Preparation of tissues for microscopic examination

Permanent microscopic preparation

Examining cells, fragments of tissues or organs, after their vital processes have been stopped by fixation Allows the examination in light microscopy of the structure and main characteristics of cells

Permanent microscopic slide

Purpose preserving cellular structures and the relations between them indefinitely Used in: - clinics - morphopathology - forensics - research purposes - educational purposes

Steps in obtaining permanent microscopic slides1. 2. 3. 4. 5. 6. 7. 8.

Recoltation Fixation Clearing Embedding Sectioning Staining Mounting Labeling

Recoltation

Obtaining the sample to be examined from humans, living animals or corps Recoltation:Biopsic Necropsic

Biopsic recoltation

Obtaining the sample from living humans or animals Diagnostic purpose Obtained by: - harvesting cells from the mucous layer -needle biopsy -aspiration -surgery

Necropsic recoltation

After sacrificing the animal; in specialised laboratories, in research or educational purposes Recoltation from human corps in morphopathology (anatomopathology) or forensics laboratories

Conditions for a good recoltation

As soon as possible from the moment of death, in order to avoid autolysis processes Sample dimension no more than 5 mm Quality of instruments sharp, to avoid loosing the parallelism between the plans Adequate to the organ type changes in the shape, volume will be avoided

Fixation

The decisive step in obtaining a permanent microscopic slide A physical or chemical process that stops as soon as possible vital cellular processes, maintaining with minimal alteration the shape, volume, spatial and molecular relation between elements

Fixation

By fixation we intend:1.

2. 3. 4.

To conserve the tissue from autolysis and bacterial attack To prevent the loss of cellular constituents To increase optical differentiation of cellular structures To increase tissue consistency, in order to facilitate their going through the other steps of the technique especially slicing

Fixatives classification

Physical heat, microwaves Chemical:Aldehydes formaldehyde, glutaraldehyde, acrolein Oxidizing agents osmium tetraoxide, potassium permanganate, potassium dichromate Protein denaturating agents acetic acid, methyl alcohol, ethyl alcohol Miscellaneous mercuric chloride, picric acid, non aldehyde containing fixatives

Fixatives classification

Simple formaldehyde, glutaraldehyde, acetic acid, metyl alcohol, osmium tetraoxide Mixtures used due to their unequal affinity for various structural elements

Fixatives classification

Mixtures of fixatives:

By chemical nature:Aqueous Alcoholic

By purpose:Universal / topographic Cytologic

Fixation mechanism

Formes cross-links between proteins, thereby crossforming a gel keeps structures in their in vivo relations to one another Soluble proteins are fixed to structural proteins insoluble gives mechanical strength for next steps Protein denaturation

Qualities of a good fixative

Good tissue penetration Stabilizes the tissue, preserving the character and distribution of cellular components Prevents fixation artefacts Prevents structure deformation maintaining shape and volume Preservs cellular constituents

Qualities of a good fixative

Destroys microorganisms Extracts inactivated autolytic enzymes Increases tissue consistancy Confers optical differentiation Maintains its chemical composition Cheap, nontoxic, nonflamable, nonirritant

PostPost-fixation treatments

Decalcification for bone Sample dissociation Mordansation helps both fixation and staining

Embedding

Process that creates optimal conditions for slicing the samples in thin sections ( m), transparent and with parallel sides, in order to examine them by light microscope

Embedding

Embedding masses:

By the embedding process:Penetrates the cell Remains in the intercellular space

By water solubility:Anhydrous: paraffin, celloidin, paraplast paraffin, Aqueous: gelatin, polyethylene glycol

Paraffin embedding previous steps

Dehydration

Acohol in increasing concentrations, from 50 degrees to absolute alcohol, each in 3 succesive baths Removing the alcohol from the sample, using clearing agents, miscible with paraffin wax; they give the sample a translucent appearance. Most used xylene, toluene, chloroform

Clearing

Paraffin embedding

Impregnation Soaking the sample in melted paraffin, than solidification in block by cooling to confer an omogenous consistency The solid paraffin block is mounted on an object holder

Sectioning

The embedded or frosen sample is cut in thin slices ( m), using a microtome Steps:Modeling the block for sectioning Mounting the block on the object holder Sectioning Mounting the sections on slide

Staining

Intends to increase the contrast between cell or tissue components by modifying refraction indexes of the morphologic substrate, using dyes Staining technique depends on fixative nature and on the tissue

Mounting

Protection of fragile sections , dye preservation, offering an omogenous and transparent optic environment necessary to microscopic examination The specimen is fixed between the slide and the cover glass within different mounting environment

Mounting

Mounting environment:Aqueous: glycerine, Apathy syrup Anhydrous: Canada balsam

Labeling

It will be noted:Celullar type Recoltation source The fixative The staining Date