dr. Alya Amila Fitrie, M.Kes dr.

Lokot Donna Lubis Department of Histology, Medicine Faculty University of Sumatera Utara Medan 2010

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Explain in general how to study the cell

Describes methods to study cells with tissue processing and staining, and the benefits of cell and tissue staining

Describes the various types of microscopes, how microscope works, and the benefits of them

Explains the terms used in the observation of histological preparations

Microscopic
Biochemistry

Cell culture

Histology is the study of the microscopic structure of tissue

Histotechnique

method of making histological preparations of a particular specimen through a series of processes to become the preparations were ready for analysis.

Tissue source: Human Animal

Fiksasi (Fixation);

Dehidrasi (Dehydration)

Pembeningan (Clearing)

Pemotongan Jaringan (Sectioning)

Pengecoran (Blocking/Casting)

Pembenaman (Infiltration/Impregn ation/Embedding)

Pewarnaan (Staining)

Perekatan (Mounting)

Pelabelan (Labelling)

To preserve tissue architecture, tissue must be first be fixed Chemical reagents are used to hold tissue components in their normal places during the subsequent processing They also prevent tissue from rotting and auto-digestion by leaked lysosomes Protective from microbial attack Fixative only preserve protein

Fixation effect to the processed tissues:

Inhibit the process of decay and autolisis Tissue preservation Tissue hardening Solidified colloid Optical differentiation Effect of staining

Things that must be observed in histological tissue fixation is :

Tissue

Thickness Tissue part to demonstrate

Fixation solution

Volume. Type of fixation solution pH

 Aim

: Eliminating the water in cells

Alkohol
Alkohol 70%, 80%, 90%, masing-masing 1 hari; Alkohol 95% 2 hari ( 2x ganti); Alkohol 100 % 2 hari ( 2x ganti).

Spiritus atau metanol Sukrosa 20% Aseton

jarang digunakan tetapi dapat digunakan seperti etanol;

Sukrosa 20% selama 2 hari;

3 x 20 menit.

 As

wax and resin arent miscible with alcohol either, the alcohol is replaced with an organic substances called xylene and the tissue goes transparent

 Substances

that are used:

Benzene/ benzol Benzyl benzoat Xylene/xylol

Chloroform

Cedar wood oil

Methyl benzoat

Embedding tissue provides them with physical supports so they can be sectioned thinly.

The process involves hardening the tissues by replacing the water with resin or wax.

Neither resin nor wax miscible with water however, so water is gradually replaced with alcohol in process called dehydration.

Tools : Histoplate from plastic (casette) and metal plate (mold).

L shaped piece of metal (Leuckhart) Arrange 2 pieces of the iron pieces of sheet metal to form a

Spatula for attaching paraffin blocks. Label this tissue identity when blocking.

cuboid space; Pour a little liquid paraffin at the edges to prevent leaking; Put the tissue; Pour paraffin enough to cover the whole tissue; Avoid air bubble formation.

 For

optimum viewing, the embedded wax block must be sectioned as finely as possible The ideal thickness of the section would be no wider than the diameter of the cells (57) for light microscopy. Sectioning done by microtome

 The

thinly cut sections of ribbon are placed onto warm water to flatten them out A glass slide is placed towards the thinly ribbon which picks it up from the water

Cells are virtually colourless must be stained to be visualised

Routine stain distinguish the nucleus from the cytoplasm

Spesific stains : identify spesific cellular, chemical or enzyme components such as carbohydrate.

Immunohistochemistry (IHC) : identify spesific protein in the cell.

First process to stain de-waxing

Most conventional stain are water based and cant mix with wax the wax must be removed from the tissue in order for the stain to penetrate into the tissue
The process is the reverse of before :
Section dipped into xylene to remove the wax Dipped into alcohol Dipped into water

Eosin

a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fiber for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic.

Hematoxylin

a basic dye which binds to nucleic acid (binds to ribosomes and rough endoplasmic reticulum due to DNA/RNA content) it stains them a purple-blue.

Collagen fiber

Light microscope Fluorescent microscope Phase contrast microscope Dark field microscope Electron microscope

 To

be viewed under a microscope cells must be within the microscopes limit or resolution. The rsolving power of an electron microscope (EM) is around 400x of a light microscope (LM).

The nature and character staining

Asidofilik, Basofilik

Shape
Similarities to other forms the position of something on the polarity of the cells

Cuboidal, Columnar, spindle, squamous, reticular Star-shaped appearance, Umbrella sign, Ladder like, Signet ring cell Apical, Basal, Centric, Eccentric, Adluminal, Fairy nuclear

 Histology.

Tissue Processing. Download from www.jameswatts.co.uk Zulham. Bahan kuliah : Dasar-dasar memahami sel dan jaringan. Alberts B et al. 2002. Molecular Biology of the Cell 4th Ed. New York. Garland Science. Young B, Heath JW. Histology A Text and Atlas. 4th Ed. H&E Staining. ABNOVA, http://www.youtube.com/watch?v=2D0rj0m6 dVs

Thank you