FIXATION Flashcards
trans based
killing, penetration, and hardening of tissues
fixation
first and most critical step in tissue processing
fixation
what is the purpose of fixation
Preserve the morphologic and chemical integrity of the cell in a life-like manner as possible
factors affecting fixation
fixative of choice
time
tissue-to-fixative ratio
penetration rate
thickness of specimen
tissue components
pH
temperature
what is fixative of choice in fixation?
10% neutral buffered solution
time used to perform in fixation
20-30 mins after blood supply is cut off
thickness of specimen in light microscopy
2cm2 x 0.4cm
thickness of specimen in electron microscopy
1-2mm2
what tissue components have longer fixation time
fibrous tissue
mucus
fat
blood
what tissue components have shorter fixation time
small of loosely textures tissues (example: appendix)
optimal pH of fixation
6 to 8
optimal temperature
45 degrees celsius
temp for tissue processors
40 degrees celsius
temp for microwave processing
65 degrees celsius
temp for electron microscopy
0-4 degrees celsius
temp for tuberculosis
100 degrees celsius
temp for rapid biopsy
60 degrees celsius
hypertonic is?
cell shrinkage
hypotonic and isotonic is?
cell swelling
tissues are remained at what solution?
slightly hypertonic solution (400-450 mOsm)
hastens fixation
agitation, vacuum
types of fixatives based on composition
simple
compound
types of fixative based on action
microanatomical
cytological
histochemical
General study of tissues w/o
structure alteration
microanatomical
pH is < 4-6 and has glacial acetic acid has affinity to nuclear chromatin
nuclear
has a pH > 4-6 and HAc destroys mitochondria and golgi bodies
cytoplasmic
Preserves chemical constituents if cells and tissues
histochemical
an aldehyde fixative that is produced by oxidation of methanol
formaldehyde
percent concentrations of formaldehyde in:
gas form
stock concentration
working solution
what pH level
100%
37-40%
10%
pH 7
Best general tissue fixative; pH 7
10% Neutral Buffered Formalin (NBF)
Recommended for fixation of CNS tissues and general post mortem tissues for histochemical examination
10% Formol-Saline
what is the fixation time of NBF
4 to 24hrs
Recommended for routine post mortem tissues and Silver Reticulum staining methods
Formol-Corrosive (Formol Mercuric Chloride)
good for microincineration
techniques; Fixes sputum
Gendre’s (Alcoholic Formalin)
For gastrointestinal (GI) tissues, prostate biopsies, and bone marrow (BM)
Hollande’s
Made up of 2 formaldehyde residues linked by 3 carbon chains; Container must be refrigerated
Glutaraldehyde
concentrations of glutaraldehyde for:
immune electron microsopy
small TSE fragments
most common
large TSE fragments
0.25%
2.5%
3%
4%
Polymer of Formalin; For ultrathin and electron microscopy
Paraformaldehyde
Acrolein in glutaraldehyde or formalin; For Electron Histochemistry & Electron
Immunocytochemistry
Karnovsky’s Paraformaldehyde /
Glutaraldehyde
no smudging of nuclei and
distortion of staining compared with formalin
40% Aqueous Glyoxal
Most common metallic fixative; 5-7%
mercuric chloride
remedy for mercuric chloride
dekenkerization
Good general fixative for adequate preservation of all kinds of tissues
zenker’s fluid
Brown pigments are removed with saturated alcoholic picric acid or NaOH
Zenker-Formol (Helly’s Solution)
for skin tumor biopsy; minimum cell shrinkage and tissue hardening due to counter-balance effect of acids and mercury
Heidenhain’s Susa solution
Recommended for hematopoietic and lymphoid tissues
B-5 Fixative
fixation time for B-5 fixative
4-8 hours
Recommended for making smears of loose cells on slides
Shaudinn’s Fluid
Preserves carbohydrates; precipitates all protein
Chromic Acid 1-2%
For chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid containing tissues
Regaud’s / Muller’s Fluid
Study of early degenerative processes and necrosis; Rickettsiae and other bacteria; Preserves myelin
Orths’ Fluid
Preserves lipids & mitochondria at pH 4.5 to 5.2; cytoplasm, chromatin and chromosome are fixed
Potassium Dichromate
Preserves acid mucopolysaccharide
lead fixatives
remedy for lead fixatives
add drops of acetic acid to
dissolve residue
Normally used in strong aqueous solution (1%); Glycogen demonstration; has yellowing effect
picric acid fixatives
For embryo and pituitary biopsies, and tissues to be stained with Masson’s Trichrome
Bouin’s
Better and less messy than Bouin’s
Brasil’s Alcoholic Picroformol
Incorporated in compound fixatives; Destroys mitochondria and Golgi elements, thus not for cytoplasmic fixation
Glacial Acetic Acid
it is good and causes polarization to glycogen
alcohol fixatives
Most rapid tissue fixative (1-3 hrs); Fixing brain tissues for rabies diagnosis
Carnoy’s Fluid
Enzyme studies; does not fix but preserves glycogen
Ethanol (70 - 100%)
Dry and wet smears, Bone Marrow smears, bacterial smears
Methanol / Wood Alcohol (100%)
Touch prep smears to be Wright-stained
Isopropanol
For mucopolysaccharide (12-18 hrs)
Newcomer’s Fluid
For sputum (4 - 18hrs)
Gendre’s (Alcoholic Formalin)
tissue to fixative ratio of osmotic fixatives
1:5
Most common osmic acid fixative and is a nuclear fixative
Flemming’s Solution w/ GAC
a cytoplasmic fixative
Flemming’s Solution w/o GAC
○ Incorporated into compound fixatives
○ Marked swelling effect on tissues
○ Poor penetration thus for small pieces of
tissues or bones
○ Weak decalcifying agent, this has
softening effect on dense fibrous tissues
Trichloroacetic Acid
Use at ice cold temperature (-5 to 4oC)
Acetone
Involves thermal coagulation of tissue proteins for rapid diagnosis
heat fixation
optimum temperature for heat fixation
45-55 degrees celsius
To improve the demonstration of particular substances
secondary fixation
Technique whereby a primary fixed tissue is placed in aq. Solution of 2.5% - 3%
potassium dichromate for 24hrs
post-chromatization
process of removing excess fixatives (washing out)
tap water
50%-70% alcohol
alcohol iodine
