CHAPTER 16: PRINCIPLES of STAINING

PROCESS: DR NUCLEAR STAIN DB ACTUAL STAINING PROCESS - immerse the

COUNTERSTAIN DC sample [BEFORE or AFTER fixation and
mounting] in dye sol.
Deparaffinization
Rehydration: descending alc PARTS OF CELL:
Nuclear Stain (HEMATOXYLIN) ● acidic (stains blue/purple)
Differentiation ○ DNA (heterochromatin and
Bluing nucleolus) in NUCLEUS
Counterstain (EOSIN) ○ RNA in RIBOSOMES and in RER
Dehydration: ascending alc ● basic (stains red/pink)
Clearing and Mounting ○ CYTOPLASM
○ COLLAGEN
STAINING - process whereby tissue ○ CONNECTIVE TISSUE
components are made visible in microscopic ○ other structures that supports
sections by: cell
● direct interaction with:
○ dye or staining sol. MANY dyes require the use of a MORDANT
● chemical compound; reacts w/ stain →
● HEMATOXYLIN and EOSIN staining - forms insoluble, colored precipitate
ROUTINE histology staining on the tissue.
○ why? ● REMEMBER: dye solution washed
■ cheap, quick, informative away = MORDANT STAIN REMAINS
○ basic principle:
■ H - stains nuclear detail NOTE: color of stains, not real color of tissue
■ E - cytoplasmic detail of and structure. it depends on the stain
cell and tissue architecture
STAINING of PARAFFIN SECTIONS
COLORED COMPOUND is used to produce a PARAFFIN WAX - poorly permeable to most
CONTRAST btw dif. tissues and cellular staining sol.
components. BASED ON: ● SHOULD be removed from the section
● varying affinities prior to staining
○ How?
MAIN RESON why cells are stained: ○ immerse prffn sect. → solvent
● to ENHANCE contrast and visualization (XYLENE) 2 times at 1-2mins.
of cell or certain components under for sect. up to 10microns thick
microscope. ■ XYLENE (not miscible w
aq. sol and low conc alc.),
NOTE: Cells may be stained to: MUST BE REMOVED
● highlight metabolic processes USING ABSOLUTE
● differentiate between live and dead cells ALCOHOL → descending
● demonstrate the relationship between conc. of alc.
internal and external structures
● this PREVENTS
● identify different types of cells.
damage and
detachment of
HISTOLOGIC STAIN - purified form of coloring
sections
agent or crude dye that is applied in an
○ Alcohol → replaced w H2O b4
aqueous sol.
actual staining of sect.
● procedure is REVERSE of
IMPREGNATION. “sections to water”
 ● then the section is MOUNTED then ■ MUSCLE
dried. ■ CONNECTIVE TISSUE
if DRYING NOT COMPLETE = sect. esp. ■ NEUROLOGIC
BONE and NERVOUS TISSUE is detached
from slide during staining (after adding acid METHODS of STAINING
differentiator) 1. DIRECT STAINING
- process of giving color to sections by
use of ALCOHOLIC STAIN = NO need to using aq. or alcoholic dye sol.
REPLACE alcohol w H2O. - ONLY ONE DYE used
- washing: 30-60 seconds prior to drying
AFTER deparaffinization (w/ xylene), section and examination
transferred to decreasing conc. of alcohol = - BASIC DYE = (+) charge
"Sections to Alcohol" - CELL WALL and CYTOPLASM = (-)
charge
AFTER STAINING, section is dehydrated with - METHYLENE BLUE
INCREASING conc. of alcohol → cleared w/ 2 - example of simple stain
changes of xylene for mounting. - blue stains will color all cells blue
- stand out against bright
NOTE: background of LM
● most mountants are miscible with xylene
● second change of xylene = raise 2. INDIRECT STAINING
refractive index(RI) of glass slide = - process where action of dye is
reduced light refraction INTENSIFIED by ADDING ANOTHER
● if stained sect. will not be mounted let it REAGENT or a MORDANT
stay in XYLENE. - mordant, serves as link or bridge
btw tissue and dye
PROLONGED IMMERSION in ALCOHOL = - form a colored “lake” →
stains are removed combines with tissue →
“tissue-mordant-dye-complex”,
SECTIONS FLOAT OFF THE SLIDE when: which is insoluble in ordinary aq.
● slide is dirty or greasy and alcoholic solvents.
● sections not left in paraffin oven long - allows subsequent
enough to dry and fixed COUNTERSTAINING and
○ what to do? DEHYDRATION to be carried out
■ sections must be left in the - by ITSLEF = weak staining
oven for min. of 30 - mordant
minutes before staining. - applied to tissue before stain, or
- may be included as part of
HISTOLOGICAL STAINING staining technique, or
RECALL: - may be added to dye sol. itself
● HISTOLOGICAL STAINING is the - EXAMPLES of mordants:
process whereby tissue constituents are - potassium alum w/
demonstrated in sects by: hematoxylin in Ehrlich’s
○ direct interact w dye or staining Hematoxylin
sols. - iron in Weigert’s hematoxylin
■ result: coloration of
ACTIVE TISSUE
COMPONENT
● includes:
○ micro-anatomic stains
○ bacterial stains
○ specific tissue stains:
 - IN SOME TECHNIQUES
- applied separately
- or applied as combined
stain

*DIFFERENTIAL STAIN
- more than one chemical stain
- done by washing section in simple sol
- ACCENTUATOR, not essential to
(h2o or alc.) or by use of acids and
chemical union of tissue and dye.
oxidizing agents
- does not participate in reax. BUT
- usually controlled by following exact
ACCELERATES the reax.
times specified for staining, or by
- EXAMPLES of accentuators:
examination under the microscope.
- potassium hydroxide in
- if PRIMARY STAIN: basic dye
Loeffler’s methylene blue
- differentiation: acid solution
- phenol in carbol thionine
- if acidic dye
and carbol fuschin
- alkaline medium is used after
acidic dye
3. PROGRESSIVE STAINING
- ALCOHOL
- tissue elements stained in DEFINITE
- both differentiator for both basic
sequence
and acidic dyes
- staining solution applied for SPECIFIC
- how? dissolve excess dye
PERIODS of TIME
- MORDANTS
- not washed or decolorized
- act as differentiating agent
- relies on SELECTIVE AFFINITY of dye
- e.g., iron alum can oxidize
for diff. cell elements
hematoxylin
- uses:
4. REGRESSIVE STAINING
- WBC differential (abnormalities
- tissue is FIRST: OVERSTAINED →
in WBCs)
excess STAIN is REMOVED or
- Gram stain
DECOLORIZED.
- uses to dyes: crystal violet
- H&E stain
and fuschin or safranin
- most common
(counterstains)
- for MICRO-ANATOMICAL
- differentiate: G+ and G-
STUDIES using regressive
bacteria based on
staining
peptidoglycan layer of the
- consists of overstained
surface cell of bacteria
nuclei
- superfluous and excessive
METACHROMATIC STAINING
color of tissue constituent
- stain ortho chromatically (in color
by acid differentiation
shades that are similar to the color dye
itself)
5. DIFFERENTIATION (DECOLORIZATION)
- use of specific dyes which
- SELECTIVE removal of excess stain
DIFFERENTIATE PARTICULAR
from tissue during regressive staining
SUBSTANCES by staining w color thats
- this procedure DIFFERENTIATES /
different from stain itself
DISTINGUISHES types of staining
(metachromasia)
impart the same color to all bacteria and
- employed for staining:
other biological material. - CARTILAGE
- DIFFERENTIAL STAINING METHODS - CONNECTIVE TISSUES
- impart DISTINCTIVE COLOR - EPITHELIAL MUSCINS
only to certain types of bacteria - MAST CELL GRANULES & AMYLOID
 - involve IMMERSING SAMPLES → SUPRAVITAL STAINING
rinsing → observation - stains living cells removed from an
- METHYL VIOLET (e.g., crystal violet) organism.
give metachromatic staining - It differs from intravital staining,
- NOT CONSIDERED to be most - which introduces stains into the
effective for the purpose body.
- AZURES OR TOLUIDINE BLUE - Supravital stains, like:
- MORE EFFECTIVE - New Methylene Blue
- exception: amyloid when - Brilliant Cresyl Blue
significant metachromasia is - are toxic to the organism
given by amyloid deposits using and may create unique
crystal violet or methy violets. staining patterns within
cells.
METALLIC IMPREGNATION - Dilute solutions are used
- uses COLORLESS SOL. OF METALLIC to achieve desired effects
SALTS to highlight specific tissue (1:5,000 to 1:50,000).
elements by: - Common dyes include:
- creating an opaque, typically - NEUTRAL RED (best vital dye)
black deposit on the tissue or - JANUS GREEN
bacteria surface. - for mitochondria
- Unlike stains, these metallic - TRYPAN BLUE
impregnating agents are NOT - one dye dissolved in
ABSORBED 100mL of dH2O
- but form a physical precipitate or - used immediately
reduction product on the tissue. - DANGEROUS: stand
- Care is required due to potential more than 1hr (bcs toxic to
explosiveness of ammoniacal silver cell)
solutions. - Nile blue, Thionine, and Toluidine
blue.
VITAL STAINING
- selectively stains living cell constituents,
either by:
- phagocytosis of dye particles or
- staining pre-existing cellular
components.
- It helps REVEAL CYTOLOGICAL
DETAILS and CHEMICAL REAX within
cells.
- Vital stains are excluded by living cells
but taken up by dead cells, with
resistance in staining the nucleus of
living cells indicating cell death.

INTRAVITAL STAINING
- involves injecting dyes into the animal
body to color certain cells, particularly
those of the RETICULO-ENDOTHELIAL
SYSTEM.
- Common dyes include:
- lithium, carmine, and India ink.