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Histology Fall 2019 > Fixation > Flashcards

Fixation Flashcards

1
Q

what is a fixative?

A

alters the tissue by stabilizing protein so it is resistant to further changes

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2
Q

what do fixatives do? (4)

A

prevent autolysis by deactivating enzymes, kills bacteria and molds that can cause putrefaction, makes tissues more receptive to dyes and can act as a mordant, stabilizes cellular structures to resist change during subsequent processing and staining

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3
Q

autolysis

A

breakdown of tissue due to enzyme activity

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4
Q

putrefaction

A

breakdown of tissue by bacteria

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5
Q

direction of fixation

A

outer layer of the tissue moving inwards

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6
Q

appropriate tissue size

A

3-4mm thick and 2x2cm square

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7
Q

reasons for choosing a fixative

A

rate of penetration, length of storage, pH for electron microscopy, osmolality for EM, looking for a specific structure that may be adversely affected by certain fixatives

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8
Q

non-coagulant fixative

A

creates a gel that is difficult for subsequent solutions to penetrate, preferred for electron microscopy due to a more life-like fixation

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9
Q

coagulant fixative

A

establish a network in tissue that allows solutions to readily penetrate the interior of the tissue

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10
Q

non-additive fixative

A

act on tissue without chemically combining with it, often by dissociating water molecules from protein

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11
Q

additive fixative

A

add themselves onto the protein and cause a change

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12
Q

black acid hematin

A

occurs when formalin is used and the pH drops below 6, especially on tissues rich in blood
can be removed by treating sections with alcoholic picric acid or alkaline alcohol

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13
Q

physical agents of fixation

A

heat and desiccation

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14
Q

volume of fixation fluid

A

15 to 20x the volume of tissue

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15
Q

4 factors affecting fixation

A

time in fixative, temperature of fixative, size of tissue, volume ratio of fixative solution

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16
Q

4 functions of fixatives

A

kill the tissue, maintain proper relationships between cellular structures, increase contrast between tissue elements via differing refractive indexes, stabilize the tissue physically and chemically

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17
Q

list of simple fixatives

A

Acetic Acid, formalin, glutaraldehyde, glyoxal, mercuric chloride, osmium tetroxide, picric acid, potassium dichromate, zinc salts

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18
Q

list of compound fixatives

A

B-5, Bouin, Gendre, Hollande, Zenker, Helly, Orth, Zamboni, Zinc Formalin

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19
Q

Acetic Acid

A

non coagulant (coagulates) cytoplasm and nucleic acids
does not fix or destroy carbs or lipids
penetrates rapidly
leaves tissue soft
precipitates DNA
Increases protein swelling (disadvantage)
Lyses red blood cells (often counteracted in compound fixatives)

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20
Q

B-5

A

Good nuclear detail for staining blood forming and lymph tissue
good for many special stains

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21
Q

Bouin

A

great for soft delicate structures and trichrome stains
acetic acid swelling is countered by picric acid shrinkage
hardening of formaldehyde is countered by soft fixation of picric acid
yellow color is removed with 50-70% EtOH
don’t use for EM because it does not preserve nucleic acids

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22
Q

Gendre Solution

A

great preservation of carbs, esp glycogen

excess picric acid is removed with 80% EtOH

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23
Q

Hollande Solution

A

can decalcify small bone specimens
good for GI biopsies
cupric acetate counters the lysis of RBCs and eosinophil granules caused by acetic acid

24
Q

Zenker

A

tissues must be treated for mercury pigment with iodine followed by sodium thiosulfate
makes nuclei less basophilic and cytoplasm more acidophilic, increase time in hematoxylin and reduce eosin
good for nuclear chromatin, connective tissue
very toxic and carcinogenic

25
Q

Helly

A

same as Zenker except it preserves red blood cells because the acetic acid is replaced with formalin
good for bone marrow, spleen, and kidney

26
Q

Orth Solution

A

good for demonstrating chromaffin granules in the adrenal glands

27
Q

Zamboni Solution

A

very stable, good general use fixative

allows secondary fixation with osmium tetroxide, sometimes preferred for EM over glutaraldehyde

28
Q

Zinc Formalin

A

no loss of antigenicity for long term storage

prevents cross-linking

29
Q

non-aqueous fixatives

A

acetone, methanol, ethanol, carnoy, clarke

30
Q

acetone

A

non-additive coagulant (no gel matrix)
demonstrates enzymes, esp acid and alkaline phosphatase
used on frozen tissue for IHC
acts fast but causes shrinkage, distortion and hardening

31
Q

methanol and ethanol

A

methyl alcohol is for touch preps and blood smears, ethyl alcohol is used to preserve glycogen and urate crystals, which are water soluble
preserves most pigments, dissolves fat, and causes shrinkage

32
Q

Carnoy

A

lyses RBCs, sometimes used for cytology
fast, preserves glycogen, good nuclei
excessive shrinkage and hardening

33
Q

Clarke

A

good for blood smears and frozen sections

34
Q

Michel Transport Medium

A

pH 7-7.2

ingredients: Anhydrous citric acid, ammonium sulfate, N-ethylamide, Magnesium sulfate

35
Q

Remove Formalin pigments

A
  1. absolute alcohol saturate with picric acid, then wash with water or
  2. 70% alcohol with ammonium hydroxide, wash, then rinse with 1% acetic acid
36
Q

Remove Mercury pigments

A

treat with iodine, wash in running water, put in sodium thiosulfate, wash in water

37
Q

Remove Chrome pigments

A
  1. wash tissue in running water before dehydration or

2. can be partially removed from the section using acidic alcohol

38
Q

common fixation problems

A

autolysis, incomplete fixation

39
Q

troubleshooting autolysis

A

get tissue into fixative ASAP
pin open uterus and GI samples to expose mucosa to fixative
gross thin sections and keep in large volume of fixative
bisect lymph nodes for fixative

40
Q

troubleshooting incomplete fixation

A

increase time in fixative
change to another fixative
use formalin alcohol in the 1st 3 stages of processing
gross sections thin with 15 to 20x fixative
change formalin solution frequently to prevent depletion of fixative
agitate cassettes in fixative solution
do not pack cassettes tightly in processing holders

41
Q

B5 ingredients

A

mercuric chloride, sodium acetate, formaldehyde

42
Q

Bouin ingredients

A

picric acid, formaldehyde, acetic acid

43
Q

Gendre ingredients

A

95% EtOH saturated with Picric acid, formaldehyde, acetic acid

44
Q

Hollande ingredients

A

copper acetate, picric acid, formaldehyde, acetic acid

45
Q

Zenker ingredients

A

mercuric chloride, potassium dichromate, acetic acid

46
Q

Helly ingredients

A

mercuric chloride, potassium chromate, formaldehyde

47
Q

Orth ingredients

A

potassium dichromate, sodium sulfate, formaldehyde

48
Q

Zamboni ingredients

A

paraformaldehyde, picric acid, phosphate buffer

49
Q

Zinc formalin ingredients

A

zinc sulfate, formaldehyde

50
Q

Carnoy ingredients

A

absolute elthyl alcohol, chloroform, acetic acid

51
Q

Clarke ingredients

A

absolute ethyl alcohol, acetic acid

52
Q

list of coagulants

A

zinc salts, mercuric chloride, cupric sulfate, ethanol, methanol, acetone, picric acid, potassium dichromate<3.5

53
Q

list of additive fixatives

A

mercuric chloride, chromium tetroxide, picric acid, formaldehyde, glutaraldehyde, osmium tetroxide, zinc sulfate, zinc chloride, glyoxal, potassium dichromate

54
Q

list of non-coagulant fixatives

A

formaldehyde, potassium dichromate>3.5, osmium tetroxide, glutaraldehyde

55
Q

list of non-additive fixatives

A

ethanol, methanol, acetone

Histology Fall 2019 (31 decks)

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