ALDEHYDE FIXATIVE Flashcards
Chemically alter the tissue by binding with it and adding themselves to the tissue
Additive Fixative
Types of additive fixative
Formaldehyde
Mercuric chloride
Chromium trioxide
Picric acid
Glutaraldehyde
Osmium tetraoxide
Zinc Sulfate
Acts on tissue without chemically combining with it
Non-Additive Fixative
Examples of Non-Additive Fixative
Acetone
Alcohol
Historically defined as the killing, penetration, and hardening of tissues
Fixation
Currently defined as the alteration of tissues by stabilizing __
Protein
Funtions of Fixation
- change the soluble contents of cells into insoluble structures
- fixation stops autolysis putrefaction and decay
- stabilize structures to maintain the proper relationship of cells and their stoma
- Fixation enhances staining
- hardens tissue making it firm for proper grossing and easy cutting of thin sections for processing
- affects refractive index
Effects of Fixation
- Safer handlingand processing
- prevents bacterial decomposition
- tissue resistance to the effect of subsequent processing
Chemically alter the tissue by binding with it and adding themselves to the tissue
Additive fixative
Examples of additive fixative
*Formaldehyde
*Mercuric chloride
*Chromium trioxide
*picric acid
*glutaraldehyde
*osmium trioxide
*zinc sulfate
Act on tissue without chemicallyvcombining with it
Non-additive fixative
Examples of non-additive fixative
*Acetone
*alcohol
Acts by creating a network that allows solutions to readily penetrate anterior of the tissue
Coagulant fixative
Examples of coagulant fixative
*zinc salts
*mercuric chloride
*picric acid
*ethyl alcohol
*methyl alcohol
*acetone
-create gel that makes it difficult to penetrate subsequent solutions
-sections should be cut thinly
Non-coagulant fixative
Section tissues should be cut very thinly
Non-Coagulant Fixative
Preserve specific cellular constituents
cytologic fixatives
What is the additive of nuclear fixatives
Glacial acetic acid
Ph of nuclear fixative with glacial acetic acid
4.6pH or less
What type of cytologic fixative is without glacial acetic acid
Cytoplasmic fixative
pH of cytoplasmic fixative
4.6pH or more
what type of cytologic fixative is with glacial acetic acid
Nuclear fixative
Fixative that preserve the chemical constituents of cells and tissue
Histochemical Fixative
Fixative that permits the general microscopic study of tissue structure without altering the structural integrity
Microanatomical Fixative
Factors affecting fixation
- Temperature
- Specimen size
- Volume ratio
- Type of tissue
- Time of fixation
- Penetration
- pH
8.osmolality
Ideal fixative to specimen ratio
15-20:1
Ratio of osmium tetraoxide to specimen
5-10:1
Ratio of fixative for museum material
50-100:1
Temp at which fixation is mostly done
20-22C
temperature at which RNA is done
45C
Temp at which DNA is done
65C
Temperature at which formalin is heated for urgent Biopsy
60C
temp at which formalin is heated for tissue with TB
100C
Recommended specimen size
2cm^2 x 4mm
specimen size for electrom microscopy
1 to 2mm^2
specimen size for ung edema
1 to 2 cm
Types of tissues
A. Hollow organs
B. Air Filled Lungs
C. Human Brains
D. Whole Organs
2 methods to fix hollow organs
- Place cotton (submerged with fixative) in the hollow space of the organ
- Cut the organ
what to do for air filled lungs
Place layers of gauze layering the tissue
what solution is used to flush blood from the brain
Ringer’s Lactate solution
What part of the brain do we use to tie string in a solution of fixative
Circle of Willis
ideal time to perform fixation after the interruption of blood supply
20-30 mins
formalin + alcohol
Formol Alcohol
Tissue must not be immersed for no longer than
60 mins
formalin produces a dark pigment which can obscure cellular detail at what pH?
Low pH
recommended pH for fixation
6-8 pH
What is the ideal osmolality of fixatives
Isotonic
What osmolality is used as holding solution for frozen section and kidney biopsy
Isotonic solutin
Fixation is retarded by:
- size and thickness of tissue
- presence of mucus
- presence of fats
- presence of blood
- cold temp
fixation is enhanced by:
- size and thickness of tissue
- agitation
- heat
- concentration and penetration
- buffering
- time interval
what is used to flush mucus before fixation
Normal saline solution
what is used to flush blood before fixation
Saline
Aldehyde Fixatives
A. Formaldehyde
B. 10% Formol Saline
C. 10% Neutral Buffered Formalin or Phosphate - Buffered Formalin (pH7)
D. Formal-Corrosive (Formal-Sublimate)
E. Alcoholic Formalin/ Gendre’s Fixative/ Gendre’s Fluid
F. Glutaraldehyde
Aldehyde Fixatives are used in what sections of the lab
- routine paraffin sections
- Electron Microscopy
- histochemical and enzyme studies
cheapest fixative
Formaldehyde
Colorless gas commonly available at 37-40% solution in water
Formaldehyde
Most widely used fixative in histopatology today
10% Formalin
what can 10% formaldehyde preserve?
Lipid
what can’t 10% formaldehyde preserve?
Carbohydrates
Simple microanatomical fixative
10% formol saline
10% formol saline is recommended for
- central nervous tissue
- genal post-mortem tissue
- preserves enzymes and nucleo proteins
- demonstrate fat and mucin
formula of 10% formol saline
- formadehyde, 40%
- NaCl
- Distilled H2O
fixation time of 10% formol saline at 35C
24 hours
fixation time of 10% formol saline at 20-25C
48 hours
for preservation and storage of surgical post-mortem and research specimen
10% neutral buffered formalin or phosphate buffered formalin
Formula of 10% neutral buffered formalin
- Sodium dihydrogen phosphate
- disodium hydrogen phostpahte
- formaldehyde 40%
- distilled H2O
other name for 10% neutral bufferd formalin
Phosphate buffered formalin
fixation time for 10% neutral buffered formalin
4-24 hours
for routine post mortem tissues
Formal- corrosive or formol- corrosive
Fixation for cytological structures and blood cell
Formal - corrosive
fixation for neutral fats and phospholipids
Formal - corrosive
Other name for formal - corrosive
Formal- sublimate
Formula of formal-corrosive fixation
-Saturated aq. Mercuric chloride
-formaldehyde 40%
fixation time of formal - corrosive
3-24 hours
other name of alcoholic formalin
Gendre’s Fixative or Gendre’s Fluid
for glycogen and micro incineration technique
Gendre’s fixative
Fixative that is also for clearing
Formal - corrosive
why is alcoholic formalin recommended for sputum
It coagulates mucus
Advantage of alcoholic fixative
- fxation will be reduced1/2 of he typical fixation time
- for rapid diagnosis
- fixing and dehydrating at the same time
Formula of alcoholic formalin
- 95% ethyl alcohol saturated with picric acid
- strong formaldehyde solution
- glacial acteic acid
For electron microscopy and enzyme histochemistry
Glutaraldehyde
Small tissue fragments and needle biopsy
2.5% sltn
Percent of. Concentration for Large tissues less than 4mm thick when using glutaraldehyde
4% solution
Fixation time for glutaraldehyde for small specimens
30 mins - 2hrs
Fixation time of glutaraldehyde for large tissues
6-8 hours up to 24 hours
what nuclear structure does nuclear fixative preserve
chromosome
glacial acetic acid destroys cytoplasmic elements such as _
golgi apparatus and mitochondria
Preserves mucoploysaccharides
Histochemical fixative
NUCLEAR FIXATIVES
- FLEMMING’S FLUID
- CARNOY’S FLUID
- BOUIN’S FLUID
- NEWCOMER’S FLUID
- HEIDENHAIN’S SUSA
CYTOPLASMIC FIXATIVES
- FLEMMING’S W/O HAC
- KELLY’S FLUID
- FORMALIN WITH POST CHROMING
- REGAUD’S FLUID (MULLER’S FLUID)
- ORTH’S FLUID
HISTOCHEMICAL FIXATIVES
- FORMAL SALINE
- ABSOLUTE ETHYL ALCOHOL
- ACETONE
- NEWCOMER’S FLUID
form of secondary fixation whereby a primarily fixed tissue is placed in an aqueous solution of 2.5-3% potassium dichromate for 24 hours
post-chromatization
process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from the tissues
washing out
cross linking reagent
paraformaldehyde
