Fixatives HPCT Flashcards
Microanatomical fixatives example
10% Formol Saline
10% Neutral buffer formalin
Heidenhein’s susa
Zenker’s solution
Zenker’s Formol (Helly’s solution)
Bouin’s solution
Brasil Solution
Preserver CYTOPLASMIC structure
No glacial acetic acid
pH is more than 4.6
Cytoplasmic fixatives
Nuclear fixatives example
Flemming’s fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenhain susa
Cytoplasmic fixatives example
Flemming’s fluid without acetic acid
Helly’s Fluid
Regaud’s Fluid (Muller’s fluid)
Orth’s Fluid
Histochemical Fixatives example
Formol saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid
Fixatives for satisfactory for routine paraffin sections
For electron microscopy
For Histochemical and enzyme studies
Aldehyde Fixatives
Most widely used concentration for this fixative is 10%
A gas produced by the oxidation of METHYL ALCOHOL
Pure stock solution of this fixative is 40% which is unsatisfactory for routine fixation
Dilution is 1:10 or 1:20
usual fixation time of this fixative is 24 hours
Buffered to pH 7 with PHOSPHATE BUFFER
Formaldehyde
Cheap, Readily available, easy to prepare, Relatively stable
Compatible with most stain
Preservers fats, glycogen, and mucin
Allows tissue enzymes to be studied because it does not precipitate proteins
Recommended for nervous tissue preservation
Allows natural tissue colors to be restored; recommended for colored tissue photography
Tolerant fixative used for mailing specimen
Advantages of formaldehyde
Disadvantages of formaldehyde
May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis
May produce considerable shrinkage of tissues
A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
Advantages of formalin
Cheap, Readily available, easy to prepare, Relatively stable
Compatible with most stain
Preservers fats, glycogen, and mucin
Allows tissue enzymes to be studied because it does not precipitate proteins
Recommended for nervous tissue preservation
Allows natural tissue colors to be restored; recommended for colored tissue photography
Tolerant fixative used for mailing specimen
May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis
May produce considerable shrinkage of tissues
A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
Disadvantages of formalin
Microanatomical fixative
Recommended for fixation of CNS and general postmortem tissues for histochemical explanation
Preserves enzymes and nucleoproteins
Demonstrates fats and mucin
10% Formol saline
Recommended for preservation and storage of SURGICAL, POST-MORTEM, and RESEARCH specimen
Fixation time is 4-24 hours
Best fixative for tissues containing iron pigments and for elastic fibers
10% Neutral buffered formalin or Phosphate-buffer formalin
Recommended for routine POST-MORTEM TISSUES
Fixation time of this fixative is 3-24 hours
Penetrates SMALL PIECES of TISSUES RAPIDLY
Excellent for many staining procedures including SILVER RETICULUM METHODS
Formol-Corrosive or Formol-Sublimate
Fixation of this fixative is FASTER
for RAPID DIAGNOSIS because it FIXES AND DEHYDRATES at the same time
Good for preservation of GLYCOGEN and for MICRO-INCINERATION technique
Used to fix SPUTUM since it COAGULATES mucus
Produces GROSS HARDENING of TISSUES
Causes partial LYSIS of RBCs
Preservation of iron-containing pigments is POOR
Alcoholic formalin or Gendre’s fixative
contains 95% ethanol saturated with picric acid, Formaldehyde, and Glacial acetic acid
Made up of 2 formaldehyde residues, linked by 3 carbon chains
For ROUTINE LIGHT MISCROCOPIC WORK
Buffered glutaraldehyde, followed by secondary fixation in osmium tetroxide is satisfactory for ELECTRON MICROSCOPY
Fixation time of this fixative is 1/2 hour to 2 hours
Preserves PLASMA PROTEINS
Produces LESS TISSUE SHRINKAGE
EXPENSIVE
LESS STABLE
Penetrates tissue SLOWLY
Tends to make tissue more BRITTLE
Reduces PAS (Periodic acid–Schiff) positivity of reactive mucin
Glutaraldehyde
List of aldehyde fixatives
Formaldehyde (Formalin)
10% Formol Saline
10% NBF or Phosphate-buffered formalin
Formol- corrosive / Formol sublimate
Alcoholic formalin / Gendre’s Fixative
Glutaraldehyde
Most common metallic fixative; used in 5-7%
Penetrates poorly and produces shrinkage of tissues
May form BLACK PRECIPITATES of MERCURY
Precipitates ALL PROTEIN
Recommended for RENAL TISSUES, FIBRIN, CONNECTIVE TISSUES, and MUSCLES
Rapidly HARDENS the OUTER LAYER of the TISSUE with incomplete fixation of the center
Trichrome staining is excellent. Permits brilliant metachromic staining of cells
Mercuric Chloride
Mercuric chloride stock solution + GLACIAL ACETIC ACID
Recommended for fixing small pieces of LIVER, SPLEEN, CONNECTIVE TISSUE FIBERS, and NUCLEI
Fixation time is 12 - 24 hours
RECOMMENDED FOR TRICHROME STAINING
Permits BRILLIANT STAINING of NUCLEAR and CONNECTIVE TISSUE FIBERS
COMPATIBLE with MOST stains
May ACT as a MORDANT
PENETRATION is POOR
Zenker’s Fluid
Fixation time of this fixative is 12-24 hours
EXCELLENT MICROANATOMIC FIXATIVE for PITUITARY GLAND, BONE MARROW, and BLOOD containing organs such as SPLEEN, and LIVER
PRESERVES CYTOPLASMIC GRANULES well
Zenker-Formol / Helly’s solution
PBB (Pituitary gland, Bone marrow, BLOOD containing organ)
Recommended mainly for TUMOR BIOPSIES especially of the skin
Excellent CYTOLOGIC FIXATIVE
Fixation time : 3-12 hrs
Produces brilliant results with SHARP NUCLEAR and CYTOPLASMIC details
Permits EASIER sectioning of large blocks
of FIBROUS CONNECTIVE TISSUES
RBC preservation is POOR
Some CYTOPLASMIC granules are DISSOLVED
Weigert’s method of staining elastic fibers is not possible in Susa-fixed tissues
Heidenhain’s Susa Solution
commonly used for BONE MARROW BIOPSIES
Rapid fixation can be achiever in 1 1/2 - 2 hours
B-5 Fixative
Use in 1-2% aqueous solution
Precipitates ALL PROTEINS and ADEQUATELY PRESERVES CARBOHYDRATES
A STRONG OXIDIZING AGENT
Not used because IT IS HAZARDOUS
Chromic Acid
Used in 3% Aqueous solution
PRESERVES LIPIDS AND MITOCHONDRIA
Potassium Dichromate
Fixation time of this fixative is 12-48 hours
HARDENS TISSUES BETTER and MORE RAPIDLY than Orth’s Fluid
Recommended for DEMONSTRATION OF CHROMATIN, MITOCHONDRIA, MITOTIC FIGURES, GOLGI BODIES, RBC, AND COLLOID-CONTAINING TISSUES
Must always be FRESHLY PREPARED
GLYCOGEN penetration is POOR
NUCLEAR STAINING is POOR
DOES NOT preserve FATS
Intensity of PAS reaction is REDUCED
Regaud’s Fluid/ Muller’s Fluid
Preserves GMRC
Golgi bodies, Mitochondria and mitotic fluid, RBC, Colloid-containing tissue
Fixation time is 36-72 hours
RECOMMENDED for STUDY of EARLY DEGENERATIVE PROCESSES AND TISSUE NECROSIS
Demonstrates RICKETTSIAE and OTHER BACTERIA
Preserves MYELIN better than BUFFERED FORMALIN
Orth’s Fluid
Used in 4% aqueous solution of basic lead acetate
Recommended for ACID MUCOPLYSACCHARIDES
Fixes CONNECTIVE TISSUE MUCIN
Takes up CARBON DIOXIDE to FORM INSOLUBLE CARBONATE especially on PROLONGED STANDING
Lead Fixatives
Normally used in strong saturated aqueous solution (1%)
Excellent Fixative for GLYCOGEN DEMONSTRATION
Also DYES the tissue. ALLOWS Brilliant staining with the TRICHROME method
Precipitates ALL proteins
STABLE
causes RBC HEMOLYSIS and REDUCES the amount of DEMONSTRABLE FERRIC IRON in TISSUES
Must NEVER be washed in water before dehydration
HIGHLY EXPLOSIVE when DRY
ALTERS AND DISSOLVES LIPIDS
SUITABLE for ANILINE Stains
Causes shrinkage of tissue (Slightly Hypertonic)
Picric Acid
recommended for FIXATION of EMBRYOS and PITUITARY BIOPSIES
Excellent Fixative for preserving SOFT and DELICATE structures
Fixation time of this fixative is 6-24 hours
PRESERVES Glycogen
Does NOT need washing out
Bouin’s Solution
BETTER and LESS MESSY than Bouin’s Solution
EXCELLENT FIXATIVE for GLYCOGEN
Brasil’s Alcoholic Picroformol Fixative
Solidifies at 17C
FIXES and PRECIPITATES NUCLEOPROTEINS
Precipitates CHROMOSOMES and CHROMATIN materials
Causes tissue to SWELL (Hypotonic)
Glacial Acetic Acid
Must be used in concentrations ranging from 70-100% because less concentrated solution will produce lysis of cells
Alcohol fixatives
Used to fix and preserve GLYCOGEN PIGMENTS, BLOOD, TISSUE FILMS, and SMEARS
Ideal for SMALL TISSUE FRAGMENTS
Excellent for GLYCOGEN PRESERVATION
Preserves NUCLEAR STAINS
Lower concentrations will cause RBC HEMOLYSIS and INADEQUATELY preserve leukocytes
DISSOLVES fats and Lipids
Absolute alcohol
Excellent for fixing DRY and WET smears, BLOOD SMEARS, and BONE MARROW TISSUES
FIXES and DEHYDRATES at the same time
Penetration is SLOW
Tissues may be OVERHARDENED and DIFFICULT to cut if left for more than 48 HOURS
Methyl Alcohol
Used for fixing TOUCH PREPARATIONS
95% Isopropyl alcohol
Used at 70-100% concentration
a SIMPLE FIXATIVE
Fixation time is 18-24 hours
Preserves but DOES NOT fix glycogen
Ethyl Alcohol
Used to fix BRAIN TISSUES for the diagnosis of RABIES
Fixation time is 1-3 hours
Considered as the MOST RAPID FIXATIVE
Fixes and dehydrates at the SAME TIME
Preserves NISSL’s granules and Cytoplasmic granules WELL
Preserves NUCLEOPROTEINS and NUCLEIC acids
Excellent fixative for GLYCOGEN
Carnoy’s Fluid
CURLS -Chromosomes, Urgent Biopsies, Rabies (Brain), lymph nodes/Lymph glands
Histochemical fixative and nuclear fixative
Produces BETTER reaction in FEULGEN STAIN than Carnoy’s Fluid
Recommended for fixing MUCOPOLYSACCHARIDES and NUCLEAR PROTEINS
Fixation time is 12-18 hours at 3c
Newcomer’s Fluid
Most common chrome-osmium acetic acid fixative
Fixation time is 24- 48 hours
Excellent fixative for NUCLEAR STRUCTURES
PERMANENTLY fixes FATS
Flemming’s Solution
Made up of only chromatic acid and osmic acid
Recommended for cytoplasmic structures particularly the mitochondria
Fixation time is 24-48 hours
Flemming’s solution w/o acetic acid
Precipitates proteins
WEAK decalcifying agent
Softening effect on DENSE FIBROUS TISSUES facilitates preparation of such sections
POOR penetrating agent
Suitable only for SMALL PIECES OF TISSUES or BONES
Trichloroacetic acid
Used at ice cold temperature ranging from -5c to 4c
Recommended for study of WATER DIFFUSIBLE ENZYMES especially PHOSPHATES and LIPASES
Used in fixing brain tissues for diagnosis of RABIES
Used as solvent for certain METALLIC SALTS to be used in FREEZE SUBSTITUTION techniques for tissue blocks
Evaporates RAPIDLY
Acetone
Involves thermal coagulation of tissue protein for rapid diagnosis
HEAT FIXATION
A process of placing an already fixed tissue in a second fixative
SECONDARY FIXATION
Form of secondary fixation
2.5-3%K dichromate for 24 hrs to act as mordant for better staining and aid in cytologic preservation of tissues
Post-Chromatization
The process of removing excess fixative from the tissue after fixation
Washing out
Solution used for washing out Helly’s solution, Zenker’s Solution, Flemming’s solution, Formalin, Osmic acid
Tap Water
Solution used for washing Picric’s acid (Bouin’s Solution)
50-70% Alcohol
Solution used for washing out Mercuric fixation
Alcoholic Iodine
Fixative of choice and Fixative to avoid when your target to study is PROTEIN
Fixative of choice: NBF, Paraformaldehyde
Fixative to avoid: Osmium Tetroxide
Fixative of choice and Fixative to avoid when your target to study is Enzymes
Fixative of choice: Frozen section
Fixative to avoid: Chemical Fixatives
Fixative of choice and Fixative to avoid when your target to study is Lipids
Fixative of choice: Frozen section, Glutaraldehyde, Osmium tetroxide
Fixative to avoid: Alcoholic and NBF
Fixative of choice and Fixative to avoid when your target to study is Nucleic acid
Fixative of choice: alcoholic fixatives
Fixative to avoid: Aldehydes
Fixative of choice and Fixative to avoid when your target to study is Mucopolysaccharides
Fixative of choice: Frozen section
Fixative to avoid: Chemical
Fixative of choice and Fixative to avoid when your target to study is Biogenic amines
Fixative of choice: Bouin’s solution and NBF
Fixative of choice and Fixative to avoid when your target to study is Glycogen
Fixative of choice: Alcoholic fixatives
Fixative to avoid: Osmium tetroxide
Fixatives for Electron Microscope (GOPKZ)
Glutaraldehyde
Osmium tetroxide
Paraformaldehyde
Karnovsky’s Fixative
Zamboni’s Fixative
Used in ELECTRON MICROSCOPY
Preserves CYTOPLASMIC STRUCTURES well such as GOLGI BODIES and MITOCHONDRIA
Produces BRILLIANT NUCLEAR STAINING with SAFRANIN
Adequately fixes materials for ULTRATHIN sectioning in EM
VERY EXPENSIVE
POOR penetrating agent, suitably ONLY for SMALL PIECES of tissues
INHIBITS hematoxylin and makes counterstaining DIFFICULT
EXTREMELY VOLATILE
Can IRRITATE the EYES producing conjunctivitis or may cause deposition of BLACK OSMIC OXIDE in the cornea leading to blindness
Osmium Tetroxide
