Fixation Flashcards
BOC Study Guide Question
To adequately remove calcium from a specimen containing areas of microcalcification, the tissue could be fixed in:
A. Hollande
B. Bouin
C. Gendre
D. Zamboni
HT level
A. Hollande
It is an acidic solution but is balanced with the cupric acetate it contains. Solution is specifically described as “will decalcify small specimens of bone”
See Hollande Solution fixative profile flash card
BOC Study Guide Question
Which of the following fixatives contains copper acetate?
A. Hollande
B. Bouin
C. Gendre
D. Zamboni
HT level
A. Hollande
Its ingredients are copper acetate, piric acid, formaldehyde, acetic acid (blances shrinkage), DI water
See Hollande Solution fixative profile flash card
BOC Study Guide Question
The problem with the image is the result of:
A. Incomplete fixation
B. Excessive dehydration
C. Incomplete clearing
D. Poor paraffin infiltration
HT level
A. Incomplete fixation
-cracks
-fuzzy nuclei
BOC Study Guide Question
Bouin solution is contraindictated for:
A. Small tisue biopsies
B. Tissue intended for subsequent trichrome stains
C. Tissue to be stained by the Feulgen reaction
D. Routine tissue sections
HT level
C. Tissues to be stained by the Feulgen reaction
Picric acid is a sufficiently strong acid to hydrolyze nuclei. So if stains for DNA (Feulgen) or RNA are anticipated, then any fixative containing picric acid (Bouin, Gendre, or Hollande) should be avoided
See Feulge Reaction or Bouin Fixative Profile flash cards
BOC Study Guide Question
Formalin pigment can be removed from tissue sections by treatment with 10% ____:
A. HCL in 70% alcohol
B. Nitric acid in 70% alcohol
C. Sulfuric acid in 70% alcohol
D. Ammonium hydroxide in 70% alcohol
HT level
D. Ammonium hydroxide in 70% alcohol
Formalin pigment can be dissolved with alcoholic solutions containing picric acid, sodium hydroxide, or ammonium hydroxide. Ammonium hydroxide is the only alkaline alcohol option.
See Formalin Pigment flash card
BOC Study Guide Question
One action of acetic acid is to:
A. Exert a shrinking effect on tissue
B. Render nucleoprotein acidophilic
C. Form salt linkages between protein chains
D. Coagulant for nucleoproteins
HT level
D. Coagulate nucleoproteins
Acetic acid coagulates & preserves nucleic acids/nucleoproteins, swells tissue, only exposes hydrophilic groups, and does not create salt links.
See Acidic Acid flash card
BOC Study Guide Question
A fixative containing potassium dichromate:
A. is suitable when histochemical techniques are planned
B. will result in excellent subsequent silver staining
C. is preferred for preservation of argentaffin cells
D. will make tissue more receptive to eosin staining
HT level
D. will make tissue more receptive to eosin staining
See Potassium Dichromate flash card
BOC Study Guide Question
A biopsy of whichof the following tissues should be sectioned at 2um?
A. bladder
B. heart
C. kidney
D. liver
HT level
C. kidney
General rule kidney bxs should be sectioned @ 2um to conserve tissue & avoid re-bx of organ sentive to bxing
See Microtomy flash card deck
BOC Study Guide Question
Which of the following fixatives may give false positive results in some carbohydrate techniques?
A. NBF
B. formic acid
C. paraformaldehyde
D. glutaraldehyde
HT level
D. Glutaraldehye
It is a dialdehyde, and the extra aldehyde group does not form crosslinks. The open aldehyde group is free which contributes to background staning on PAS where the principle reaction is based on the oxidation of certain tissue elements to aldehydes by periodic acid.
See Glutaraldehyde flash card
BOC Study Guide Question
The problem seen in this imageis known as:
A. cell shrinkage
B. smudgy nuclei
C. pyknotic nuclei
D. nuclear bubbling
HT level
D. Nuclear bubbling
See additonal example image
BOC Study Guide Question
Tissue stored for long periods of time in unbuffered formalin or acetate formalin may show brown, cyrstalline pigment in stained sections. To remove the pigment prior to staining it is necessary to treat the microscopic section with:
A. saturated alcoholic picric acid
B. alcoholic lithium chloride
C. iodine and sodium thiosulfate
D. potassium permanganate and oxalic acid
HT level
A. saturated alcoholic picric acid
The fixative solution’s pH dropped lower than 6.0, formalin/hematin pigment is dissolved by alcoholic basic solutions (ammonium hydroxide) or alcoholic picric acid
See Formalin Pigment flash card
BOC Study Guide Question
For good fixation of tissue with osmium tetroxide for EM, it is recommended that the tissue segment be no larger than:
A. 1 mm3
B. 2 mm3
C. 1 cm3
D. 2 cm3
HT level
A. 1 mm3
Osmium tetroxide penetrates very poorly. 1mm cubes are ideal for EM and thin at microtmoy for fat demonstartion in paraffin sections.
See osmium tetroxide flash card
BOC Study Guide Question
It is necessary to adjust the pH of most formalin solutions because of the presence of:
A. methanol
B. formic acid
C. paraformaldehyde
D. carbon dioxide
HT level
B. Formic acid
Formaldehyde solutions become acidic by recting with atmospheric oxygen=> make formic acid. So they require pH raising (or prefectly buffering) to neutrality. Formic acid will form formalin pigment.
See formic acid and formalin pigment flash cards
BOC Study Guide Question
Carnoy solution is recommended for the preservation of:
A. acid-fast bacilli
B. nucleic acids
C. lipids
D. RBCs
HT level
B. Nucleic acids
Carnoy solution has good nuclear preservation but RBCs lyse, dissloves lipids, and definitely not recommended for acid-fast bacilli demonstrations.
See Clark Solution flash card
BOC Study Guide Question
Which of the following factors affects fixation for light microscopy?
A. temperature
B. volume ratio
C. penetration rate
D. pH
HT level
D. pH
pH form 4 to 9 makes little difference in fine structure formalin fixation. pH< 4 will produce pigment however. So aso long as the volume ratio, temp, & rate of penetratoin are good, LM images will be okay.
See the Factors Affecting Fixation flash card
BOC Study Guide Question
Very bloody cytology smears are often treated with:
A. 10% formalin
B. Hollande solution
C. Clark solution
D. Acetone
HT level
C. Clark Solution
Hollande stablizes RBC membranes, the rest lyse RBCs but only Clark solution is commonly used for lysing of RBC (removing obscurity) in cytology specimens.
See the Clark Solution flash card
BOC Study Guide Question
Glyoxal is one of the newer fixatives which has the added advantage of:
A. ability to crosslink
B. rapidity of action
C. enhanced staning
D. preservation of erythrocytes
HT level
B. Rapidity of action
Glyoxal is rapid acting, crosslinks specifically, lyses RBCs, and has a slight reduction in staining especially after longer periods of storage
See the Glyoxal flash card
BOC Study Guide Question
If a tissue section was fixed in a solution different from that required for a staining procedure, microscropic sections frequently can be stained anyways if they are:
A. soaked in a solution of lithium carbonate prior to staining
B. revitalized by washing in a solution of sodium bisulfate
C. postfixed in the appropriate fixative prior to staining
D. treated with hydrogen peroxide
HT level
C. Postfied in the appropriate fixative prior to staining
Sections can usually be mordanted or postfixed in the fixative required for yielding the best staining results
See the Mordant vocab flash card
BOC Study Guide Question
B-5 fixative contains:
A. mercuric chloride, sodium acetate, and glacial acetic acid
B. mercuric chloride, potassium dichromate, and glacial acetic acid
C. mercuric chloride, sodium acetate, and 37% to 40% formaldehyde
D. mercuric chloride, potassium dichromate, and 37% to 40% formaldehyde
HT level
C. mercuric chloride, sodium acetate, and 37% to 40% formaldehyde
B-5 Ingredients: mercuric chloride, sodium acetate, DI, dilute with formaldehyde
See Mercuric Chloride flash card
BOC Study Guide Question
Zamboni PAF refers to a fixative containing:
A. potassium dichromate, acetic acid, and formaldehyde
B. potassium aluminum sulfate and paraformaldehyde
C. buffered picric acid and formaldehyde
D. picric acid, acetic acid, and formaldehyde
HT level
C. Buffered picric acid and formaldehyde
Zamboni is also know as “buffered picric acid-formaldehyde” solution that may be used as a general fixative. It allows secondary fixation with osmium tetroxide and preserves the morphologic characteristics accurately; therfor it is useful for both LM & EM
See Zamboni fixative flash card
BOC Study Guide Question
For most fixatives, the volume of fixing fluid in relation to the volume of tissue should be:
A. 2 to 5 times
B. 6 to 9 times
C. 10 to 14 times
D. 15 to 20 times
HT level
D. 15 to 20 times
The golden ratio range for volume fix: volume tissue
15:1 to 20:1
BOC Study Guide Question
The preferred fixative when tissue is to be stained for the presence of simple fats is:
A. Zenker
B. Helly
C. Hollande
D. NBF
HT level
D. NBF
More specifically, stains for fats are done on frozen sections and the preferred fixative right off the cyrostate is NBF
See NBF flash card
BOC Study Guide Question
Fixation of cytology smears should occur within:
A. 1-2 seconds
B. 10-15 seconds
C. 40-45 seconds
D. 1 minute
HT level
A. 1-2 seconds
If longer an air-drying artifact will occur
BOC Study Guide Question
A good fixative for routine use is one that:
A. makes tissue more permeable to fluids
B. is hypotonic to the tissue constituents
C. Enhances putrefaction of tissue components
D. Promotes autolysis
HT level
A. Makes tissue more permeable to fluids
So that all subsequent process can occur.
Think processing with alcohol and infitration
BOC Study Guide Question
A pigment caused by chromate-containing fixatives can be prevented by treating the tissue prior to processing with:
A. running water
B.iodine
C. piciric acid
D. Potasium permanganate
HT level
A. Running water
Chromate-containing fixatives shoud be washed (with water) before processing becuase the dehydrating alcohol can couase an insoluble pigment to form
See Potassium chromate flash card
Fixative Profile
Hollande Solution
(Part 1)
Properties & functions
From Self Instruction textbook, notes & etc
Picric acid-based >modified Bouin solution
* Protein coagulant additive +
* Nucleoprotein ppts, DNA soluble/partially hydrolyzed+/-
* Nill lipid and carbohydrate reaction -
* Very slow penetration -
* Inhibits enzyme activity +
* Poor EM/ultrastructural preservation -
* Stablizes (cupric acetate) RBC membranes, granuels of eosinophils & endocrine cells +
* Excellent for GI, testies, endocrine, & connective
Piric acid = exploseive dry
See Part 2
Fixative Profile
Hollande Solution
(Part 2 with Ingredients)
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Picric acid-based >modified Bouin solution
- Will decal small bone spec. +
- *Should not be used with Feulgen reaction** becasue of false pasitve with Schiffs due to DNA hydrolysis-
* - Fixative must be washed out before phosphate buff formalin (insoluble phosphate ppt)
**Ingredients: **copper acetate, piric acid, formaldehyde, acetic acid (blances shrinkage), DI water
Piric acid = exploseive dry
See Part 1
Fixative Profile
Zamboni (PAF)
(Part 1)
Properties & functions
From Self Instruction textbook, notes & etc
Picric acid-based
* Protein coagulant additive +
* Nucleoprotein ppts, DNA soluble/partially hydrolyzed+/-
* Nill lipid and carbohydrate reaction -
* Very slow penetration -
* Inhibits enzyme activity +
* Good EM/ultrastructural preservation if postfixed with osmium +/-
* Good general fixative+
Piric acid = exploseive dry
See Part 2
Fixative Profile
Zamboni (PAF)
(Part 2)
Properties & ingredients
From Self Instruction textbook, notes & etc
Picric acid-based
* pH 7.3 so somewhat stable +
* Should not be used with Feulgen reaction* -
* The paraformaldehyde needs to be slightly alkaline in order to dissociate it into formaldehyde
**Ingredients: **paraformaldehyde, saturated aqueous picric acid, sodium hydroxide, phospate buffer, & DI
Piric acid = exploseive dry
See Part 1
Fixative Profile
Acetic Acid
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Not usually used alone
Nonadditive fixative
* Nucleoprotein/DNA coagulant nonadditive +
* Nill protein, lipid, &carbohydrate reactions -
* Rapid penetration +
* Counter acts shrinking + (protein swelling more than anyother fixative, collagen swells because the acid breaks protein links exposing hydrophilic which pulls water in)
* Unknown enzyme activity +/-
* Poor EM/ultrastructural preservation -
* Lysis RBC -
See Acetic Acid chemial structure
Fixative Profile
Potassium Dichromate
(Part 1)
Properties & functions
From Self Instruction textbook, notes & etc
Not Used Alone
Additive fixative
Orth Solution (only real example)
* Basic compared to chromic acid
* pH > 3.5 specific use for chromaffin granuels (orange/brown) in cytoplasm of adreanl medulla; diag phenochromocytoma+
* Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining +
* Tissue must be washed (with water) after fixation-
* Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+
* Attaches to some lipids & makes insoulble +/-
* Nil carbohydrate reaction
* Rapid penetration +
See also chromic acid flash card
Fixative Profile
Potassium Dichromate
(Part 2)
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Not Used Alone
Additive fixative
Orth Solution (only real example)
* Chromium is hightly toxic-
* Inhibits enzyme activity+
* Poor/distorted EM/ultrastrucutral preservation
* Protein noncoagulant additive+
* Dissolves DNA/precipitates nucleoprotein +/-
* Attaches to some lipids & makes insoulble +/-
Orth Ingredients: potassium dichromate, sodium sulfate, DI, dilute with formaldehyde
See also chromic acid flash card
Fixative Profile
Chromic acid
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Not Used Alone
Additive fixative
* Potassium dichromate in an acidic solution
* Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining +
* Tissue must be washed after fixation-
* Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+
* Chromium is hightly toxic-
* Inhibits enzyme activity+
* Poor/distorted EM/ultrastrucutral preservation
* Protein coagulant additive+
* Nucleic acids coagulant+
* Oxidizes unsat. fatty acids-
* Oxidizes carbohydrates to aldehydes-
* Slow penetration-
See potassium dichromate chemial structure
Fixative Profile
Glyoxal
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Dialdehyde
Additive fixative
** Noncogulant fixative**
* Much less toxic than formaldehyde+
* Protein additive (form n-hydroxymethl adduct with 2 carbon atoms)+
* Nucleic acid additive +
* Nil lipid & carbohydrate reactions -
* Rapid penetration ( with little crosslinking) +
* Enzyme activity inferior to formaldehyde-
* Unkown EM/ultrastructural preservation -
* Chromatin & membranes perserved with excellent clarity+
* Stains for H. pylori (silver) unsatisfactory-
* Antigen retrieval not needed except if arginine is th epitope +/-
* Despite one free aldehyde group, no PAS reaction problems +
* Lysis RBCs -
* Crosslinks specifically +/-
* Slight reduction is staning especially after longer periods of storage
Fixative Profile
Carnoy Solution
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Nonaqueous fixative
Nonadditive fixative
Noncogulant fixative
* Rapid acting + (should not go longer than 4hrs)
* Preserves glycogen +
* Good nuclear preservation +
* Excessive shrinkage -
* Excessive hardening -
* Lysis RBCs -
* Helps strip RBCs for cyology spec +
* Prolonged exposure can damage CNS, liver, kidneys, & eyes
* Carcinogenic due to chloroform
Carnoy Ingredients: absolute alcohol, chloroform, acetic acid
Fixative Profile
Glutaraldehyde
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Aqueous fixative
Additive fixative
Noncogulant fixative
! The extra alddehyde not involved in crosslinking reacts tot PAS stain causing false positives!
* Penetrates slowly & poorly (thin gross sections)
* Crosslinking rapidly
* Good EM/ultrastructure preservation +
–>usualy post fixed with osmium
* Excessive hardening if prolonged -
!Unstable= breaks down when oxygen exposed
* Noncoagulant of nucelic acids+
* Perserves lipids with gradual loss-/+
* Carbohydrates nill-
* Inhibits enzyme activity >formaldehyde+
Glutaraldehyde Ingredients: sodium phosphate/monobasic, sodium hydroxide, DI, & 25% gluaraldehyde
Fixative Profile
Mercuric Chloride (HgCl2)
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Additive fixative
Coagulant fixative
B-5 Solution (only real example)
* Protein coagulant +
* Nucleoprotein/DNA coagulant +
* Nill carbohydrate reactions -
* “Unmaskes” some lipids +/-
* Rapid penetration +
* Inhibits enzyme activity -
* Organelles preserved so EM/ultrastructural preservation +
-Not used alone-
B-5 Ingredients: mercuric chloride, sodium acetate, DI, dilute with formaldehyde
See B-5 solution flash card
Fixative Profile
Osmium tetroxide
(OsO4 and aka Osmic acid)
Properties & functions
From Self Instruction textbook, notes & etc
Additive fixative
Noncoagulant fixative
Primarly postfixative for EM to ensure preservation of lipids
* Protein additve noncoagulant +
* Nucleoprotein/DNA noncoagulant +
* Nill carbohydrate reactions -
* Reacts with and adds to doulbe bone in lipids makes them insoluble +
* Very slow penetration (1mm cubes) -
* Inhibits enzyme activity +
* Excellent organelle & membrane preservation so excellent EM/ultrastructural +
-Used alone-
!Vaporizes easily, so expsensive & hazardous
–> TWA 0.002ppm
Vocab
Mordant
From Self Instruction textbook, notes & etc
A reagent used to link the stain or dye molecules to the tissue. Many in histo are metals espeically those linking hematein to tissue
Example: Hematoxylin is an acid, but as it is almost always used in conjunction with alum or iron (the mordant) it becomes a basic stain
Vocab
Additive
From Self Instruction textbook, notes & etc
A chemical or substance that adds on to/links/combines with, another substance altering it usally improving/strengthening. The electrical charge at attachment site changes. Many add on to tissue proteins.
Examples:
1. Mercuric chloride-proteins
2. Picric acid-proteins
3. Potassium chromate-proteins
4. Formaldehyde-proteins
5. Glutaraldehyde-proteins
6. Glyoxal-proteins & nucleic acids
7. Osmium tetroxide-proteins
8. Zinc sulfate/chloride-proteins
See the mercuric chloride, picric acid, potassium chromate, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc sulfate/choride flash cards
Vocab
Aqueous Fixatives
From Self Instruction textbook, notes & etc
Waster based fixatives. Lipid preservation is possible with only a few.
Examples:
1. Acetic acid
2. Formaldehyde-some gradual lipid loss
3. Glutaraldehyde-some gradual lipid loss
4. Glyoxal-
5. Mercuric chloride-Unmasks some lipids
6. Osmium tetroxide-reacts & adds to double bond in lippids making them insoluble
7. Picric acid
8. Potassium dichromate low pH-oxidizes unsaturated fatty acids (so no preservation)
9. Potassum dichromate hi pH- attacehds to some & makes them insoluble
10. Zinc sulfate/chloride
Vocab
Nonadditive
From Self Instruction textbook, notes & etc
A chemical or substance that act on tissue without chemically combining with it. Mostly organic compouds that primarly dissociate bound water molecules from tissue protein groups, protein solubility is then lost and it precipitates (PPT). Excessive water removal shrinks & harden tissue.
Examples:
1. Alcohols (nonaqueous, can be used alone)
2. Acetone (nonaqueous, can be used alone)
3. Acetic acid (noted the only sweller)
Compare to Additive fixatives
Vocab
Coagulant
From Self Instruction textbook, notes & etc
“Metal Mesh”
Creates a network in tisue that allows solutions to gain entry into the interior of the tissue easily (easy penetration).
Examples:
1. Zinc salts
2. Mercuric chloride
3. Cupric sulfate
4. Eythl alcohol
5. Acetone
6. Picric Acid
7. Acetic acid- Nucleic acids
Hold mesh ball when learning above examples
Compare to Noncoagulant fixatives
Vocab
Noncoagulant
From Self Instruction textbook, notes & etc
“Jello”
Fixatives that create gel that make penetration by subsequent solutions difficult.
Examples:
1. Formaldehye
2. Gulteraldehyde
3. Glyoxal
4. Osmium tetroxide
5. Postassium dichromate
Draw jello cup with all six fixatives above
Compare to Coagulant fixatives
Vocab
Pyknosis
From Self Instruction textbook, notes & etc
The irreversible condensation of Chromatins in nucleii. It can happen when the solution temperature in microwave exceeds 68C.
Trouble Shooting
Formic Acid
Cause, Prevention, & Correction
From Self Instruction textbook, notes & etc
Cause: Unbuffered formaldehyde solutions (acidic pH4-5, includes unbuff-zinc formaldehye solutions too) react to atmospheric oxygen which forms formic acid. The solutions forms black acid hematin/ formalin pigment when added to blood rich tissue.
Prevention: Maintain solution pH closer to 7 (buffering) & appropriate volume ratio of fixative solution.
Correction: Treat tissue sections with alcoholic picric aid or alkaline alcohol.
Trouble Shooting
How do you remove chromium pigment?
From Self Instruction textbook, notes & etc
Correction: It can partially be removed by the addition of acid alcohol.
Trouble Shooting
How do you remove mercury pigment?
From Self Instruction textbook, notes & etc
Correction: It can be removed by the addition of Iodine and sodium thiosulfate.
Think “MIS” aka Mercury = Iodine & soium thiosulfate
Trouble Shooting
How do you remove formalin pigment?
From Self Instruction textbook, notes & etc
Correction: It can be removed by the addition of alcoholic picric acid and alkaline alcohol.
Trouble Shooting
All Factors Affecting Fixation
From Self Instruction textbook, notes & etc
- Temperature
- Size
- Volume Ratio
- Time
- Choice of Fixative
- Penetration
- pH
- Osmolality
- Storage
See respective flash cards for each factor
Fixation Factor
Temperature
From Self Instruction textbook, notes & etc
Can affect tissue morphology
Increasing it also increases rate of fixation but also rate of autolysis
45C max
See respective flash cards for each factor
Trouble Shooting
Formalin Pigment
Cause, Prevention, & Correction
From Self Instruction textbook, notes & etc
Also: Black acid hematin
Cause: Formic acid added to blood rich tissue from unbuffered formaldehyde solutions (acidic pH4-5, includes unbuff-zinc formaldehye solutions too) that react to atmospheric oxygen.
Prevention: Stop formic acid from forming. So maintain solution pH closer to 7 (buffering) & appropriate volume ratio of fixative solution.
Correction: Treat tissue sections with alcoholic picric acid or alkaline alcohol.
Fixative Group
Picric Acid-Based Solutions
From Self Instruction textbook, notes & etc
Additive fixative
!Contraindicated for tissues destine for Feulgen reaction as fixation hydrolyzes the nuclei excessively so stain will be unsatisfactory!
-Bouin
-Gendre
-Hollande
-Zamboni (PAF or picric acid formalin)
See Picric Acid chemical structure
See Bouin, Gendre, Hollande, & Zamboni flash cards respectively
How are Zinc-formalin fixatives different than “normal” formalin?
From Self Instruction textbook, notes & etc
Give poor ultrastructural preservation because Zinc is a coagulant.
See coagulant & formalin flash cards
Fixative Profile
Potassium Dichromate
(aka chromic acid)
Properties, functions & ingredients
From Self Instruction textbook, notes & etc
Not Used Alone
Orth Solution (only real example)
* (pH > 3.5) Specific use for chromaffin granuels (orange/brown) in cytoplasm of adreanl medulla; diag phenochromocytoma+
* Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining +
* Tissue must be washed after fixation-
* Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+
* Chromium is hightly toxic-
* Inhibits enzyme activity+
* poor/distorted EM/ultrastrucutral preservation
* pH effects alot of characteristics
- <3.5 pH: protein coagulant additive+, nucleic acids coagulant+, oxidizes unsat. fatty acids-, oxidizes carbohydrates to aldehydes-, & slow penetration-
- > 3.5 pH: protein noncoagulant additive+, dissolves DNA/precipitates nucleoprotein +/-, attaches to some lipids & makes insoulble +/-, nil carbohydrate reaction, rapid penetration +
Orth Ingredients: potassium dichromate, sodium sulfate, DI, dilute with formaldehyde
See Potassium dichromate chemial structure
Fixation Factor
Size
From Self Instruction textbook, notes & etc
“thickness”
Effects reagent penetration, as the more distanceto cross the longer it will take but autolysis of deeper/inner tissue will occur first
See respective flash cards for each factor
Fixation Factor
Volume Ratio
From Self Instruction textbook, notes & etc
Volume of fixative must be at least 15x-20x > tissue volume
If not,the fixative composition will be compromised as water & serum proteins are displaced
See respective flash cards for each factor
Fixation Factor
Time
From Self Instruction textbook, notes & etc
Ischemic (blood supply cut off) & duration
Inadequate duration will not preserve morph
Avoid autolysis, putrefication, & desquamation of epithelium
See respective flash cards for each factor
Fixation Factor
Choice of Fixative
From Self Instruction textbook, notes & etc
Appropriate choice, as often no correction possible
I.F. , enzyme study, or fat = frozen
Staining reaction might require post fixation (ex trichrome)
Water soluble element (urate cyrstals) = 100% ROH
See respective flash cards for each factor
Fixation Factor
Penetration
From Self Instruction textbook, notes & etc
Deepth =k sq(t)
k= diffusibility coefficient (unique to each fixative)
Type of tissue influences it (muscle fiber, capillaries, & high surface area
Heat
Coagulant vs noncoagulant
See respective flash cards for each factor
Fixation Factor
Tissue Storage
From Self Instruction textbook, notes & etc
Additional studies wet tissue reserve
NBF=indefinet storage but IHC ideal is to move to 70% ROH to stop crosslinking
See respective flash cards for each factor
Fixation Factor
Osmolality
From Self Instruction textbook, notes & etc
the # of particles in solution
Critical/Important in ultrastructural/EM studies
Most body fluids 0.3 Osm
1 Osm = 1 formula weight of nondissociating compound per 1000g solution (ex sucrose)
Isotonic [sol particles] = cytosol
Hypo [sol particles] < cytosol
Hyper [sol particles] > cytosol
See respective flash cards for each factor
Fixation Factor
pH
From Self Instruction textbook, notes & etc
Effects reactivity of fixative and EM/ultrastructural preservation
Remember, pickling in high acidity does not bode well for EM
See respective flash cards for each factor
Microscopic evaluation of a tissue section reveals a brown pigment lying on top of the tissue. Adjacent sections are treated with: 1. iodine and sodium thiosulfate 2. potassium ferrocyanide and hydrochloric acid 3. saturated alcoholic solution of picric acid. All sections still show the brown pigment. This pigment could have resulted from improper washing following fixation in:
A. Zenker fluid
B. formalin
C. B-5 fluid
D. Bouin fluid
BOC HT Question Pool
A. Zenker fluid
The sections must have been fixed in Zenker fluid, whichcontains chromium trioxide, and not washed carefully before placing the tissue in alcohol. The alcohol will reduce the chromium and** forms an insoluble pigment in the tissue.**
Zenker Ingredients: DI, Mercuric chloride,
Potassium dichromate, Glacial acetic acid,
Fixation time: 4 – 24 hours
