VEM 023- LESSON 1.pdf

VEM 023: VETERINARY MICROSCOPIC ANATOMY (HISTOLOGY)
MICROSCOPY
Light Microscopy Electron Microscopy
1. Examination of living cells and tissues
2. Examination of killed cells and tissues
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)

Electron Microscopy
Uses a beam of electrons and
their wave-like characteristics
to magnify an object's image,
unlike the optical microscope
that uses visible light to
magnify images.

• Transmission Electron Microscope (TEM)

• Scanning Electron Microscope (SEM)

Light Microscopy
• Examination of living cells and tissues • Examination of killed cells and tissues
Live cell imaging through time-lapse
microscopy.
a. Smear
b. Teasing
c. Paraffin Technique

Human cancer cells under a
live cell microscope.

Time-lapse microscopy of
neurons growing in a cell
culture.

1. SMEAR
- Usually used for blood and
sperms.
PROS: A few cells are damaged
CONS: Uneven thickness and
staining

2. TEASING
- for separating nerve bundles
• Nerve bundle is placed on the
glass slide.
• With a needle point, the nerve
fibers in the bundle are
separated (teased apart) and
spread over the slide.

Slide Preparation
• Paraffin Technique
Tissues are fixed and embedded in wax,
making it easier to cut sections from
which are then stained.
1.Excision
2.Fixation
3.Washing
4.Dehydration
5.Clearing
6.Embedding
7.Sectioning
8.Staining
8 STEPS

Eat Fries With Delicious
Cheese, get Extra SauceS!
• Eat: Excision
• Fries: Fixation
• With: Washing
• Delicious: Dehydration
• Cheese: Clearing
• Extra: Embedding
• Sauces: Sectioning
• sauceS: Staining

1. Excision
• Initial and most critical step
• Samples collected through biopsy
or within 5 mins after death
• Tissues are cut into small pieces
Tissue slices should not exceed 2.5 × 2.0 × 0.4 cm in a standard
tissue processing cassette.

2. Fixation
• To preserve the tissue and
stops post-mortem autolysis
by inactivating autolytic
enzymes.
MOST COMMON FIXATIVE:
10% Neutral Buffered Formalin
Adrenal gland, brain, eyes, and few
other soft delicate structures: Bouin’s
or Karnovsky’s are preferred.

2. Fixation
• Takes 24 to 72 hours
• Softer tissues = shorter period
Fixatives must fully diffuse through
the tissues to preserve all cells
Cut into small fragments to
facilitate penetration

3. Washing
• Washing of fixed samples
• Remove excess fixative
• Cassette/gauze is immersed in
gently running tap water from
15 to 30 mins.

4. Dehydration
• To remove 75% of water in tissues
• Water is not miscible with
paraffin wax
Dehydrating agent:
ETHANOL (ABSOLUTE ALCOHOL)
in increasing concentration.

Done gradually in increasing concentration (from 50% to 100%) to prevent
tissue shrinkage which occurs during sudden withdrawal of water.
70%
ETOH
90%
ETOH
100%
ETOH
100%
ETOH
100%
ETOH
100%
ETOH
15
mins
15
mins
15
mins
15
mins
30
mins
45
mins

5. Clearing
• To remove alcohol introduced
during dehydration
Ø Alcohol not miscible with
paraffin
Clearing agent: XYLENE

Xylene
(I)
20
mins
20
mins
45
mins
Xylene
(II)
Xylene
(III)
Xylene removes alcohol. It is essential before embedding the tissue with
paraffin because alcohol and paraffin do not mix.

6. Embedding
To support the tissue during the
cutting process without altering the
morphology of the specimen.
Infiltration agent:
XYLENE-SOFT PARAFFIN MIXTURE

Wax
(I)
30
mins
30
mins
45
mins
Wax
(II)
Wax
(III)
Samples are placed in a mold
(“boats”) and allowed to solidify for
24 hrs.

7. Sectioning
The hardened block with tissue and
surrounding embedding medium is
trimmed and placed for sectioning in an
instrument called a MICROTOME.

Hundreds of sections can be obtained
from a single specimen. This process is
called SERIAL SECTIONING, since all the
slides are generated from the same
tissue.
Each section: 3.0 to 6.0 um

Ribbon of sections Cut section is then to be float in the
water bath for it helps to remove
wrinkles and spread the specimen.

Glass slide is then kept on a slide
warmer at 58 Deg C temp for 15-20
min to ensure adhesion.
Floated section is then picked
up on an adhesive coated
glass slide.

8. Staining
Stains are used to enhance the
visualization of cellular
components, each will highlight a
different cellular structure.

o Paraffin is removed by immersing tissue sections in 2 changes of xylene
o Sections prepared for rehydration by immersing in decreasing conc. of
ethanol (removes xylene)
o Sections are rehydrated by immersing in gently running water
Xylene
(I)
20
mins
20
mins
Xylene
(II)
100%
ETOH
95%
ETOH
85%
ETOH
70%
ETOH
30
mins
15
mins
15
mins
15
mins
15 - 30
mins

• Hematoxylin and Eosin
Hematoxylin Eosin
Localizes to negatively charged
cell structures
Localizes positively charged cell
structures
DNA, RNA , carbohydrates in
cartilage, collagen
Proteins, mitochondria,
cytoplasm
Basophilic Eosinophilic
Blue dye Red/pink dye

Hepatocytes Renal tubular cells Hyaline Cartilage Cardiac Muscle

Cells with abundant cytoplasmic proteins show more intense
eosinophilia.

• Periodic Acid-Schiff (PAS)
PAS
Highlights carbohydrates
Golgi apparatus, plasma
membrane, and the goblet cells
PAS positive (PAS+)
Bright magenta (purple)

• Special Stains
Osmic Acid
- reacts with fat to give grey-
black color
Golgi Silver Staining
- visualization technique for
nervous system cells.
-black stain to neurons

Stain for Elastin
- stain elastin-rich structures brown or shades of
purple
1. Weigerts’ resorcin fuchsin
2. Aldehyde fuchsin
3. Orcein
Stain for Lipid
- demonstrate lipid droplets and myelin
1. Oil red O
2. Sudan black
3. Osmic acid (Osmium
tetroxide)

o Stained sections are washed by water to remove excess stains.
o Stained sections are dehydrated by immersing in increasing conc. of
ethanol (removes water)
o Stained sections are cleared by immersing in 2 changes of xylene
Xylene
(I)
20
mins
20
mins
Xylene
(II)
70%
ETOH
85%
ETOH
95%
ETOH
100%
ETOH
15
mins
15
mins
15
mins
30
mins

o Stained sections are mounted to prevent drying of tissue and enhances resolution

Fixation using Formalin
Tissue Processing
Washing
Dehydration
Ethanol
Xylene
Clearing
Paraffin
which makes it easier to
Embedding
Sectioning
Staining

Frozen Sections
• used for FASTER RESULTS
(e.g. during surgery)
• Tissue samples analyzed
quickly before completing
the surgery: 10 MINUTES

• the mass is embedded in
OPTIMAL CUTTING
TEMPERATURE COMPOUNT
(OCT)

Interpretation of Slides
• Types of tissue sections:
LONGITUDINAL SECTION CROSS SECTION OBLIQUE SECTION
- cut along the longest direction of
an organ
- cut perpendicular to the length
of an organ
- cut at an angle between cross
and longitudinal section

Jejunum, in H&E
[x.s.] / [c.s.] [l.s.]

Artifacts
- features of a prepared slide that are not
present in the original sample but are
introduced during tissue preparation.
- Just one flawed laboratory technique can
spoil the final result.

Tissue sections
can appear “jarred”
or out of focus
Elongations and
distortions in
cells/tissues
PINCHING/CRUSHING
o an artifact as a result of using a dull knife or forceps when
handling tissue samples.

POSTMORTEM CHANGE
o an artifact due to delayed fixation
Shrunken/faded
structures or cells
Diffused gaps
around
cells/structures
There may be
presence of bacteria
Poorly define/absent
nucleus

Cracking in center of
liver caused by
inadequate fixation.

DARK PRECIPITATE / FORMALIN STAINS
o an artifact due inadequate washing of tissues
Black/brown stains
that appears in
different levels of
resolution

SHRINKAGE
o an artifact that is mainly due to sudden dehydration.
Irregular spaces between
portions of thin/compressed
tissues

SCRATCHES
o an artifact that appears
due to flaws, nicks, or
dirt on the cutting edge
of the microtome blade
Straight slashes or ragged tears
that are parallel with each other

CHATTER / “VENETIAN BLINDS”
o an artifact that appears due loosened microtome knife, resulting in
vibrations.
Narrow parallel bands
that are evenly
spaced

FOLDS AND WRINKLES
o an artifact that results due
to failure of the tissue
section to stretch out
completely when they are
picked up and mounted on
glass slides.
FOLDS: dark bands of
overlapping tissues
WRINKLES: tiny
corrugations/waves
across tissue

Black arrow: tear
Blue arrow: debris

VEM 023- LESSON 1.pdf

Recommended

Recommended

More Related Content

Similar to VEM 023- LESSON 1.pdf

Similar to VEM 023- LESSON 1.pdf (20)

Recently uploaded

Recently uploaded (20)

VEM 023- LESSON 1.pdf