[M] Week 8: Gross Room / Surgical Cut- up - Part 2 Flashcards
MIXTURE OF FIXATIVES
two aldehyde fixative mixtures have been particularly useful for electron cytochemistry
KARNOVSKY’S PARAFORMALDEHYDEGLU ARALDEHYDE SOLUTION
MIXTURE OF FIXATIVES
another aldehyde which has been introduced as a mixture
with glutaraldehyde or formaldehyde
ACROLEIN
ALDEHYDE FIXATIVES
- 10% Formalin
- usual fixation time 24 hrs
- one of the most widely used
○ compatible with many stains, penetrates tissues well
Disadvantages:
- irritating fumes and upon contact to skin
- forms pigment granules (precipitates may be removed by filtration or addition of 10% methanol)
- unsuitable for electron microscopy
Formaldehyde (Formalin)
ALDEHYDE FIXATIVES
- saturated formaldehyde diluted to 10% with NaCl
- recommended for fixation of CNS & general post-mortem tissues for histochemical examination
- slow fixative → >24 hrs
10% Formal-Saline
ALDEHYDE FIXATIVES
- recommended for preservation and storage of surgical, post-mortem & research specimen
- prevents precipitation of pigments (Commonly used by laboratories)
- best fixative for tissues containing iron pigments and elastic fibers
- 10% solution with sulfate or phosphate
10% Neutral Buffered Formalin or Phosphate-Buffered Formalin
ALDEHYDE FIXATIVES
- recommended for routine post-mortem tissues or autopsy
- excellent for many staining procedures including silver reticulum methods
Formol-Corrosive (Formol-Sublimate)
ALDEHYDE FIXATIVES
- can be used for rapid diagnosis, fixes & dehydrates at the same time
- good for the preservation of glycogen & for micro incineration
technique - fix sputum
Alcoholic Formalin (Gendre’s) Fixative
ALDEHYDE FIXATIVES
- Like buffered glutaraldehyde, followed by secondary fixation in osmium tetroxide is satisfactory for electron microscopy
- 2-5% for small tissue fragments
- 4% for* larger tissues*
- Recommended for enzyme histochemistry and electron microscopy
Glutaraldehyde
Enumerate the ALDEHYDE FIXATIVE
- Formaldehyde (Formalin)
- 10% Formal-Saline
- 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin
- Formol-Corrosive (Formol-Sublimate)
- Alcoholic Formalin (Gendre’s) Fixative
- Glutaraldehyde
Enumerate the Metallic Fixatives
- Mercuric Chloride
- Zenkers Fluid
- Zenker-Formol (Helly’s Solution)
- Heidenhains Susa Solution
- B-5 Fixative
METALLIC FIXATIVES
- Most common metallic fixative (5 7% aqueous solution)
- Mercury deposits are removed from deparaffinized sections before staining, by treating with 0.5% iodine solution in 70% ethanol for 5-10 minutes
- Routine fixative of choice for preservation of cell detail in tissue photography
Mercuric Chloride
METALLIC FIXATIVES
- with glacial acetic acid → prevent turbidity & formation of dark precipitate
- recommended for fixing small pieces of liver, spleen, connective tissue fibers & nuclei
- recommended for trichrome staining
- De-zenkerization is performed by adding alcoholic iodine
Zenkers Fluid
METALLIC FIXATIVES
- microanatomic fixative for pituitary gland, bone marrow & blood containing organs
- It also produces pigments that can be removed by saturated alcoholic picric acid or NaOH
Zenker-Formol (Helly’s Solution)
METALLIC FIXATIVES
- recommended mainly for tumor biopsy, especially skin
- excellent cytologic fixative
Heidenahain’s susa solution
METALLIC FIXATIVES
Bone marrow biopsies
B-5 Fixative
Enumerate the CHROMATE FIXATIVES
- 1-2% Chormic acid
- 3% Potassium Dichromate
- Regard’s (mueller’s) fluid
- Orths Fluid
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CHROMATE FIXATIVES
recommended for demonstration of chromatin, mitochondria,
mitotic figures, Golgi bodies, RBC, and colloid-containing
tissues
Regard’s (mueller’s) fluid
CHROMATE FIXATIVES
- recommended for study of early degenerative processes & tissue necrosis
- demonstrates Rickettsiae & other bacteria
- preserves myelin better than buffered formalin
Orth’s Fluid
Fixative
In 4% aqueous solution is recommended for acid mucopolysaccharides
- It fixes connective tissue mucin
- It takes up CO2 to form insoluble lead carbonate removed by filtration or adding acetic acid
Lead Fixatives
Fixative
Enumerate the PICRIC ACID FIXATIVES
- Bouin’s Solution
- Brasils’s Alcoholic Picroformol Fixative
- Excellent fixative for glycogen demonstration
- Yellow color – removed by treatment with another acid dye or lithium carbonate
Picric Acid Fixatives
Picric Acid Fixatives
- Recommended for embryos & pituitary biopsies
- Excellent fixative for preserving soft & delicate tissues
- Preferred fixative for tissues to be stained by Masson’s trichrome for collagen, elastic, or connective tissue
Bouin’s solution
Picric Acid Fixatives
- Better and less “messy” than Bouin’s solution
- Excellent fixative for glycogen
Brasils’s Alcoholic Picroformol Fixative
- Normally used in conjunction with other fixatives (A compound fixative)
- Solidifies at 170C
- Very useful in the study of nuclear components of the cell
- Causes tissue to swell which is used to counteract the shrinkage produced by other components (mercury)
- Contraindicated for cytoplasmic fixation
Glacial Acetic Acid
- May be both as a fixative & dehydrating agent
- Excellent for glycogen demonstration
- Contraindicated when lipids are to be studied
Alcohol Fixatives
Enumerate the Alcohol Fixatives
- 100% Methyl Alcohol
- 95% Isopropyl Alcohol
- 70-100% Ethyl Alcohol
- Carnoy’s Fluid
- Newcomer’s Fluid
ALCOHOL FIXATIVES
Excellent for fixing dry & wet smears, blood smears & bone
marrow tissues
100% METHYL ALCOHOL
ALCOHOL FIXATIVES
Used for fixing touch preparations, for certain special
staining procedures such as Wright-Giemsa
95% ISOPROPYL ALCOHOL
ALCOHOL FIXATIVES
Used as a simple fixative but more frequently incorporated
into compound fixatives
70%-100% ETHYL ALCOHOL
ALCOHOL FIXATIVES
- Recommended for fixing chromosomes, lymph glands & urgent biopsies
- Considered to be the most rapid fixative
- Also used to fix **brain tissue for diagnosis of rabies **
CARNOY’S FLUID
ALCOHOL FIXATIVES
- Recommended for fixing mucopolysaccharides and nuclear proteins
- Acts both as nuclear & histochemical fixative
NEWCOMER’S FLUID
- Fixes conjugated fats & lipids permanently
- Used extensively for neurological tissues
- Adequately fixes electron microscopy
- Poor penetration. The tissue should only be 2-3mm thick
- Addition of saturated aqueous mercuric chloride will prevent its reduction with the formation of black deposits
Osmium Tetroxide (Osmic Acid)
Enumeratre the Osmium Tetroxide (Osmic Acid) Fixatives
- Flemmings Solution
- Flemmings solution without acetic acid
- Most common chrome-osmium acetic acid fixative
- Excellent for nuclear structures
- Permanently fixes fat
FLEMMING’S SOLUTION
Osmium Tetroxide (Osmic Acid)
Recommended for cytoplasmic structures, particularly
mitochondria
FLEMMING’S SOLUTION WITHOUT ACETIC ACID
Fixative
- Precipitates proteins & poor penetrating agent
- A weak decalcifying agent with softening effect on dense fibrous tissues (Causes marked swelling effect on tissues serve to counteract shrinkage produced by other fixatives)
- Sometimes incorporated into compound fixatives
TRICHLOROACETIC ACID
Fixative
- Used it at ice cold temp from – **5 deg C to 40 deg C **
- Study of water-diffusible enzymes especially phosphatases & lipases
- Fixing brain tissues for diagnosis of rabies
- Dissolves fat
- Preserves glycogen poorly
Acetone
Fixative
- Thermal coagulation of proteins
- Suitable for frozen tissue sections
(Fresh tissue is used. If we need to fix, it should be heat-fixed.) - Preparation of bacteriologic smears
- Produces considerable tissue shrinkage & distortion
- Destroys RBC
- Dissolves starch and glycogen
Heat Fixation
Heat fixation should be used with precaution
- Placing an already fixed tissue into a second fixative
-
Before dehydration & on deparaffinized section before staining
○ Facilitate & improve the demonstration of particular substances
○ Special staining techniques
○ Ensure further & complete hardening and preservation of tissues
Secondary Fixation
- Form of secondary fixation
- A primarily fixed tissue is placed in 2.5 to 3% potassium dichromate for 24 hours
○ Act as mordant for better staining effects
○ Aid in cytologic preservation of tissues
Post-Chromatization
Removing excess fixative to improve staining and remove artifacts
Several solutions may be used by submerging specimen in:
- tap water or running waterr
- 50-70% alcohol
- alcoholic iodine
Washing out
Improper Fixation causes?
- Autolysis
- Removal of substances; loss or inactivation of enzymes
- Artifacy pigments
- Soft & feather-like
- Brittle & hard
IMPROPER FIXATION
Match
- Autolysis
- Removal of substances; loss or inactivation of enzymes
- Artifacy pigments
- Soft & feather-like
- Brittle & hard
A. Wrong choice of fixative
B. Delay fixation or insufficient fixative, For large tissues in small containers , Solid organs not serially cut, Hollow organs that float
C. Incomplete fixation
D. Over fixation
E. Incomplete washing
- B
- A
- E
- C
- D
- Eliminated or reduced by fixation in phenol formalin
- Use of neutral buffered formalin and phenol formalin almost completely stops the formation of formalin pigment
Fixation Artificats
FIXATION ARTIFACTS
Partial coagulation of partially fixed protein by ethanol or
incomplete wax impregnation
CRUSH ARTIFACT
FIXATION ARTIFACTS
- used to accelerate staining, decalcification, immunohistochemistry & EM
- Blocks are placed in a microwave
○ Alternative: regular kitchen microwave
○ 450 watts, 550C for 1.5 to 4 minutes - Useful in preserving neurochemical substances, such as acetylcholine
- Rapid fixation
- Reduces time taken for immunohistochemistry (IHC) and in-situ hybridization (ISH)
Microwave Technique
Enumerate the IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE
TECHNIQUES
- Enzyme Histochemistry
- Electron Microscopy
IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES
- To preserve the maximum enzyme activity at its original localization, while also preserving structural integrity
- 4% Formaldehyde or Formal saline
- Acetone or Formaldehyde for fresh frozen cryostat sections
Enzyme HIstochemistry
IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES
- Osmium tetroxide
- Glutaraldehyde
- Paraformaldehyde
- Karnovsky’s paraformaldehyde-glutaraldehyde
Electron Microscopy
