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

Heaviest particles

Particles with lower specific densities

Centrifuge

1g liver

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