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Simple fixatives
Simple aqueous fixatives or fixative ingredients
| Simple aqueous fixatives and fixative ingredients are: | Acetic acid, formaldehyde, glutaraldehyde, glyoxal, mercuric chloride, osmium tetroxide, picric acid, potassium dichromate, zinc salts |
| What is the molecular structure of Acetic Acid? | CH3COOH |
| What household chemical is acetic acid in dilute form? | Vinegar |
| What is another name for concentrated acetic acid? Why? | Glacial acetic acid because of its unusual freezing point of 16.6 C or 61.9 F |
| This fixative does not fix or destroy carbohydrates or lipids. | Acetic Acid |
| The rate of penetration for acetic acid | very rapidly and leaves tissue soft |
| Major use of acetic acid | the precipitation and preservation of nucleoproteins and DNA |
| Hyrdophillic | water loving |
| Storage protocol for acetic acid | stored at room temperature and away from strong oxidizers, nitric acid, and strong caustics |
| __________________ causes increased protein swelling more than any other fixative. | Acetic Acid |
| The swelling caused by acetic acid considered a disadvantage unless..... | added to compound fixatives to counteract the shrinking effect of another ingredient |
| Swelling is a characteristic of acetic acid when the pH is | <4.0 |
| __________________ swells at pH 2.5. Why? | Collagen, because links in the proteins are broken and any hydrophillic(waterloving) groups present are exposed. |
| Acetic Acid does what to red blood cells? | Red blood cells are lysed by acetic acid making their preservation poor. |
| Health Hazards of Acetic Acid | severe burns, PEL of 10 ppm, should be transported in acid container and used under a hood |
| True or False: Acetic acid should be added to water and not the other way around. | True |
| Acetic Acid Aqueous v nonaqueous, additive v nonadditive, coagulant v noncoagulant | Aqueous, nonadditive, noncoagulant |
| What is formaldehyde? | Colorless gas and is sold as a solution 37-40% gas by weight in water. |
| Most widely used fixative | 10% formalin |
| What happens to formaldehyde in aqueous solutions? | In aqueous solution, formaldehyde combines chemically with water to form methylene hydrate(methylene glycol) |
| The longer, insoluble polymeric form of formaldehyde deposited as a white powder in 37-40% formaldehyde | Paraformaldehyde |
| How is paraformaldehyde formed? | Paraformaldehyde forms with time or when the formaldehyde solution is exposed to low temperature(32 F) |
| ______________________ is added to formaldehyde to help prevent the formation of paraformaldehyde. | 10-14% methanol |
| When is paraformaldehyde used? | Paraformaldehyde is used in electron microscopy because it yields a pure formaldehyde solution. |
| 4% paraformaldehyde =________ formalin | 10% |
| Penetration of formaldehyde/methylene hydrate | penetration occurs quickly into the tissues, however fixation reactions(addition/crosslinking) are slow |
| What do crosslinks in well fixed tissue in formaldehyde do? | Prevent alteration during processing and staining; however, tissue that is poorly fixed can be re-denatured by alcohol and heat |
| Are lipids preserved in formaldehyde? | yes, but they are not made insoluble and prolonged storage in formalin leads to a gradual loss of lipids |
| What is dissolved in alcohol and xylene? | Lipids |
| What is added to aqueous formalin solutions that can preserve lipids? | Calcium salts, The calcium ions preserve the phospholipids by reducing their solubility. |
| Fixative that hardens tissue more than any other fixative except ethanol and acetone | Formaldehyde |
| What fixative is used for diagnosis on morphologic criteria ? | The morphologic criteria used for diagnosis have been established primarily on NBF fixed tissues. |
| What is another name for formalin pigment? | Black acid hematin |
| Formalin pigment | A microcrystalline dark brown pigment when the acid aqueous solution of formaldehyde acts on tissues rich in blood |
| Where does formalin pigment lie? | Formalin pigment lies on top of the Erythrocytes(RBCs) |
| Can formalin pigment be prevented, removed, or both? | Both |
| How is formalin pigment prevented? | Maintaining a solution pH near neutrality and using the appropriate volume ratio of fixative solution |
| How is formalin pigment removed? | Treating tissue sections with alcoholic picric acid or alkaline alcohol |
| Formalin pigment is undesirable. Why? | It may react during the staining procedure to mask or simulate microorganisms and pathologically relevant pigments. |
| Silver solutions used in procedures for staining melanin, fungi, reticulin, and spirochetes for reduced by what artifact? | Formalin pigment |
| True or False: Acetic acid can be used in a simple aqueous solution(unbuffered) or with the addition of sodium chloride to achieve the correct osmolality. The solutions are acidic(pH 4-5) and react with oxygen to form formic acid. | False, Formaldehyde |
| The formalin pigment is birefringent(seen) and can be monitored by_____________ before silver staining or bleached or removed if necessary. | polarization |
| Formaldehyde solutions are hypertonic but is osmotically active. Why is this important? | The buffer vehicle is important and the tonicity and composition of buffer ions influence morphology. |
| What is formaldehyde solutions neutralized with to prevent pigment formation? | Calcium or magnesium carbonate |
| Common formalin solutions (9 of them) | 10% aqueous formalin, 10% formalin saline, acetate formalin, calcium formalin, formalin ammonium bromide, 10% neutralized formalin, 10% neutral-buffered formalin, modified millonig formalin, phosphate buffered paraformaldehyde |
| is recommended for the fixation and preservation of phospholipids | Calcium formalin |
| What are the ingredients for 10% aqueous formalin | formaldehyde 37-40% 100ml Distilled water 900 mL |
| 10% formalin saline consists of | formaldehyde 37-40% 100 mL Sodium Chloride 9g Distilled water 900 mL Distilled water 900 mL |
| Is 10% formalin saline hypotonic, isotonic, or hypertonic? | isotonic |
| What makes up Calcium formalin | Formaldehyde 37-40% 100mL Calcium chloride 10 g Distilled water 900 mL Distilled water 900 mL |
| Action of phospholipids | take up water and extend their surface by growing outward wormlike myelin forms |
| Calcium ions prevent_____________________ | The gradual solution and distortion caused by the outgrowths formed by phospholipids. |
| What fixative contains formaldehyde 37-40% (150 mL), ammonium bromide (20g), and distilled water (850 mL)? | Ammonium bromide formalin |
| Ammonium bromide formalin are recommended for tissue specimens of______________________ especially when what procedure is to be performed? | central nervous system, Cajal astrocyte |
| What does acetate formalin contain? | Formaldehyde 37-40% 100mL, Sodium acetate 20g, Distilled water 850 mL *recommends 2% solution of calcium acetate instead of sodium |
| The anhydrous compound yields a solution pH of 7.3 and the trihydrate yields a solution pH of 7.0 | Acetate formalin |
| Formaldehyde 37-40% 100 mL, distilled water 900 mL, and calcium or magnesium carbonate to excess | 10% neutralized formalin |
| Why is 10% neutralized formalin not recommended? | the solution becomes acid after withdrawal from the storage bottle |
| Most widely used solution for routine formalin fixation. Has a pH of 6.8-7.2 and is hypotonic in the buffer ions present | 10% neutral buffered formalin |
| 10% neutral buffered formalin ingredients | formaldehyde 37-40% 100 mL Distilled water 900 mL Sodium phosphate mono basic 4g Sodium phosphate dibasic 6.5 g Sodium phosphate monobasic 4g sodium phosphate dibasic 6.5g |
| fixative that consists of formaldehyde 37-40% 100mL, distilled water 900mL, Sodium phosphate, monobasic 18.6g, and Sodium hydroxide 4.2 g | Modified millonig formalin |
| True or false: Modified Millonig formalin can be used as a dual-purpose fixative, allowing electron microscopy on stored tissue. | True |
| Phosphate buffered paraformaldehyde | 2.26% monobasic sodium phosphate solution 83 mL 2.52% sodium hydroxide solution 17 mL Mix well Add 4g of paraformaldehyde and heat the solution to depolyermize the paraformaldehyde |
| Commonly used for ultrastructural studies | phosphate buffered paraformaldehyde |
| Why is paraformaldehyde not used as a dual purpose fixative? | the difficulty of penetration |
| What is the molecular structure of Glutaraldehyde? | C5H8O2 |
| What is the difference between glutaraldehyde and formaldehyde? | It is a dialdehyde, with 1 aldehyde group on each end of the molecule. It crosslinks like formaldehyde however the extra aldehyde group is not involved. It is left free to react in any method using schiff reagent for the detection of aldehydes. |
| Techniques using Schiff reagent, such as the periodic acid- Schiff stain, cannot be used on ______________________fixed tissue. Why? | Glutaraldehyde, false positive |
| Gultaraldehyde uses | used for fixation of specimens for electron microscopy because it preserves ultrastructure. The best of any aldehyde |
| Should fixation be prolonged in Glutaraldehyde? why or why not? | Fixation should not be prolonged because it tends to overharden tissue. |
| What is the recommended fixation time for glutaraldehyde? | 2 hours |
| penetration rate of Glutaraldehyde | fixes and penetrates at the same rate=slowly and poorly |
| Not widely accepted as a fixative for light microscopy | Glutaraldehyde |
| Correct the statement Formaldehyde is an unstable substance that breaks down on exposure to oxygen, with a resultant spike in pH. | Glutaraldehyde, drop |
| What is the concentration range of glutaraldehyde solutions used? | 2-4% |
| How to prepare glutaraldehyde for electron microscopy? | To use for electron microscopy, small vials of glutaraldehyde that were sealed under inert nitrogen should be used and prepared just before use |
| What fixative is the smallest dialdehyde and how is it typically supplied? | Glyoxal, 40% aqueous solution |
| Glyoxal has largely replaced formaldehyde in several industries and in some laboratories. Why? | textile- imparting wrinkle resistance and permanent press creases, its ability to crosslink, and in laboratories because it is much less toxic. |
| Is phosphate buffered glutaraldehyde a sensitizer? | yes, it can cause irritation to the respiratory tract, digestive tract, and skin |
| Sodium phosphate, monobasic 1.9g Sodium hydroxide 0.43g distilled water 100 mL mix well add Glutaraldehyde, 25% solution 18mL | Phosphate buffered glutaraldehyde |
| True or false: Phosphate buffered glutaraldehyde is compatible with oxidizers and alkalis. | false, incompatible |
| What should glyoxal fixative solutions be buffered to and why may it contain a small amount of alcohol? | pH of 4.0 and to catalyze reactions with proteins |
| True or false: Glyoxal fixative solutions are supplied commercially as either alcohol or water based solutions. | True |
| What happens to glyoxal at a high pH? | Glyoxal converts into glycolic acid at a high pH |
| ______________________ reacts with oxygen containing end groups at one pH and amines over a different pH range. | Glyoxal |
| What happens to prolonged storage of glyoxal? | Prolonged storage of tissue in glyoxal fixatives lead to a slight reduction of staining, but this can usually be compensated for prolonged staining times. |
| What happens to erythrocytes and granules of eosinophils and paneth cells in glyoxal formation? | RBCs are lysed and granules of eosinophils and paneth cells do not stain with eosin |
| Safety hazards of glyoxal | can cause skin irritation and has a threshold limit value of 0.1 mg |
| Is glyoxal a carcinogen | no, but it does give off vapors(no odor) |
| Time range for surgical specimens fixed in glyoxal | 4-6 hours |
| Penetration rate of glyoxal | slightly faster than NBF |
| What happens to prolonged storage of tissue in glyoxal fixative? | leads to a slight reduction of staining |
| _______________is leached by the acidity of glyoxal. | Iron |
| What is the silver staining of ______________________ is unsatisfactory in Glyoxal. | Helicobacter pylori |
| Safety hazard of Mercuric Chloride | Extremely toxic |
| What is mercuric chloride referred to as | corrosive sublimate or bichloride of mercury |
| Mercury fixation pigment of mercury can not be prevented but can be removed with____________________. | Iodine followed by sodium thiosulfate |
| How does iodine and sodium thiosulfate remove mercury pigment? | The iodine oxidizes mercury to mercuric iodide which is soluble and sodium thiosulfate removes the excess iodide from the section. |
| Mercury safety hazards | affects the central nervous system, poisoning by skin absorption and can cause acute nephritis, corrosive |
| What fixative do laboratories substitute zinc for? | Mercuric Chloride |
| Molecular structure of Mercuric Chloride | HgCl2 |
| How is mercury used? | in compound fixatives because it is a protein coagulant enhances staining leaving the tissue very receptive to dyes. |
| Its presence in tissue prevents freezing, so frozen sections are difficult to prepare. | Mercuric Chloride |
| How do mercuric salts penetrate? | Mercuric salts penetrate poorly, will produce shrinkage or allow shrinkage in the subsequent processing steps, and can harden excessively with prolonged exposure. |
| Disposal of mercury | do not dispose of in sanitary sewer system(sink) must be collected for appropriate disposal |
| Molecular structure of Osmium tetroxide | OsO4 |
| True or false: Potassium dichromate is not typically used for fixation in histology | False, Osmium Tetroxide |
| Primary use of Osmium Tetroxide | Electron microscopy |
| Actions of Osmium Tetroxide | chemically combines with lipids making them insoluble; 93% of the lipids can be extracted after formaldehyde fixation, but only 7% can be extracted after fixation |
| Where should osmium tetroxide be used and why? | Under a fume hood and vaporizes readily |
| What is the TWA of osmium tetroxide? | 0.002 ppm |
| What will Osmium tetroxide fix? | Conjunctiva, cornea, nasal mucosa |
| What happens to membranes after fixation in osmium tetroxide? | Membranes become black and electron dense |
| After fixation will osmium tetroxide readily accept anionic(acid) dyes or cationic(basic) dyes? | cationic(basic)dyes |
| Molecular structure of picric acid | C6H3N3O7 |
| Another name for picric acid | trinitrophenol or TNP |
| a fixative that is the only substance used as a fixative ingrededient and as a stain | Picric Acid |
| Picric acid Aqueous v. Nonaqueous, Additive v. nonadditive, coagulant v. noncoagulant | Aqueous, additive, coagulant |
| Rate of penetration for picric acid | slow |
| Special uses of picric acid | mordant for trichrome procedures |
| Characteristics of picric acid | precipitates nucleoprotein, leaves DNA soluble and partly hydrolyzed, should not be used with Feulgen reaction or alone, looks like damp sand, explosive must remain 10% moisture |
| How should picric acid be washed? | washing with 50% alcohol is still recommended |
| If picric acid is not completely removed from the tissue, what happens? | the results will be the eventual distortion or obliteration of almost all cellular structures |
| Molecular structure of potassium dichromate | K2Cr2O7 |
| What is special about potassium dichromate?(Think about coagulant v noncoagulant) | It is a noncoagulant unless used in an acid solution; then it will act like chromic acid. This changeover happens at a pH of 3.4-3.8. |
| Characteristics of potassium dichromate | fixed tissue is soft, but shrink more after processing for paraffin embedding than tissue fixed in other solutions, preserves mitochondria by rendering lipid component of membranes insoluble in alcohol, but dissolves DNA, oxidizes unsaturated fatty acids |
| Rate of penetration of potassium dichromate | slow (pH<3.5) fairly rapid (pH>3.5) |
| Special use of potassium dichromate | pH<3.5 none pH>3.5 preserves chromaffin granules(used in orth solution for fixation of pheochromocytomas) |
| Aqueous v. Nonaqueous, Additive v Nonadditive of potassium dichromate | Aqueous and Additive |
| Zinc Salts is a replacment of what fixative ingredient and why? | Mercury because it does not have associated hazards and preserves tissue antigenicity |
| True or False: Zinc Sulfate is more corrosive zinc chloride | False. Zinc Chloride is more corrosive than zinc sulfate. |
| Fixative that preserves immunoreactivity, gives good morphologic preservation, used for routine fixation and substitutes mercury in B-5 solution | Zinc Salts |
| molecular structure of Zinc Salts | ZnSO4 |
| Zinc salts Aqueous v nonaqueous Additive v nonadditive coagulant v noncoagulant | Aqueous, additive, and coagulant |
| Characteristics of Zinc salts | inhibits enzyme activity, poor electron microscopy(ultrastructural preservation), can precipitate in the processor |
| According to Dapson and Dapson, what causes zinc to precipitate? | carbonates present in tap water, phosphates from buffered formalin, pH altering influences from tissue, heat, pressure, vacuum from processor, and alcohol |
| why does zinc formalin fixatives have to be buffered? | to prevent the formation of formalin pigment |
| Health hazards of zinc sulfate | moderate, inhalation can cause irritation to respiratory tract and salts may hydrolyze into acid if swallowed. 10g can cause fatality, skin and eye irritant |
| Health hazard of zinc chloride | severe, corrosive, will cause burns harmful if swallowed, inhaled, or comes into contact with the skin or eyes. |
| PEL of zinc chloride | OSHA 1mg/m3 as a fume |
| What other reagents have been investigated for use in fixative solutions(8 of them)? | carbodiimides, diisocyanates, diazonium compounds, tannic acid, cationic surfactants, diazolidinyl urea, bronopol, and bis-carbonyl compounds. |
| What does Dapson and Dapson mention about the true character of a fixative? | Dapson and Dapson warn that if a fixative works, it cannot be completely safe, because if it fixes specimens it will also fix your skin and cornea. If it does not then it is not a true fixative. |
| Rate of penetration of 10% auqeous formalin? | slow |
| 10% aqueous formalin Additive v nonadditive, aqueous v nonaqueous, and coagulant v noncoagulant | additive, aqueous, noncoagulant |
| Special uses of 10% aqueous formalin | routine specimen and immunohistochemical (IHC) |
| Characteristics of 10% aqueous formalin | not osmotically active, hypotonic, may produce formalin pigment due to its acidic pH |
| 10% formalin saline aqueous v nonaqueous, additive v nonadditive, coagulant v noncoagulant | aqueous, additive, noncoagulant |
| Special uses of calcium formalin | used for fixation and preservation of phospholipds in tissue |
| Characteristics of calcium formalin | calcium changes phospholipid solubility |
| is calcium formalin aqueous or non-aqueous? Additive or non-additive? Coagulant or non-coagulant? | aqueous, additive, coagulant |
| Characteristics of ammonium bromide formalin | acidic, lyses RBCs, causes nuclei to give a direct Fuelgen reaction due to hydrolysis during fixation |
| Aqueous v nonaqueous, additive v nonadditive, coagulant v noncoagulant of formalin ammonium bromide | aqueous, additive, noncoagulant |
| What is Acetate formalin used for? | phospholipid fixation |
| Acetate formalin Aqueous v nonaqueous, additive v nonadditive, coagulant v noncoagulant | aqueous, additive, and noncoagulant |
| 10% of NBF aqueous v nonaqeous, additive v nonadditive, and coagulant v noncoagulant | aqueous, additive, noncoagulant |
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