Microtechnique

Lecture 1
Dr. Esam Qnais
Hashemite University
 Microtechnique
Microtechnique: The art of preparing objects for
examination under the microscope (in our course
light microscope).

Steps of tissue preparation for light microscopic

examination:
1- Obtaining the specimen (Biopsy (medical removal
of tissue from living subjects) or Autopsy (medical
removal of tissue from dead subjects (corpse))
2- Preparing the specimen: Smear, squash, whole
mount, or sectioning.
3- Fixation
4- Dehydration: Gradual removal of water from the tissue using
ascending grads of ethyl alcohol to prevent tissue shrinking

5- Clearing: Replacement of alcohol in tissue by clearing fluid

like xylene, benzene, or acetone

6- Embedding: Tissues are impregnated in paraffin

7- Sectioning: Cutting with microtome

8- Mounting and staining

9- Labeling
Fixation: preservation the physical
structure of the tissue.
Methods of fixation:
1- Physical fixation:
A- Heat
B- Freezing
C- Drying
2- Chemical fixation

Advantages of chemical fixation:

1- to avoid tissue digestion (autolysis or microbial digestion)-
2- hardening of tissue
3- chemical fixation prevents shrinkage and swelling of tissue
during dehydration and embedding.
4- chemical fixation enhance tissues affinity for staining
5- chemical fixation makes tissue more resistant for heat during
embedding
6- chemical fixation enhances index of refraction for tissues
Criteria of good fixatives:
1- rate of penetration ()
2- shrinkage and swelling ()
3- hardens tissue
4- effects on staining (mordant)
mordant: enhances stain binding to tissue
5- coagulation of soluble protein
6- stability
7- price
8- chemical effects on tissue
 Fixatives
1- 100% formalin= 30-40% formaldehyde (gas)
- paraformaldehyde: white precipitate
- good for nervous tissues
- brown spots in spleen and liver (disadvantage)
2- glutaraldehyde
2- Bouins fixative (Picric acid fixative)
3- Susas fixative
4- Zenkers fixative (Metallic fixative)
5- Carnoys fixative (Alcoholic fixative)
6- Altmanns fixative
7- Flemming fixative
8- formalin-acetic acid alcohol (plant).
 Two types of chemical fixatives
1- Cross-linking protein (formalin)
- rate of penetration of embedding media ()
2- Denaturate of protein (acetone) precipitaion
- rate of penetration of embedding media ()
- it damages some organelles such as mitochondria
 The number of factors affecting the
fixation process includes buffering,
penetration, volume, temperature
and concentration
 In fixation pH is critical. This is especially the
case with formaldehyde, where acidity favors
the formation of formalin-heme pigment
deposits in tissue and may lead to protein
denaturation and structural deformations.
Sources of acidity include hypoxia of tissues
and oxidation of formaldehyde stocks to
formic acid. To prevent extremes of acidity or
basicity, fixatives are generally buffered to a
pH between 6-8. Common buffers include
phosphate, bicarbonate, cacodylate, and
veronal. Commercial formaldehyde fixatives
are often buffered with phosphate at a pH of
7.
The diffusibility of each fixative will determine its rate of
penetration through tissue. Formalin and alcohol penetrate the
fastest, glutaraldehyde the slowest, with mercurials and Mirsky's
Fixative falling between these extremes. Normally, slow rates of
penetration are only a problem when dealing with whole organ
perfusion, because thin tissue sections are easily permeated and not
affected by this variable. Speed of fixation can be dramatically
improved by the use of microwave protocols.
The standard ratio of fixative to tissue volume is 10:1. Lower
volumes can be used if frequent changes of the fixative are carried
out to prevent exhaustion. Agitation will enhance the process by
ensuring that fresh fixative solution is constantly washing over the
surface of the tissue.
Increasing the temperature will increase the speed of fixation, and
hot formaldehyde is often used in automated tissue processors. The
temperature used must be selected carefully, as thermal
denaturation of tissue proteins will begin to occur at extreme
temperatures. Generallconcentration of the fixative can affect the
rate of fixation and the y 60C is used with formaldehyde fixatives.
The total penetration of fixative into the sample. Too high a
concentration will lead to hardening of the tissue and the formation
of excessive artifacts. Too low a concentration will allow exhaustion
of the fixative before the process is complete
Duration of Fixation-
- Formalin (10%) 12hr-several weeks
- Flemming: 24hr

- Bouins fixative 12-24 hr

Methods of Fixation

1- immersion
2- perfusion
3- by leaving small or thin pieces in the vapour
of fixative
4- injection ( anatomy labs, museum)
5- phase partition fixation
 Methods of Fixation
perfusion
 Factors Affecting Fixation

In fixation pH is critical. This is especially the case with

formaldehyde, where acidity favors the formation of
formalin-heme pigment deposits in tissue and may lead
to protein denaturation and structural deformations.
Sources of acidity include hypoxia of tissues and
oxidation of formaldehyde stocks to formic acid. To
prevent extremes of acidity or basicity, fixatives are
generally buffered to a pH between 6-8. Common buffers
include phosphate, bicarbonate, cacodylate, and veronal.
Commercial formaldehyde fixatives are often buffered
with phosphate at a pH of 7.
See you later