Histological Techniques

HISTOLOGICAL
TECHNIQUES
Histology
  G. “histos” – tissue
  study of group of similarly group of cells or tissues

bound together by varying amount of intercellular
substances for the performance of specific function/s
Preparation of Tissues for Light
Microscopic Examination
1.  Obtaining the specimen
- surgical specimen that

are normal or diseased
tissue
- forceps and sharp
razor blade
2. Fixation
- tissue immersed in
chemical (fixative)
- preserve the protoplasm
Disadvantage: precipitation of proteins
- best fixative produce fine precipitates
ex. Neutral formalin, osmic acid
  choice of fixative depends on the cellular component that

should be preserved
3. Processing
a. Dehydration
- immerse in increasing concentrations of

alcohol
- removes water from the tissue w/o cellular

damage
b. Clearing
- tissue is made transparent
- xylol, chloroform, benzene
or cedarwood oil
c. Embedding
- killed and dehydrated tissue
encased in block of solid paraffin or
celloidin
d.Sectioning
- small block of paraffin

containing the tissue is
mounted in microtome
Microtome
Designed to cut thin slices

(thickness is bet. 2 and 10
microns or 0.002-0.010 µm)
Paraffin affixed to a slide

(dissolve)
Remove dissolved paraffin

e.Staining
-unstained tissue (transparent)

-dyeing or metallic coating
of different tissue component
- H&E is used
hematoxylin – basic
eosin – acid dye
Eosinophils, rbc, cytoplasm of

parietal cells stain with acid
dye
Hyaline matrix, cytoplasm of
glandular cells, neurons bind
with basic dyes
f. Mounting
- stained sections placed on a

slide with a medium that
hardens
- coverslip is placed over
the section
Purpose:
 Protection
 Make a permanent preparation

1. Direct observation of living tissues
and cells
  Cells are studied while still alive
  Colorless
  Phase contrast microscope
  Amoeboid movement
  phagocytic activity of blood cells

a.  Exteriorization and transillumination of organs
- brought outside the body cavity
- sterile moist chamber for transillumination
and direct microscopic examination
ex.
Release of secretory material from
pancreatic cell
ovulation
2. Cell, Tissue and Organ Culture
  in vitro “in glass” cultivation
Cell Culture
- non-adherent, dividing cells are

transferred from vessel to vessel
ex.
Short term culture of
wbc
Tissue Culture
- ( immature tissue
culture) a.k.a. “explant”
- embedded in coagulum
of blood plasma +
embryonic juice
- incubated
- cells proliferate in the
zone of outgrowth
  Organ Culture
- maintenance of mature tissue or organ
fragments
Use: study direct effects of drugs or hormones
on various tissue
3. Mechanical Micromanipulation and
Microdissection
Instrument: Microminipulator
Application:
- moves glass needles or pipettes so a

single cell can be manipulated or
directed
-  moves fixed cells while studied

under SEM
-  granules, vacuoles , mitochondria

can be moved
4. Use of Radiation Probes
  selective staining of organelles with colored dye
  use of high power organ lasers
  beams of protons and beams of UV irradiate small areas

of living cells
  specific cell organelles are destroyed to assess its effect

on the cell
5. Cinematography
•  motion pictures taken thru the objectives of
microscope
Use/Application:
1. record cell activity
ex. movement of cells
and organelles
mitosis
phagocytosis
muscle contraction
movement of cilia
6. Differential Centrifugation
  One of the most valuable & widely used methods in biological chemistry.
Cells (disrupted)
homogenate collected (cell organelles and inclusions)
Layered in sucrose
Centrifuge at high speed (maintained above freezing temp.)
  Heaviest particles are sedimented first
  Lower specific densities are brought down by successive centrifugation
  Centrifugal methods isolate fractions of nuclei, nucleoli, mitochondria, lysosomes ,

ribosomes, pigment and secretory granules
1g liver
Heaviest particles
Centrifuge
Particles with lower

specific densities
7. Microincineration
  Tissue slices leaves ash which retain fine structural
details
  Minerals can be identified within the cells

8. Frozen Section Method
•  tissue is placed directly on
a stage of special
microtome with an outlet
for carbon dioxide.
•  Use: for sectioning biopsy

material
•  operation for examination

of cancer cell.
9. Freeze drying technique
  Tissue is frozen &
dehydrated at low
temperature in high
vacuum
  Reagents are added to

make protein content of
tissue insoluble
10. Use of Stain
Purpose:
-differentiate tissue elements as
certain cellular elements to
produce a contrast
Classification of Stains
1. Acid stain
- cytoplasmic stain
- affect the cytoplasm of the cell
ex. eosin
2. Basic Stain
- nuclear stain
- special affinity for nuclei
  Histological sections are stained with basic and acidic
stain
ex. hematoxylin and eosin (H&E)
nuclear structures – stained dark purple or blue
intercellular material – stained pink
Methods of Staining
1.  Staining fresh on living unfixed tissue cells
- vital staining
a. Intra Vital staining
- staining of cells in living body by IV
injection of dyes
Ex. Trypan blue
b. Supra Vital staining
- cells are removed from the organism
- stain added to the medium of cells

Methods of Staining
2. Staining of fixed dead tissues
- tissues killed, embedded sectioned stained
and mounted on slide

Histological Techniques

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HISTOLOGICAL

 study of group of similarly group of cells or tissues

- surgical specimen that

- forceps and sharp

- preserve the protoplasm

Disadvantage: precipitation of proteins

- best fixative produce fine precipitates

ex. Neutral formalin, osmic acid

 choice of fixative depends on the cellular component that

- immerse in increasing concentrations of

- removes water from the tissue w/o cellular

- tissue is made transparent

- xylol, chloroform, benzene

- killed and dehydrated tissue

encased in block of solid paraffin or

- small block of paraffin

Designed to cut thin slices

Paraffin affixed to a slide

Remove dissolved paraffin

-unstained tissue (transparent)

Eosinophils, rbc, cytoplasm of

- stained sections placed on a

- coverslip is placed over

 Make a permanent preparation

 Phase contrast microscope

 phagocytic activity of blood cells

- brought outside the body cavity

- sterile moist chamber for transillumination

and direct microscopic examination

Release of secretory material from

- non-adherent, dividing cells are

Short term culture of

culture) a.k.a. “explant”

- cells proliferate in the

- maintenance of mature tissue or organ

Use: study direct effects of drugs or hormones

- moves glass needles or pipettes so a

- moves fixed cells while studied

- granules, vacuoles , mitochondria

 use of high power organ lasers

 beams of protons and beams of UV irradiate small areas

 specific cell organelles are destroyed to assess its effect

1. record cell activity

ex. movement of cells

homogenate collected (cell organelles and inclusions)

Centrifuge at high speed (maintained above freezing temp.)

 Heaviest particles are sedimented first

 Lower specific densities are brought down by successive centrifugation

 Centrifugal methods isolate fractions of nuclei, nucleoli, mitochondria, lysosomes ,

Particles with lower

 Minerals can be identified within the cells

• Use: for sectioning biopsy

• operation for examination

 Reagents are added to

a. Intra Vital staining

- staining of cells in living body by IV

Ex. Trypan blue

b. Supra Vital staining

- cells are removed from the organism

  study of group of similarly group of cells or tissues

  choice of fixative depends on the cellular component that

 Make a permanent preparation

  Phase contrast microscope

  phagocytic activity of blood cells

-  moves fixed cells while studied

-  granules, vacuoles , mitochondria

  use of high power organ lasers

  beams of protons and beams of UV irradiate small areas

  specific cell organelles are destroyed to assess its effect

  Heaviest particles are sedimented first

  Lower specific densities are brought down by successive centrifugation

  Centrifugal methods isolate fractions of nuclei, nucleoli, mitochondria, lysosomes ,

  Minerals can be identified within the cells

•  Use: for sectioning biopsy

•  operation for examination

  Reagents are added to