Fixatives HPCT Flashcards by Potato Patata

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

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Preserver CYTOPLASMIC structure
No glacial acetic acid
pH is more than 4.6

Cytoplasmic fixatives

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Nuclear fixatives example

Flemming’s fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenhain susa

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Cytoplasmic fixatives example

Flemming’s fluid without acetic acid
Helly’s Fluid
Regaud’s Fluid (Muller’s fluid)
Orth’s Fluid

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Histochemical Fixatives example

Formol saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid

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Fixatives for satisfactory for routine paraffin sections
For electron microscopy
For Histochemical and enzyme studies

Aldehyde Fixatives

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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)

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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

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commonly used for BONE MARROW BIOPSIES
Rapid fixation can be achiever in 1 1/2 - 2 hours

B-5 Fixative

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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

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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