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