FIXATION Flashcards
The first and most critical step in histotechnology
Fixation
PRIMARY AIM OF FIXATION:
to preserve the morphologic and chemical integrity of the cell in as lifelike manner as possible
SECONDARY GOAL OF FIXATION:
to harden and protect the tissue from the trauma of further handling, so that it is easier to cut during gross examination
Satisfactory fixation occurs between the pH of
pH 6 and 8
Fixation of surgical specimens is traditionally carried out at
room temperature
Many laboratories use tissue processors that work at
40°C
Formalin heated to ______ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased
60°C
Can be used to fix tissues with tuberculosis
Formalin at 100°C
Brain is usually suspended whole in ____________ for 2-3 weeks to ensure fixation and some hardening prior to sectioning
10% buffered formalin
For fixation, the best results are usually obtained using what type of solution?
slightly hypertonic solutions (400-450 mOsm)
Causes polymerizationof the aldehyde, with consequent decrease in its effective concentration
presence of a buffer
This have been found to be an ideal concentration for immuno-electron microscopy
Low concentrations of glutaraldehyde (0.25%)
Primary fixation using buffered formalin should be for how hours?
2-6 hours
The maximum effectiveness of fixation is noted to be _____ the tissue volume
20 times
Traditionally, the amount of fixative used has been ________the volume of tissue to be fixed.
10-25 times
What is the adequate fixation time?
4 - 6 hours
What is the recommended size of the tissue?
2 cm2 and no more than 4 mm thick
Used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately
Refrigeration
Continues to undergo mitosis (growth) up to 30 minutes after death when refrigerated
bone marrow
This deteriorate very quickly
Brain cells
Are made up of only one
component substance
Simple Fixatives
Are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
Compound Fixatives
Are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question
Microanatomical Fixatives
Enumerate the fixatives under the aldehydes of Simple fixatives
- Formaldehyde
- Glutaraldehyde
Enumerate the fixatives under metallic fixatives of simple fixatives
- Mercuric chloride
- Chromate fixatives ( potassium dichromate, and chromic acid)
- Lead fixatives (Picric acid, acetic acid, acetone alcohol, and osmium teroxide or osmic acid)
- Heat
Enumerate the fixatives under microanatomical fixatives
- 10% Formol saline
- 10% Neutral buffered formalin
- Heidenhain’s Susa
- Formol sublimate (formol corrosive)
- Zenker’s solution
- Zenker-formol (Helly’s solution)
- Bouin’s solution
-Brasil’s solution
Enumerate the fixatives that have a ph of 4.6 or less ( hint: Nuclear fixatives)
- Flemming’s fluid
- Carnoy’s fluid
- Bouin’s fluid
- Newcomer’s fluid
- Heidenhain’s fluid
Enumerate the fixatives that have a pH of more than 4.6 (hint: Cytoplasmic fixatives)
- Flemming’s fluid without acetic acid
- Kelly’s fluid
- Formalin with “post-chroming”
- Regaud’s fluid (Muller’s fluid)
- Orth’s fluid
These fixatives preserve the chemical constituents of cells and tissues
Histochemical fixatives
Enumerate the fixatives under histochemical fixatives
- Formol saline 10% or 10% Formol Saline
- Absolute Ethyl alcohol
- Acetone alcohol
- Newcomer’s fluid
For lipid fixation, fixatives that contain ___________ and _________ can be effective for preservation of lipids in cyrostat sections
mercuric chloride and potassium dichromate, respectively
These fixatives are generally recommended for glycogen fixation
Alcoholic fixatives
These are the most commonly used fixatives for amino acid histochemistry
Neutral buffered formol saline or formaldehyde vapor
The most useful fixatives for preserving glycogen are alcohol-based such as
Rossman’s fluid or cold absolute alcohol
One of the most widely used fixatives is________ which is made from formaldehyde, a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume
10% formalin
A simple microanatomical fixative made up of saturated formaldehyde (40% by weight volume) diluted to 10% with sodium chloride.
10% Formol- Saline
It is recommended for fixation of central nervous tissues and general post-mortem tissues for histochemical examination.
10% Formol-Saline
Recommended for preservation and storage of surgical, post-mortem and research specimens.
10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
This aldehyde fixative is recommended for routine post-mortem tissues.
Formol-Corrosive (Formol-Sublimate)
Post-fixation with ________ for 6 hours or more can enhance immunoperoxidase studies on the tissues, and in some cases, electron microscopy, if it is necessary at a later time to establish a diagnosis.
phenol-formalin (Alcoholic Formalin/ Gendre’s Fixative)
It acts in a manner similar to formaldehyde and is sometimes utilized for routine light microscopic work.
Glutaraldehyde
Enumerate the six fixatives under Aldehyde Fixative
- Formaldehyde (Formalin)
- 10% Formol-Saline
- 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
- Formol-Corrosive (Formol-Sublimate)
- Alcoholic Formalin (Gendre’s) Fixative
- Glutaraldehyde
Enumerate the fixatives under Mercuric Chloride (Metallic fixative)
- Zenker’s fluid
- Zenker-formol (Helly’s solution)
- Heidenhain’s Susa solution
- B-5 Fixative