Basic Histology and Histological Techniques (MLS-HIST 222Histopathology department

L2:Methods of prepration of tissue and cells-15/12/2019Musa Omer Musa

Contact no 0912918273

The Aim of histology

• Histology is the study of tissues (from Greek.histos=tissue).

• Firstly: This involves the examination of the architecture and

relationship of the different types of tissue.

• Secondary: The detailed investigation of the structure of the

individual cells(cytology).

• By combining this knowledge of microscopic anatomy and

cellular composition much has been learnt about the

physiological function of tissues.

• The aim of histology is to is to obtain the greatest possible

amount of information from the tissue by various

histopathological techniques.

• Normal histology: Is the study of normal tissue and cellular

composition.

• Histopathology: Is the study of abnormal tissue and cellular

composition. Or the changes occurs to tissue morphology and

cellular details due to pathogen.

• Cytology: the study of normal individual cellular structure

• Cytopathology: the study of abnormal individual cellular

structure

Anatomy

Macro

Histology

Micro

cytology

• Histology:

• Normal histology General tissues: epithelial tissue,

connective tissue, muscular tissue and nervous tissue

• And systemic organs

• Pathology:

• General and systemic pathology

• Cytology:

• Gynecological cytology and non gynecological cytology

Methods of preparation of tissues and cells

• Can be classified into:

• 1.Prepration of fresh (living) tissues and cells.

• 2.Prepration of fixed (dead) tissues and cells

1.prepration of fresh (living) tissues and cells.

1. Preparation of Fresh cells and tissues

The demonstration and examination of tissues and cells in their

living state.

Can be sub divide into:

a. Direct examination.

b. Dissociation or teasing method.

c. Vital stain

a. Direct examination

• Cells are suspended in a fluid ,such as blood or lymph in a drop

of fluid

• .The fluid may required dilution with an isotonic solution such as

normal saline (0.85%).

• Then exam under microscope.

• Why isotonic solution?

b. Dissociation or teasing method

• Cells that are grouped together in a loose tissue as in

subcutaneous connective tissue may also be examined

directly if the tissue is thin .if the tissue is thick or in

case of a solid organ .cells can be separated from one

another in a fluid medium like normal saline(isotonic

solution).

C. The vital staining method

• The staining and demonstration of living tissues and cells

Can be sub divide into:

• I. Intra vital stain: the injection of a dye into the living

organism then examined by ultra sound also kuffer’s cell in liver

can be exam by the same way in which the cells engulf the

foreign body (vital stain) and takes the stain,

• II. Supra vital stain: staining the living cells of an organism out

side the body in vitro .

• These cells are dissociate in the staining solution.

• Example of vital stains Janous green , Indian ink and methylene

blue.

• Demonstrated cell like reticulocytes(RBCs) and cellular

structure like mitochondria.

• Nuclear is resistant to vital stain Explain?

• Vital stain is consider as cytoplasm stain?

Intra & supra vital stain

2.The preparation of fixed (dead ) tissues and cells

• Cab be divided into :

• Cytological preparation

• Sectional methods

• Cytological preparation :fluids containing cells or tiny fragments of tissue

such as aspirated bone marrow ,are smeared upon a microscope slide ,the

adherent cells fixed (killed) in order to preserve their appearance.

• The smear stained to demonstrate their structure (nuclear and cytoplasm

) structure ! ?. Finally mounted and examine under microscope .

• Cytological or smear preparation methods includes:

• Scrap method :(exfolative cytology) cells smear fixed in

95%ethanol stained then exam under microscopy.

• Crush method: impress tissue between two slides then smeared

fixed in 95%ethanol stained then exam under microscope.

• Impression method: done by touch the cut surface of soft

tissue cells adhere into the slide fixed in 95% ethanol stained and

exam under microscope useful in study of soft tumor rapidly.

• Body fluid: like CSF ,peritoneum ,pleural and fine needle

aspiration these fluids centrifuge smear sediment cells and tissue

fix in 95% ethanol then exam under microscopy

• It is preferred that to fix smear will it is wet in 95% ethanol.

Smear preparation

Sectional method

• These involve the cutting of specimen into a very thin translucent

slices or sections and have the advantages preserve the micro

anatomical relation ship of tissue to each other .section cut by

microtome which produce thin section 3-5 um in thickness.

• Sectioning method done after the following steps fixation in (10%

formalin) , processing , embedding then cutting or sectioning .

• After that sections are staining , mount and then exam under

microscope.

• Sectioning methods can be divide into:

• Embedded sectional method: in which embedding medium use to

support tissue to enable cut thin section by microtome the most widely

use embedding medium is paraffin wax others like , celloidine , gelatin,

water soluble wax , agar and resin.

• Non embedding sectional method: the tissue fluid use as

supporting medium after frozen the tissue example freezing

microtome and cryostat techniques.

Embedded and non embedded section

Different between smear and sectioning preparation

Smear preparation Sectional preparation

Lack micro anatomical relation ship

micro anatomical relation ship preserve

Two dimensional cellular structure

Three dimensional structure

Large ,flattened cells Small cells

Cellular details easily seen Cellular details not easily seen

Smear preparation include impression ,crush, scrap

Divide into embedded and non embedded

Easley and rapid in preparation Take long time and needs skills

Section & smear preparation

Methods of examination

• Macroscopic examination: naked eye examination

• Microscopic examination :light microscope ,electron

microscope transmission E.M and scanning E.M

Normal liverHistology Anatomy

PathologySection Gross

Macroscopic examination

Microscopic examination

THE END