● Blueing solutions are:

● (_____________)

● (_____________)

● (______________) (potassium carbonate, magnesium

sulphate and water)

(Iron Hematoxylin Stains) (Iron salts) (nuclei black or dark gray) (Ferric chloride) (Regressive) (Muscle and connective tissue fibers) (Ferric ammonium chloride) (Regressive) (Mitochondria. muscle striations. chromatin, myelin) (Phosphotungstic acid) (Natural and/or Potassium permanganate) (Stains muscle blue) (red-brown ) (spermatogenesis)

(_______________)

● Mordant: (_____) (Ferric chloride, Ferric ammonium sulfate)

● Appearance: Stains (__________)

● Uses: Preferred for elastic fibers, mitochondria, muscle striations, and

nuclear detail in cytolog

	Mordant	Staining & Remarks
Weigert’s	(________)	Staining:(________) Remarks: (____________) Add to Van Gieson Stain: for collagen, E histolytica
Heidenhain’s	(________)	Staining:(_________) Remarks:(________)

Tungsten Hematoxylin Stains (Phosphotungstic acid)

	Mordant	Ripening Agent & Remarks
Mallory's (PTAH)	(________)	Ripening Agent:(_______) Remarks: (___________), collagen (_______) For muscle striations, fibrins

Copper Hematoxylin Stains

● For (__________)

(Secondary stain, counter stain, cytoplasmic stain) (AFTER) (BEFORE) (Eosin Y) (Eosin B) (Eosin ES)

(_________, __________, _______________)

● Acidic stain

● Counterstain (_______) hematoxylin

● Counterstain (_________) methylene blue

● 3 forms:

○ (________)- most common

○ (________)

○ (________)- ethyl eosin

(blue to blue black) (dark blue Cytoplasm, proteins in) (bright

orange-red) (purplish pink) (pink or light blue to dark blue) (purplish blue) (pink) (deep pink)

EXPECTED STAINING RESULTS

Nuclei: (____________)

Karyosome: (__________,_________)

edema fluid: pale pink

RBCs, eosinophilic granules, keratin: (________)

Basophilic cytoplasm, plasma cells, osteoblast:

(____________)

Cartilage: (____________)

Calcium and calcified bone: (_______)

Decalcified bone matrix, collagen, osteoid: (_____)

Muscle fibers: (___________)

(Van Gieson Stain) (Acid fuchsin and picric acid ) (DNA) (RNA)

Other Staining Solutions

(____________)

• (_______&_______)

• Demonstrate connective tissues

-Acid Fuchsin (Masson stain)

• stain collagen, smooth muscle, mitochondria

Acridine Orange

• Identify dead and living cells Green fluorescence - (____)

• Red fluorescence - (____)

• nucleic acid-selective fluorescent cationic dye useful for cell cycle determination

(Acridine Red 3B) (Alcian Blue) (chlorophyll)

(connective tissues and epithelial mucin)

Other Staining Solutions

(___________)

• Demonstrate deposits of calcium salts

and sites of phosphatase

activity.

(_____)

• Same as (______)

• Stains acid mucopolysaccharides

• Excellent stain

• Resistant to counterstains

• Specific for (_________&___________)

(Aniline Blue) (Aniline blue + distilled water + glacial acetic acid) (Basic Fuchsin ) (smooth muscle) (Feulgen's and Schiff’s)

(__________)

• Cvtoplasmic stain

• Counterstain epithelial sections

• (_________+________+_________)

(__________)

• Plasma stain

• Deep staining of acid fast organisms

• Mitochondria

• Differentiation of (__________)

• Main ingredient of (_______&________) detect aldehydes

• Van Gieson : connective, mucins, elastic tissue

(Carmine) (glycogen (Best Carmine

Solution)) (Celestine Blue) (Oxazine dye)

Other Staining Solutions

(_______)

• Chromatin stain for fresh materials

• Combined with aluminum chloride to

stain (__________________)

(___________)

• (__________), alternative to iron

hematoxylin nuclear stain

• Resistant to strong acid dyes

• Routine stain for fixed tissues

• Good nuclear definition

(Congo Red) (4% aqueous solution in) (Crystal Violet) (crystal violet + methyl violet + dexterin) (Cresyl Violet)

Other Staining Solutions

(____________)

• Indicator

• Stain for axis cylinders in embryos

• Used as (_______________) in Krajian's method - elastic tissues, amyloid & myelin

(_________)

• Nuclear stain

• Stain amyloid in frozen sections, platelets

• Gentian violet: (___________+_______+_________)

(_____________)

• stain nervous tissues

• stains neuronal cytoplasm (Nissl bodies)

(Giemsa) (methylene blue and eosin ) (Gold Sublimate) (metallic

• )

Other Staining Solutions

(____________)

• Mixture of (________&_______)

• Stain blood to differentiate leukocytes

(_________)

• Stain used for (_________)

• impregnation

• Gold chloride + mercuric chloride

(Iodine) (starch granules) (Gram's Iodine) (Lugol's Iodine

Other Staining Solutions

(______)

• Oldest stain

• Used for microscopic study of (_________)

• Stains amyloid, cellulose, starch, carotene &

glycogen

• Used for removal of mercuric fixative artifact pigments

Reagent to alter crystal and methyl violet

• (__________) - identify and

differentiate bacteria

• (__________) - turns black in the

Reagent to presence of starches

(Janus Green B) (Malachite Green) (ascaris eggs and erythrocytes ) ()

Other Staining Solutions

(_____________)

• Demonstrate mitochondria during intravital staining

(______________)

• Contrast stain for (________&______)

• Bacterial spore

• Used as a decolorizer and counterstain

(Methyl Green) (green) (Methylene Blue nuclear stain ) (Polychroming)

Other Staining Solutions

(__________)

• Stains chromatin (________)

• Gives false positive reactions mucin

(____________)

• "(_________)" - oxidation of methylene blue Basic

• Stain for plasma cells

• Cytological examinations of fresh sputum for malignant cells

• Bacterial stain

• For diagnosis of diphtheria Vital staining of tissue

(Nile Red) (Nile blue with sulfuric) (Oil Red) (neutral lipids and fatty acids)

Other Staining Solutions

(__________)

• Formed by boiling (____, ___)

• lipophilic stain, in lipid globules

(________)

• Stain for (_____________)

• Rapid and simple stain

• Useful in identifying fat emboli in the lung or clot sections in the blood

(Orcein) (Osmium Tetroxide) (black)

Other Staining Solutions

(_____)

• Excellent stain

for elastic fibers

• Recommended for

dermatological

studies

(____________)

Selective stain for unsaturated

lipids and lipoproteins like myelin

(_____)

(glycogen, mucin, mucoprotein, glycoproteins, basement membranes, capsules) (amylase pre-digestion) (reddish-purple color)

Other Staining Solutions

Periodic Acid Schiff

• Stains(______, ____, _________, ______, _____, _____), blood vessels, fungi, and intravellular carbohydrates

• Stringly stains mucus-secreting cells

• Glycogen staining can be removed with an (__________) step

• Works by periodic acid oxidation of hudroxyl groups into aldehydes, which react with Schiff reagent to produce a (______________)

(Prussian Blue) (ferric ferrocyanide) (blood and lymph vessel) (Rhodamine B)

Other Staining Solutions

(___________)

• Colored salt of (____________)

• Used for microanatomical color

contrast of specimens

• Demonstration of (_______&_____) by injection

(___________)

• Used with osmic acid to fix

• Stain blood and glandular tissues

(Silver Nitrate) (Safranin O) (Toluidine Blue) (Nissl granules)

Other Staining Solutions

(_________)

• Used 10% aqueous solution

• For spirochetes, reticulum, and counterstain other fiber stains

(___________)

• Nuclear stain

• Primarily as a counterstain in Gram staining

(__________)

• Nuclear stain for fixed tissues

• Substitute thionine frozen sections

• Staining (___________) or chromophilic bodies

(Victoria Blue) (Wright Stain) (Basophilia) (Azurophilia) (Acidophilia) (Neutrophilia)

Other Staining Solution

(___________)

• Demonstration of neuroglia in frozen

sections

(__________)

• For blood cells

4 major staining properties:

(__________)

• (affinity for methylene blue)

(__________))

• (for oxidation of methylene blue)

(____________)

• (affinity for eosin)

(__________)

• (affinity for the complex of dyes)

(Not real dyes (no auxochrome groups) (BLACK) (ORANGE) (RED)

Oil Soluble Dyes (Lysochromes

• (________________________)

• Soluble in lipid medium of tissues

O

Sudan Black B → (_____)

Sudan III → (__________)

Sudan IV (Scharlach R) → (________)

(Sudan Black) (0.5%) (70% ethanol) (blue-black)

Oil Soluble Dyes (Lysochron

(___________)

• Most sensitive of the lysochromes

• Greater affinity to phospholipids & neutral fats

• The ability of fats to adsorb the stain is related to dye concentration,

temperature and physical state of fats.

• Prepared as (_____) solution boiled in (________)

for 10 minutes under a reflux condenser

• Stains phospholipids and neutral fats

Resulting color: (________)

(Sudan IV) (2% benzoic acid (in 70% alcohol) (Benzoic acid) (intense red)

0il Soluble Dyes (ysochromes

(________)

• Scharlach R

• Not stain phospholipids

• Prepared by saturating the dye in one part

(___________(_______) and one

part of acetone.

• (______) → intensifies fat & prevents

deterioration of solution

• Neutral lipids = (__________)

(Sudan III) (lighter orange)

Oil Soluble Dyes (Lysochromes)

(_________)

• First Sudan dye to be introduced

• Not stain phospholipids

• Good fats stain for CNS

• Resulting color = (__________)