FIXATION (TYPES OF FIXATIVES)
Lead fixatives
-4% aqueous solution of basic lead acetate or lead nitrate -recommended for fixing acid mucopolysaccharides -fixes connective tissue mucin
post-chromatization
-a form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 - 3% potassium dichromate for 24 hours to act as mordant -for better staining effects -to aid in cytologic preservation of tissues
Osmic acid/Osmium Tetroxide
-a pale yellow powder w/c dissolves in water (up to 6% at 20C) to form a strong oxidizing solution -fixes conjugated fats and lipids permanently
Schaudinn's Sublimated Alcohol Solution
-best for wet smear preparation -will give better staining of the nuclei and connective tissues
Brasil's Alcoholic Picroformol Fixative
-better and less messy than Bouin's solution -excellent fixative for Glycogen
concentration of fixative
-concentration of fixative should be adjusted downto the lowest level possible, bc you will expend less money for the fixative -formalin is best at 10% -glutaraldehyde is generally made up at 0.25 to 4% -too high conc. may adversely affect the tissues and produce artefact similar to excessive heat
Flemming's fluid
-demonstrates and fixes fat permanently -small amount of fixative is required
Methyl Alcohol 100%
-excellent for fixing dry and wet smears, blood smears and bone marrow tissues -fixes and dehydrates at the same time
time interval
-from removal of tissue to fixation -the faster you can get the tissue and fix it, the better keep tissue moist with saline- will result to artefacts when dry -the longer you wait, the more nuclear shrinkage and artefactual clumping will occur
temperature
-increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue
Heat fixation
-involves thermal coagulation of tissue proteins for rapid diagnosis -usually employed for frozen tissue sections and prep of bacteriologic smears -fixation is better -preserves nuclear and cytoplasmic detail -suitable for frozen tissue
Glacial Acetic Acid
-normally used in conjunction with other fixatives to form a compound solution -it solidifies at 17 degrees celcius -fixes and precipitates nucleoproteins
Ethyl alcohol
-preserves but does not fix glycogen -fixes blood, tissue films and smears
washing-out
-process of removing excess fixative from the tissue after fixation in order to improve staining and remove artifacts from the tissues
Alcohol Fixatives
-rapidly denatures and precipitates proteins by destroying hydrogen and other bonds -must be used in concentration ranging from 70% to 100% -less concentrated solutions will produce cell lysis
10% Formol-Saline
-recommended for fixation of CNS tissues and general post-mortem tissues for histochemical examinations -ideal for fixing autopsy specimen
Bouin's solution
-recommended for fixation of testis, GI tract, and endocrine tissue -does not do a bad job n hematopoietic tissues either, and doesn't require dezenkerizing before staining
Glutaraldehyde
-recommended for fixation of tissues for electron microscopy -must be cold and buffered and not more than 3 months old -preferably sectioned at a thickness no more than 1 mm to enhance fixation
Heidenhain's Susa
-recommended for fixing biopsy and tumor of the skin -produces brilliant staining with good cytoplasmic details
Carnoy's fluid
-recommended for fixing chromosomes, lymph glands and urgent biopsies -most rapid fixative, fixes and dehydrates at the same time -permits good nuclear staining and differentiation -preserves nissl granules and cytoplasmic granules; nucleoproteins and nucleic acids -suitable for curettings and biopsy materials
Bouin's fluid
-recommended for fixing embryonic tissue (placenta, umbilical cord and fetus) -preserves glycogen -useful for fixing minute fragments of tissues
Newcomer's fluid
-recommended for fixing mucopolysaccharides and nucleoproteins -act as both nuclear and histochemical fixative -produces better reaction in Feulgen stain than Carnoy's fluid
Zenker's fixatives
-recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow -fixes nuclei very well and gives good detail -mercurey deposits must be removed (DEZENKERIZED)before staining or black deposits will result in the sections
Formol-Corrosive(Formol-Sublimate)
-recommended for routine post-mortem tissues -cytologic details and RBCs are well preserved -ideal for silver reticulum staining method
Orth's fluid
-recommended for the study of early degenerative processes and tissue necrosis -demonstrate rickettsiae and other bacteria -preserves myelin better than buffered formalin
Helley's fluid
-recommendedfor fixing pituitary, bone marrow and spleen -will produce better results for nuclear staining than Zenker's fluid -well preserved cytoplasmic granules
aldehyde fixatives
-satisfactory for routine paraffin sections -electron microscopy;when histochemical and enzyme studies are indicated
Trichloroacetic acid
-sometimes incorporated into compounds fixatives -it precipitates proteins
Alcohols
-specifically ethanol, are used primarily for cytologic smears -Etahanol (95%) is fast and cheap
Acetone
-used at ice cold temperature ranging from -5 to 4 degrees celsius -used in fixing brain tissues for diagnosis of rabies
Formalin
-used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced -mot forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly
chromic acid
-used in 1-2% aqueous solution, usually as a constituent of a compound fixative -precipitates all proteins and adequately preserve carbohydrates.
Alcoholic-Formalin (Gender's Fixative)
-used to fix sputum because it coagulates mucus -good for preservation of glycogen -can be used for rapid diagnosis because it fixes and dehydrates at the same time
volume
-volume of fixative is important 10:1 ratio of fixative to tissue -change the fixative at intervals to avoid exhaustion of the fixative -agitation of the specimen in the fixative will also enhance fixation
Hypoxia
... of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity
secondary fixation
>process of placing an already fixed tissue in a second fixative in order to: -facilitate and improve the demonstration of particular substances -make special staining possible(w/secondary fixative acting as mordant) -ensure further and complete hardening and preservation of tissues >may be done before dehydration and on deparaffinized sections before staining >usually 10% formalin or 10% formalin-saline as a primary fixative
nuclear fixatives cytoplasmic fixatives
Cytological fixatives
microanatomical cytological histochemical
according to action
simple compound
according to composition
formaldehyde/formalin 10% Formol-Saline 10% BNF or Phosphate Buffered-Formalin Formol-Corrosive/Formol-Sublimate Alcoholic-Formalin glutaraldehyde
aldehyde fixatives
compound fixatives
are made up of 2 or more fixatives w/c have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents
mercurials
are somewhere in between -one way to get around this problem is sectioning the tissues thinly (2-3 mm)
fixation(pH)
best carried out close to neutral pH, in the range of 6.0 - 8.0
commercial formalin
buffered with phosphate at a pH of 7
potassium dichromate chromic acid regaud's (Moller's) fluid orth's fluid
chromate fixatives
inhibition of autolysis hardening of tissue solidification of colloid material optical differentiation effects on staining loss of material during fixation tissue shrinkage
common effects of fixation
penetration of tissues
depends upon the diffusability of each individual fixative, w/c is a constant
Formalin-Ammonium Bromide solution
excellent fixative for brain tissues w/c the silver and gold techniques are to be used
Formalin-Sodium Acetate Solution
excellent fixative for storing whole blocks of tissue
buffer and pH temperature penetration capacity volume change agitation concentration of fixation duration of fixation
factors affecting fixation
Acidity
favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue
common buffers
include phosphate, bicarbonate, cacodylate, and veronal
Tap water
is used to remove: -excess chromates from tissues fixed in Helley's, Zenker's and Flemming's solution -excess formalin excess osmic acid
50-70% Alcohol
is used to wash out excess amount of picric acid(Bouin's solution)
simple fixatives
made up of only one component substance
mercuric chloride chromate fixatives lead fixatives
metallic fixatives
10% formalin 10% formol-saline 10%buffered neutral formalin
most commonly used fixatives
Picric acid fixatives
normally used in strong saturated aqueous solution (approx. 1%)
formalin and alcohol
penetrate the best, and glutaraldehyde the worst
Champy's fluid
preserves mitochondria, golgi elements and fats
Buffered Neutral Formalin 10%
recommended for preservation and storage of surgical, post-mortem and research specimens
Regaud's/Moller's fluid
recommended for the demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, rbcs and colloid containing tissues such as yolk materials (ovary)
aldehydes metallic fixatives picric acid acetic acid acetone alcohol osmium tetroxide (osmic acid) heat
simple fixatives
Tap water 50-7-% alcohol Alcoholic iodine
solutions used for washing-out
>10% NBF - 24-48 hours >Alcoholic formalin 1 - 1 hour >Alcoholic Formalin 2 - 1 or 2 hours
steps for fixation
mercuric chloride
the most common fixative, frequently usedin saturated Aqueous solutions of 5-7%; it is included in many compound fixatives
histochemical fixatives
those that preserve the chemical constituents of cells and tissues
microanatomical fixatives
those w/c permit the general microscopic study of tissue structures w/o altering the structural pattern and normal intercellular relationship of the tissues in question
cytological fixatives
those which preserve specific parts and particular microscopic elements of the cell itself
according to composition according to action
types of fixatives
Kaisserling's fixative
used for fixing tissue of
potassium dichromate
used in 3% aqueous solution; preserves mitochondria
Alcoholic iodine
used to remove excessive mercuric fixatives
Hot formalin
will fix tissues faster, and this is often the first step on an automated tissue processor
penetration into a thin section
will occur more rapidly than for a thick section