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