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HPCT > FIXATION > Flashcards

FIXATION Flashcards

1
Q

first and MOST CRITICAL STEP IN
HISTOTECHNOLOGY

A

Fixation

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2
Q

FIXATION
Primary goal
○ Preserve the _______ & ______
integrity of the cell in as life-like manner as possible.

➢ Secondary goal:
○ ______ & protect tissue [from _____ of further handling, so that it is easier to cut and process for microscopy]

A

morphological
chemical

harden
trauma

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3
Q

prevents degeneration, decomposition,
putrefaction & distortion of tissue.

A

Fixation

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4
Q

2 basic mechanisms involved in fixation.

A

Additive

Non-additive

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5
Q

Chemical constituent of the fixative becomes part of the tissue.

Examples [3]

A

Additive fixation

formalin
mercury
osmium tetroxide

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6
Q

fixing agent is not incorporated into the tissue

Alters tissue composition by ___________ attached to the _______ molecule.

Example [1]

A

Non-additive

removing bound water
protein

alcoholic fixatives

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7
Q

Factors involved in fixation [5]

A

Hydrogen ion concentration

Temperature

Osmolality

Concentration

Duration

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8
Q

Procedure adopted to kill, harden, and preserve materials for microscopic study by means of a substance known as a____

A

fixative

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9
Q

Fixative effects
[6]

A

– Harden soft and friable tissue
– Make cells resistant to damage & distortion
– Inhibit bacterial decomposition
– Increase optical differentiation of cells & tissue components
– Acts as mordants or accentuators to promote & hasten staining
– Reduce the risk of infection

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10
Q

In fixation,

[3] and chemical constituents of tissues are preserved by [X] degeneration, decomposition and distortion of tissues after death.

A

shape
structure
intercellular relationship
chemical constituents

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11
Q

Fixation prevents [4] of tissue.

A

degeneration
decomposition
putrefaction
distortion

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12
Q

hydrogen ion conc

osmolality

duration

A

6-8 conc
+Formali buffered w/ phosphate at 7pH

Slightly hypertonic [400-450 mmol

2-6 hrs
washed 24hrs after fixation

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13
Q

temperature

standard:
EM:
Urgent biopsies:
Not fixed immediately:

A

ROOM TEMP

0-4C
60C
Refrigeration

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14
Q

Section thickness

Standard:
EM:
LM:

Large solid tissue
- Uterus- cuts ______ → penetrate fixatives properly.
- Brain- suspended [half/whole] in [%] ____ formalin for _____weeks

A

0.4cm [small/thin]
1-2mm
2cm

anteriorly
whole
10% buffered
2-3 weeks

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15
Q

bigger/thicker=[faster/longer] fixation time vise-versa

_____ > affects penetrating ability of the fixative)

A

longer

area

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16
Q

Concentration
37-40%> dilute to
____%:COMMONLY)

-ALL DEPENDS TO THE _______
-too much =______tissue

Formaldehyde –
Glutaraldehyde – [>EM]

A

37-40%
10%

chemical
shrink/overharden

10%
3%

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17
Q

PRACTICAL CONSIDERATION OF FIXATION

A

Speed
Penetration
Volume
Duration of fixation

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18
Q

PRACTICAL CONSIDERATION OF FIXATION

Speed- _________ spx to fixative [x] autolysis/putrefaction]

Penetration-

Volume- [fixative>tissue]

Duration of fixation- depends on the fixative used
–Fibrous organs > [shorter/longer] to fix biopsies / scrapings
- Can be cut down using [4]
- can be [slowed/hastened]

A

ASAP 1hr

1mm/hr diffusion

1:20 [10-25x]

longer
heat, vacuum, agitation, microwave
hastened

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19
Q

CHARACTERISTIC OF A GOOD FIXATIVE [7]

A

-cheap, stable, safe to handle
-isotonic + make cellular components insoluble to hypotonic sol’n

-produce min. distortion of cell constituent
-produce min. shrinkage

-inhibit bacterial decomposition/ autolysis
-permit rapid/even penetration
-permit application of many staining procedures

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20
Q

TYPES OF FIXATIVE ACCDG TO COMPOSITION

A

Simple fixatives

Compound fixatives

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21
Q

Enumerate simple fixatives

[all]

A

Aldehydes- formaldehyde, glutaraldehyde

Metallic fixatives
-Mercuric chloride
-Chromate fixatives
– K dichromate, chromic acid
-Simple fixatives
–acetone, alcohol, acetic acid, picric acid, osmium tetorixide

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22
Q

Factors affecting fixation
● RETARDED [4]

A

size/thickness
[+] mucus
[+} fat/blood
cold temp

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23
Q

remedy for the presence of mucus

effect of the cold temperature

A

wash w/ NSS

enzyme inactivation

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24
Q

Factors affecting fixation
● ENHANCED [4]

A

thinner/smaller size of tissue
agitation [automatic/mechanical tissue processing used]
heat

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25
Q

placing an already fixed tissue into another fixative

A

Secondary fixation

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26
Q

secondary fixation using 2-3% ________ for [#] day to act as mordant.

A

Post-chromatization
potassium dichromate

1 day

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27
Q

removal of excess fixative in order to improve staining

A

washing out

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28
Q

Enumerate washing out components

A

tap water – [x] chromates,
helly’s, zenkers,flemming
formalin
osmic acid

50-70% alcohol: (x) excess picric acid

alcoholic iodine: (x) excess mercury fixatives.

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29
Q

a temp of 35-56C is the recommended temp for heating method and it is known to accelerate fixation BUT_____

A

hastens autolytic changes
enzyme destruction

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30
Q

Are those that permit the general microscopic study of tissue structures

A

Microanatomical fixatives

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31
Q

List all the microanatomical fixatives

A

10% formol saline
10 % neutral buffered formalin

Heidenhain’s Susa
Formol sublimate/ corrosive

Zenker’s sol’n
Zenker-formol

Bouin’s sol’n
Brasil’s sol’n

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32
Q

Are those that preserve specific parts & particular microscopic elements of the cell

A

Cytological fixatives

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33
Q

Nuclear fixatives
– preserve the _____ structure of the cell. [______]
– usually contains _________ as primary component
– pH of =/< ___.

A

nuclear
glacial acetic acid
4.6

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34
Q

– Are those that preserve the _____ structure.
– Must [also/not contain] glacial acetic acid. If not why?
- pH of =/>__

A

Cytoplasmic fixatives

cytoplasmic

can destroy the mitochondria/golgi bodies in the cytoplasm

4.6

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35
Q

are those that preserve the chemical constituents of the cells & tissues.

A

Histochemical fixatives

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36
Q

List all the nuclear fixatives

A

Heidenhain’s Susa
Bouin’s fluid

Flemming’s fluid
Newcomer’s fluid
Camoy’s fluid

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37
Q

List all the histochemical fixatives

A

10% formol saline
Newcomer’s fluid

Absolute ethyl alcohol
Acetone

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38
Q

List all the cytoplasmic
fixatives

A

Formalin w/ post chroming
Flemming’s fluid w/o acetic acid

Orth’s fluid
Regaud’s/Muller fluid
Kelly’s fluid

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39
Q

Lipid fixation
– ________ should be used in demonstrating lipid in tissue.
–[2]e can be effective for preservation of lipid in cryostat sections.
– Phospholipids are fixed w/ ______.
– Post fixing in ________ gives a better ultrastructural demonstration of lipids.
– Cholesterol is fixed w/ ______ for ultrastructural demonstration.

A

Cryostat/ frozen sections

mercuric chloride
potassium dichromate

aldehydes

imidazole osmium tetroxide

digitonin

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40
Q

CARBOHYDRATE FIXATION
– ______ fixative are recommended for _____ fixation
– Alcoholic _______ compared to neutral buffered formaldehyde is better in fixation of human ___.

A

Alcohol
glycogen

formaldehyde
skin

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41
Q

PROTEIN FIXATION
– ___________ is most commonly used fixative for amino acid histochemistry.

A

Neutral buffered formol saline

amino acid histochemistry

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42
Q

Glycogen fixation
–__________ –is the most useful fixative for glycogen
– essential when processing tissue from px w/ ______ disease.
– better retention of glycogen if the section is
coated w/ _____.

A

Rosmann’s fluid or Cold absolute alcohol

glycogen storage disease

celloidin

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43
Q

satisfactory for routine paraffin section—> ______, ______ and _______ studies

A

Aldehyde fixative

EM
histochemistry
enzyme

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44
Q

gas produced by the oxidation of methyl alcohol

A

Formaldehyde [formalin]

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45
Q

–most widely used concentration
– unsatisfactory for routine fixation concentration
– [soluble/insoluble] in water
– INC= ______.

A

10%

Pure stock solution of 40%

soluble

overharden the outer layer of the tissue

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46
Q

PROS of formaldehyde

A

cheap,readily available, easy to prepare
tolerant fixative for mailing spx

compatible w/ many stains
for fats mucin, glycogen, proteins
for nervous tissue preparation

[x] overharden tissues
[x] precipitate protein –> allows enzymes to be studied.

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47
Q

CONS of formaldehyde

A

Fumes are irritating
causes allergic rhinits/dermatitis or excessive lacrimation

soft fixative – [x] harden some syto. structures enough for paraffin embedding

If unbuffered, it [-] both eosinophilic & basophilic stain
Prolonged fixation = spx bleaching + loss of neutral color

Dispersal of fat from tissue into the fluid

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48
Q

Formaldehyde

fixation time:
buffered to what hydrogen ion conc:

A

24hrs
ph 7 w/ phosphate buffer

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49
Q

10% formol saline

– made up of saturated _______ dilute to 10% ______.

– Large spx → fixed for a [short/long] time.

– Preserves [2]
– Recommended for [2]

A

formaldehyde
sodium chloride

long time

enzymes
nucleoproteins

central nervous tissues
general post-mortem tissues

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50
Q

– Recommended for preservation and storage of surgical, post mortem and research spx

A

10% Neutral/Phosphate–buffered formalin

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51
Q

10% Neutral–buffered formalin/ Phosphate-buffered formalin

— [induces/inhibits] acid formalin pigments’ precipitation on post-mortem tissues
– Fixation time: _____.
– pH ___.

A

inhibits

4-24 hrs
7

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52
Q

10% Neutral/Phosphate-buffered formalin

PROS:

CONS:
[1]
Positivity of mucin to PAS is [increased/reduced].
Gradual loss of _______ staining of cell

A

best for tissues-containing iron pigments & elastic fibers

longer prep –> time consuming = [-] myelin to Weigdert’s iron hematoxylin stain reactivity + inert towards lipids

reduced
basophilic

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53
Q

– recommended for routine post-mortem tissues.

A

Formol sublimate/corrosive or

Formal mercuric chloride

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54
Q

Formol sublimate/corrosive fixation time

A

3-24 hrs

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55
Q

PROS of Formol mercuric chloride
– Penetrates______ rapidly.
– Excellent for [little/many] staining procedure including _______.
– It fixes _____, especially _____ & _______.
[require/does not require] washing out

A

small pieces of tissue

many
silver reticulum methods

lipids
phospholipids
neutral fats

does not require

56
Q

CONS of formol sublimate/corrosive

A

slow penetration -> [+] mercuric chloride deposits + [x] extent of tissue decalcification

57
Q

– for RAPID DIAGNOSIS → Fixes & dehydrate at the same time

A

Alcoholic formalin [Gendre’s fixative]

58
Q

Alcoholic formalin/ Gendre’s fixative
– Good for:[2]
– Coagulates _____ → Fix sputum
– [Soft/Gross]-hardening of tissues
– [Partial/Complete] RBC lysis

A

glycogen preservation
microincineration technique

mucus

gross

partial

59
Q

Alcoholic formalin/ Gendre’s fixative is poor in?

A

iron-containing pigments

60
Q

Post-fixation of gendre’s w/ ________ for [#] hrs enhance ________ studies on tissues.

A

phenol-formalin

6hrs

immunoperoxidase studies

61
Q

–made up of 2 formaldehyde residues linked by 3 carbon chains

A

Glutaraldehyde

62
Q

Glutaraldehyde
– for routine [enzyme/light] microscopic work
– [Buffered/unbuffered] → + 2ndary fixation in ______ = satisfactory for EM.
– Preserves plasma _____.
– X cause ______.

A

light

buffered
osmium tetroxide

proteins

dermatitis

63
Q

Glutaraldehyde PROS/CONS

A

[-] tissue shrinkage

[-] PAS positivity of reactive muicn

64
Q

List all the metallic fixatives [mercuric chloride]

A

-Zenker’s fluid
-Zenker-formol [Helly’s sol’n]
-Heidenhain’s susa

65
Q

– [#] metallic fixative

A

mercuric chloride

66
Q

Mercuric chloride
– _______ components → shown in fine detail

Routine fixative of choice → _____ detail in tissue _______.

– ________ staining → brilliant metachromatic cell staining + ______.

A

Nuclear

cell
photography

Trichrome staining
collagen

67
Q

– RECOMMENDED: renal tissues, fibrin, connective tissues, muscles.

A

Mercuric chloride

68
Q

In metal chloride, one must avoid wearing:

A

metal caps
jewelries

69
Q

made up of mercuric chloride W/ GLACIAL ACETIC ACID

A

Zenker’s fluid

70
Q

________
for FIXING SMALL PIECES of _____, ______, connective tissue fibers, ______.

A

Zenker’s fluid

liver
spleen
nuclei

71
Q

– fixative for pituitary gland, BM & blood-containing organs: spleen, liver

A

Zenker-formol [Helly’s sol’n]

72
Q

– Penetrates/Fixes tissues
– ________ are produced if tissue is stayed for more than 24hrs in the fixative.
Fixation time: ________

A

Zenker-formol [Helly’s sol’n]
Black pigments
12-24 hrs

73
Q

– mainly for TUMOR BIOPSIES [skin]

A

Heidenhain’s susa

74
Q

Heidenhain’s susa
– Excellent _____ fixative
– Penetrates/Fixes _____ + ______.
– Brilliant results w/ sharp ______ details

A

cytologic

rapidly
evenly

nuclear/cyto

75
Q

Heidenhain’s susa
– After fixation → tissue transferred directly to ________ = [X] swelling.

A

high-grade alcohol

76
Q

Fixation time:

3-12 hrs:
1 1⁄2 -2hrs:

A

Heidenhain’s susa

B-5 fixative

77
Q

– for BM biopsies
– form ______ on standing [X cons]

A

B-5 fixative

78
Q

List all the chromic fixatives

A

Chromic acid
Potassium dichromate

Regaud’s/Muller’s
Orth’s fluid

79
Q

– strong oxidizing agent
– precipitates: all _____ & adequately preserves: ______.
– [X] USED → because it is _____.

A

Chromic acid

protein
carbohydrates

hazardous

80
Q

used in 1-2% aqueous solution

used in 3% aqueous solution

A

Chromic acid

Potassium dichromate

81
Q

Potassium dichromate
– preserves [2]
– [X] precipitates [nuclear/cyto] structures

– If solution becomes [ACIDIFIED/ALKALINIZED]> cytoplasm, chromatin bodies, chromosomes are fixed BUT _______ are destroyed.

A

mitochondria
lipid

cytoplasmic

ACIDIFIED
mitochondria

82
Q

– Penetrates tissues well
– hardens tissues better + rapidly > Orth’s fluid. –

A

Regaud’s/Muller’s

83
Q

Regaud’s is for the demonstration of [6]

A

Golgi bodies
Chromatin
Colloid-containing tissue
RBC
Mitochondria
mitotic figures

84
Q

Fixation time

12-48hrs:
36-72 hrs:

A

Regaud’s/Muller’s
Orth’s fluid

85
Q

– for study of early degenerative processes + tissue necrosis.

A

Orth’s fluid

86
Q

untimely/cellular death toxic eposure to tissue/external factors: _________.

– demonstrates _________ +other bacteria.

A

microorganism

Rickettsiae

87
Q

Orth’s fluid is better than buffered formalin in preserving ?

A

myelin.

88
Q

–used in [%] aqueous sol’n of basic lead acetate
– for ________________.
– fixes _____________.
– takes up ________> to form= insoluble lead _____ on prolonged standing.

A

4

acid mucopolysaccharides

connective tissue mucin

carbon dioxide
insoluble
carbonate

89
Q

HOW TO REMOVE INSOLUBLE LEAD CARBONATE
[2]

A

filter paper

acetic acid drop by drop [dissolve residues]

90
Q

Picric acid
– for ______.
– used in [weak/strong] or [saturated/unsaturated] aqueous sol’n

A

glycogen

strong
saturated

91
Q

– Penetrates tissues [poorly/well] + Fixes small tissues [rapidly/slower]
– DYES the tissues → brilliant staining w/ _____ method
CONS: stains _____.

A

Picric acid

well
rapidly

trichrome

yellow

92
Q
  • for embryos + pituitary bodies
  • Excellent: soft/ delicate structures
  • Minimal distortion: ______
    structures
  • ______ staining
  • Preserves _______.
  • [require/does not] washing-out

Fixation time: _____

A

Bouin’s fluid

microanatomical structure

brilliant
glycogen

does not require

6-24 hrs

93
Q
  • better & less messy> Bouin’s
  • Excellent fixative for _____.
  • has _____ another form of fixative,
    decalcifying agent
A

Brasil’s alcoholic picroformol fixative

glycogen

TCA

94
Q

always part of fixative

A

Glacial acetic acid

95
Q

– Precipitates nucleoprotein, chromatin materials
– incorporated in compound fixative
– Solidifies: @17C

– X for cytoplasmic fixation bc it can make the sol’n _____> destroying golgi/mitochondria

A

glacial acetic acid

acidic

96
Q

LIST all the lead fixatives

A

Picric acid
Bouin’s
Brasil’s alcoholic picroformol
Glacial acetic acid

97
Q

ALCOHOL FIXATIVES
– rapidly denatures/precipitates _____.
– [%] [ < conc. = cell lysis]
– acts AS FIXATIVE + ________
– preserves ________ but dissolves _______.

A

proteins

70% - 100%

DEHYDRATING AGENT

nuclear stains
fats/lipids

98
Q

List all alcoholic fixatives

A

Methanol
Isopropyl alcohol
Ethanol

Camoy’s
Newcomer’s

99
Q

Absolute alcohol for [6]

A

glycogen
blood

pigments
tissue films
smears
nuclear stains

100
Q

Methanol
– Excellent for fixing ___ & ___ smears, ____ smears, ____ tissue.
– Fixes + ______.

– [FAST/SLOW] penetration

A

dry
wet

blood
BM

dehydrates

SLOW

101
Q

If methanol is left for 2 days what wll happen to the tissues?

Methanol is [Non-toxic/toxic] & can cause ______ when drunk.

A

→ tissues may be OVERHARDENED + DIFFICULT to CUT.

Toxic
blindness

102
Q

– for fixing touch preparations smear & for staining using Wright giemsa stain

A

Isopropyl alcohol

103
Q

– use as a simple fixative
– [X] FIX glycogen

A

Ethanol alcohol

104
Q

– for nucleoproteins/nucleic acid → for histochemistry & enzyme studies

A

Ethanol alcohol

105
Q

– fixes blood, tissue films & smears
– useful for PCR

A

Ethanol alcohol

106
Q

– MOST RAPID fixative
– Fixes + Dehydrates

A

Camoy’s fluid

107
Q

Camoy’s fluid
– for fixing [3]
– fix [organ] tissues → _____ diagnosis. [presence of_____]

A

chromosomes
lymph glands
urgent biopsies

brain
rabies
nissi granules

108
Q

– good nuclear staining + differentiation

A

Carnoy’s fluid

109
Q

Fixation time:

18-24 hrs:
1-3 hrs:

A

Ethanol alcohol

Camoy’s fluid

110
Q

– for fixing mucopolysaccharide & nuclear proteins.
– acts AS BOTH NUCLEAR & HISTOCHEMICAL FIXATIVE

A

Newcomer’s fluid

110
Q

– for conjugated fats/lipids PERMANENTLY → making them [soluble/insoluble].

A

Osmium tetroxide

insoluble

111
Q

List all osmium tetroxide

A

Flemming’s sol’n

Flemming’s w/o acetic acid

112
Q

– [#] chrome-osmium acetic acid fixative
– Excellent for [nuclear/cytoplasmic] structures

A

Flemming’s sol’n

nuclear

113
Q

– made up of only chromic/osmic acid
– for [nuclear/cytoplasmic] structures [specific _____]

A

Flemming’s w/o acetic acid

cytoplasmic
mitochondria

114
Q

Fixation time of both flemming’s sol’n/w/o acetic acid

A

24-48 hrs [1-2days]

115
Q

Trichloroacetic acid
– Precipitates ______.
– used as _____ + WEAK __________ agent
– has a [softening/hardening] effect on _______ tissue → prep of such sections.

A

proteins

fixative
decalcifying agent

softening
dense fibrous

116
Q

CONS of trichloroacetic acid:

A

– POOR PENETRATING agent
– suitable only for small pieces of tissues/bones

117
Q

– ice cold temp: -5 - 4C
– for the study of water diffusible enzymes [2]

A

Acetone
phosphatase, lipases

118
Q

2 Fixatives involved for brain tissues→ rabies diagnosis

+nissi granules:
+negri bodies:

A

Camoy’s fluid

Acetone

119
Q

Acetone as solvent includes certain ______ → in
_____________ techniques for _____ _____.

A

metallic salts

freeze substitution
tissue blocks

120
Q

– Evaporates rapidly
– POOR: dissolves fat + preserves glycogen

A

Acetone

121
Q

– this procedure involves thermal coagulation of tissue proteins for RAPID DIAGNOSIS

– for frozen tissue section + preparation of [bacteriologic/ parasitology smears]

A

Heat fixation

bacteriologic

122
Q

works as physical agent similar to vacuum, oven, agitation
– Optimum temp: 45-55C

A

microwave technique

123
Q

process of placing an already fixed tissue in a 2nd fixative.

A

SECONDARY FIXATION

124
Q

Post-Chromatization
– Mordant: __________ for [#] hrs→ better staining & aid in _____ preservation of tissues.

A

2.5-3% potassium dichromate
24 hrs
cytologic

125
Q

FIXATION ARTEFACTS [2]

A

Formalin pigment

Crush Artefact

126
Q

Formalin Pigment
– Known ______ produced under [alkaline/acid] conditions

– [-] by fixation in ________
– Under microscope: [color] stain

A

artefact
acid

phenol-formalin

black/brown

127
Q

– in surgical specimen (liver biopsies)

A

Crush artefact

128
Q

Crush artefact
– intense [basophilic/eosinophilic]staining
– Due to [full/partial] coagulation of _____ by ______.
– Incomplete: _______.

A

eosinophilic

partial
protein
ethanol

wax impregnation

129
Q

failure to arrest early autolysis of cells is caused by ?

A

failure to fix immediately

insufficient fixative

130
Q

CAUSE OF:
removal of substances SOLUBLE in fixing agent

LOSS or inactivation of enzymes needed for study

A

Wrong choice of fixative

131
Q

CAUSE OF:
[+] artefact pigments on tissue sections

A

incomplete washing of fixative

132
Q

CAUSE OF:
Tissues are soft & feather-like in consistency

A

incomple fixation

133
Q

CAUSE OF:
Shrinkage & Swelling of cells and tissue structures

A

Over fixation

134
Q

CAUSE OF:
Tissue blocks are brittle/hard

A

Prolonged fixation

HPCT (5 decks)

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