FIXATION Flashcards
first and MOST CRITICAL STEP IN
HISTOTECHNOLOGY
Fixation
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]
morphological
chemical
harden
trauma
prevents degeneration, decomposition,
putrefaction & distortion of tissue.
Fixation
2 basic mechanisms involved in fixation.
Additive
Non-additive
Chemical constituent of the fixative becomes part of the tissue.
Examples [3]
Additive fixation
formalin
mercury
osmium tetroxide
fixing agent is not incorporated into the tissue
Alters tissue composition by ___________ attached to the _______ molecule.
Example [1]
Non-additive
removing bound water
protein
alcoholic fixatives
Factors involved in fixation [5]
Hydrogen ion concentration
Temperature
Osmolality
Concentration
Duration
Procedure adopted to kill, harden, and preserve materials for microscopic study by means of a substance known as a____
fixative
Fixative effects
[6]
– 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
In fixation,
[3] and chemical constituents of tissues are preserved by [X] degeneration, decomposition and distortion of tissues after death.
shape
structure
intercellular relationship
chemical constituents
Fixation prevents [4] of tissue.
degeneration
decomposition
putrefaction
distortion
hydrogen ion conc
osmolality
duration
6-8 conc
+Formali buffered w/ phosphate at 7pH
Slightly hypertonic [400-450 mmol
2-6 hrs
washed 24hrs after fixation
temperature
standard:
EM:
Urgent biopsies:
Not fixed immediately:
ROOM TEMP
0-4C
60C
Refrigeration
Section thickness
Standard:
EM:
LM:
Large solid tissue
- Uterus- cuts ______ → penetrate fixatives properly.
- Brain- suspended [half/whole] in [%] ____ formalin for _____weeks
0.4cm [small/thin]
1-2mm
2cm
anteriorly
whole
10% buffered
2-3 weeks
bigger/thicker=[faster/longer] fixation time vise-versa
_____ > affects penetrating ability of the fixative)
longer
area
Concentration
37-40%> dilute to
____%:COMMONLY)
-ALL DEPENDS TO THE _______
-too much =______tissue
Formaldehyde –
Glutaraldehyde – [>EM]
37-40%
10%
chemical
shrink/overharden
10%
3%
PRACTICAL CONSIDERATION OF FIXATION
Speed
Penetration
Volume
Duration of fixation
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]
ASAP 1hr
1mm/hr diffusion
1:20 [10-25x]
longer
heat, vacuum, agitation, microwave
hastened
CHARACTERISTIC OF A GOOD FIXATIVE [7]
-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
TYPES OF FIXATIVE ACCDG TO COMPOSITION
Simple fixatives
Compound fixatives
Enumerate simple fixatives
[all]
Aldehydes- formaldehyde, glutaraldehyde
Metallic fixatives
-Mercuric chloride
-Chromate fixatives
– K dichromate, chromic acid
-Simple fixatives
–acetone, alcohol, acetic acid, picric acid, osmium tetorixide
Factors affecting fixation
● RETARDED [4]
size/thickness
[+] mucus
[+} fat/blood
cold temp
remedy for the presence of mucus
effect of the cold temperature
wash w/ NSS
enzyme inactivation
Factors affecting fixation
● ENHANCED [4]
thinner/smaller size of tissue
agitation [automatic/mechanical tissue processing used]
heat
placing an already fixed tissue into another fixative
Secondary fixation
secondary fixation using 2-3% ________ for [#] day to act as mordant.
Post-chromatization
potassium dichromate
1 day
removal of excess fixative in order to improve staining
washing out
Enumerate washing out components
tap water – [x] chromates,
helly’s, zenkers,flemming
formalin
osmic acid
50-70% alcohol: (x) excess picric acid
alcoholic iodine: (x) excess mercury fixatives.
a temp of 35-56C is the recommended temp for heating method and it is known to accelerate fixation BUT_____
hastens autolytic changes
enzyme destruction
Are those that permit the general microscopic study of tissue structures
Microanatomical fixatives
List all the microanatomical fixatives
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
Are those that preserve specific parts & particular microscopic elements of the cell
Cytological fixatives
Nuclear fixatives
– preserve the _____ structure of the cell. [______]
– usually contains _________ as primary component
– pH of =/< ___.
nuclear
glacial acetic acid
4.6
– Are those that preserve the _____ structure.
– Must [also/not contain] glacial acetic acid. If not why?
- pH of =/>__
Cytoplasmic fixatives
cytoplasmic
can destroy the mitochondria/golgi bodies in the cytoplasm
4.6
are those that preserve the chemical constituents of the cells & tissues.
Histochemical fixatives
List all the nuclear fixatives
Heidenhain’s Susa
Bouin’s fluid
Flemming’s fluid
Newcomer’s fluid
Camoy’s fluid
List all the histochemical fixatives
10% formol saline
Newcomer’s fluid
Absolute ethyl alcohol
Acetone
List all the cytoplasmic
fixatives
Formalin w/ post chroming
Flemming’s fluid w/o acetic acid
Orth’s fluid
Regaud’s/Muller fluid
Kelly’s fluid
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.
Cryostat/ frozen sections
mercuric chloride
potassium dichromate
aldehydes
imidazole osmium tetroxide
digitonin
CARBOHYDRATE FIXATION
– ______ fixative are recommended for _____ fixation
– Alcoholic _______ compared to neutral buffered formaldehyde is better in fixation of human ___.
Alcohol
glycogen
formaldehyde
skin
PROTEIN FIXATION
– ___________ is most commonly used fixative for amino acid histochemistry.
Neutral buffered formol saline
amino acid histochemistry
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/ _____.
Rosmann’s fluid or Cold absolute alcohol
glycogen storage disease
celloidin
satisfactory for routine paraffin section—> ______, ______ and _______ studies
Aldehyde fixative
EM
histochemistry
enzyme
gas produced by the oxidation of methyl alcohol
Formaldehyde [formalin]
–most widely used concentration
– unsatisfactory for routine fixation concentration
– [soluble/insoluble] in water
– INC= ______.
10%
Pure stock solution of 40%
soluble
overharden the outer layer of the tissue
PROS of formaldehyde
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.
CONS of formaldehyde
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
Formaldehyde
fixation time:
buffered to what hydrogen ion conc:
24hrs
ph 7 w/ phosphate buffer
10% formol saline
– made up of saturated _______ dilute to 10% ______.
– Large spx → fixed for a [short/long] time.
– Preserves [2]
– Recommended for [2]
formaldehyde
sodium chloride
long time
enzymes
nucleoproteins
central nervous tissues
general post-mortem tissues
– Recommended for preservation and storage of surgical, post mortem and research spx
10% Neutral/Phosphate–buffered formalin
10% Neutral–buffered formalin/ Phosphate-buffered formalin
— [induces/inhibits] acid formalin pigments’ precipitation on post-mortem tissues
– Fixation time: _____.
– pH ___.
inhibits
4-24 hrs
7
10% Neutral/Phosphate-buffered formalin
PROS:
CONS:
[1]
Positivity of mucin to PAS is [increased/reduced].
Gradual loss of _______ staining of cell
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
– recommended for routine post-mortem tissues.
Formol sublimate/corrosive or
Formal mercuric chloride
Formol sublimate/corrosive fixation time
3-24 hrs
PROS of Formol mercuric chloride
– Penetrates______ rapidly.
– Excellent for [little/many] staining procedure including _______.
– It fixes _____, especially _____ & _______.
[require/does not require] washing out
small pieces of tissue
many
silver reticulum methods
lipids
phospholipids
neutral fats
does not require
CONS of formol sublimate/corrosive
slow penetration -> [+] mercuric chloride deposits + [x] extent of tissue decalcification
– for RAPID DIAGNOSIS → Fixes & dehydrate at the same time
Alcoholic formalin [Gendre’s fixative]
Alcoholic formalin/ Gendre’s fixative
– Good for:[2]
– Coagulates _____ → Fix sputum
– [Soft/Gross]-hardening of tissues
– [Partial/Complete] RBC lysis
glycogen preservation
microincineration technique
mucus
gross
partial
Alcoholic formalin/ Gendre’s fixative is poor in?
iron-containing pigments
Post-fixation of gendre’s w/ ________ for [#] hrs enhance ________ studies on tissues.
phenol-formalin
6hrs
immunoperoxidase studies
–made up of 2 formaldehyde residues linked by 3 carbon chains
Glutaraldehyde
Glutaraldehyde
– for routine [enzyme/light] microscopic work
– [Buffered/unbuffered] → + 2ndary fixation in ______ = satisfactory for EM.
– Preserves plasma _____.
– X cause ______.
light
buffered
osmium tetroxide
proteins
dermatitis
Glutaraldehyde PROS/CONS
[-] tissue shrinkage
[-] PAS positivity of reactive muicn
List all the metallic fixatives [mercuric chloride]
-Zenker’s fluid
-Zenker-formol [Helly’s sol’n]
-Heidenhain’s susa
– [#] metallic fixative
mercuric chloride
Mercuric chloride
– _______ components → shown in fine detail
Routine fixative of choice → _____ detail in tissue _______.
– ________ staining → brilliant metachromatic cell staining + ______.
Nuclear
cell
photography
Trichrome staining
collagen
– RECOMMENDED: renal tissues, fibrin, connective tissues, muscles.
Mercuric chloride
In metal chloride, one must avoid wearing:
metal caps
jewelries
made up of mercuric chloride W/ GLACIAL ACETIC ACID
Zenker’s fluid
________
for FIXING SMALL PIECES of _____, ______, connective tissue fibers, ______.
Zenker’s fluid
liver
spleen
nuclei
– fixative for pituitary gland, BM & blood-containing organs: spleen, liver
Zenker-formol [Helly’s sol’n]
– Penetrates/Fixes tissues
– ________ are produced if tissue is stayed for more than 24hrs in the fixative.
Fixation time: ________
Zenker-formol [Helly’s sol’n]
Black pigments
12-24 hrs
– mainly for TUMOR BIOPSIES [skin]
Heidenhain’s susa
Heidenhain’s susa
– Excellent _____ fixative
– Penetrates/Fixes _____ + ______.
– Brilliant results w/ sharp ______ details
cytologic
rapidly
evenly
nuclear/cyto
Heidenhain’s susa
– After fixation → tissue transferred directly to ________ = [X] swelling.
high-grade alcohol
Fixation time:
3-12 hrs:
1 1⁄2 -2hrs:
Heidenhain’s susa
B-5 fixative
– for BM biopsies
– form ______ on standing [X cons]
B-5 fixative
List all the chromic fixatives
Chromic acid
Potassium dichromate
Regaud’s/Muller’s
Orth’s fluid
– strong oxidizing agent
– precipitates: all _____ & adequately preserves: ______.
– [X] USED → because it is _____.
Chromic acid
protein
carbohydrates
hazardous
used in 1-2% aqueous solution
used in 3% aqueous solution
Chromic acid
Potassium dichromate
Potassium dichromate
– preserves [2]
– [X] precipitates [nuclear/cyto] structures
– If solution becomes [ACIDIFIED/ALKALINIZED]> cytoplasm, chromatin bodies, chromosomes are fixed BUT _______ are destroyed.
mitochondria
lipid
cytoplasmic
ACIDIFIED
mitochondria
– Penetrates tissues well
– hardens tissues better + rapidly > Orth’s fluid. –
Regaud’s/Muller’s
Regaud’s is for the demonstration of [6]
Golgi bodies
Chromatin
Colloid-containing tissue
RBC
Mitochondria
mitotic figures
Fixation time
12-48hrs:
36-72 hrs:
Regaud’s/Muller’s
Orth’s fluid
– for study of early degenerative processes + tissue necrosis.
Orth’s fluid
untimely/cellular death toxic eposure to tissue/external factors: _________.
– demonstrates _________ +other bacteria.
microorganism
Rickettsiae
Orth’s fluid is better than buffered formalin in preserving ?
myelin.
–used in [%] aqueous sol’n of basic lead acetate
– for ________________.
– fixes _____________.
– takes up ________> to form= insoluble lead _____ on prolonged standing.
4
acid mucopolysaccharides
connective tissue mucin
carbon dioxide
insoluble
carbonate
HOW TO REMOVE INSOLUBLE LEAD CARBONATE
[2]
filter paper
acetic acid drop by drop [dissolve residues]
Picric acid
– for ______.
– used in [weak/strong] or [saturated/unsaturated] aqueous sol’n
glycogen
strong
saturated
– Penetrates tissues [poorly/well] + Fixes small tissues [rapidly/slower]
– DYES the tissues → brilliant staining w/ _____ method
CONS: stains _____.
Picric acid
well
rapidly
trichrome
yellow
- for embryos + pituitary bodies
- Excellent: soft/ delicate structures
- Minimal distortion: ______
structures - ______ staining
- Preserves _______.
- [require/does not] washing-out
Fixation time: _____
Bouin’s fluid
microanatomical structure
brilliant
glycogen
does not require
6-24 hrs
- better & less messy> Bouin’s
- Excellent fixative for _____.
- has _____ another form of fixative,
decalcifying agent
Brasil’s alcoholic picroformol fixative
glycogen
TCA
always part of fixative
Glacial acetic acid
– Precipitates nucleoprotein, chromatin materials
– incorporated in compound fixative
– Solidifies: @17C
– X for cytoplasmic fixation bc it can make the sol’n _____> destroying golgi/mitochondria
glacial acetic acid
acidic
LIST all the lead fixatives
Picric acid
Bouin’s
Brasil’s alcoholic picroformol
Glacial acetic acid
ALCOHOL FIXATIVES
– rapidly denatures/precipitates _____.
– [%] [ < conc. = cell lysis]
– acts AS FIXATIVE + ________
– preserves ________ but dissolves _______.
proteins
70% - 100%
DEHYDRATING AGENT
nuclear stains
fats/lipids
List all alcoholic fixatives
Methanol
Isopropyl alcohol
Ethanol
Camoy’s
Newcomer’s
Absolute alcohol for [6]
glycogen
blood
pigments
tissue films
smears
nuclear stains
Methanol
– Excellent for fixing ___ & ___ smears, ____ smears, ____ tissue.
– Fixes + ______.
– [FAST/SLOW] penetration
dry
wet
blood
BM
dehydrates
SLOW
If methanol is left for 2 days what wll happen to the tissues?
Methanol is [Non-toxic/toxic] & can cause ______ when drunk.
→ tissues may be OVERHARDENED + DIFFICULT to CUT.
Toxic
blindness
– for fixing touch preparations smear & for staining using Wright giemsa stain
Isopropyl alcohol
– use as a simple fixative
– [X] FIX glycogen
Ethanol alcohol
– for nucleoproteins/nucleic acid → for histochemistry & enzyme studies
Ethanol alcohol
– fixes blood, tissue films & smears
– useful for PCR
Ethanol alcohol
– MOST RAPID fixative
– Fixes + Dehydrates
Camoy’s fluid
Camoy’s fluid
– for fixing [3]
– fix [organ] tissues → _____ diagnosis. [presence of_____]
chromosomes
lymph glands
urgent biopsies
brain
rabies
nissi granules
– good nuclear staining + differentiation
Carnoy’s fluid
Fixation time:
18-24 hrs:
1-3 hrs:
Ethanol alcohol
Camoy’s fluid
– for fixing mucopolysaccharide & nuclear proteins.
– acts AS BOTH NUCLEAR & HISTOCHEMICAL FIXATIVE
Newcomer’s fluid
– for conjugated fats/lipids PERMANENTLY → making them [soluble/insoluble].
Osmium tetroxide
insoluble
List all osmium tetroxide
Flemming’s sol’n
Flemming’s w/o acetic acid
– [#] chrome-osmium acetic acid fixative
– Excellent for [nuclear/cytoplasmic] structures
Flemming’s sol’n
nuclear
– made up of only chromic/osmic acid
– for [nuclear/cytoplasmic] structures [specific _____]
Flemming’s w/o acetic acid
cytoplasmic
mitochondria
Fixation time of both flemming’s sol’n/w/o acetic acid
24-48 hrs [1-2days]
Trichloroacetic acid
– Precipitates ______.
– used as _____ + WEAK __________ agent
– has a [softening/hardening] effect on _______ tissue → prep of such sections.
proteins
fixative
decalcifying agent
softening
dense fibrous
CONS of trichloroacetic acid:
– POOR PENETRATING agent
– suitable only for small pieces of tissues/bones
– ice cold temp: -5 - 4C
– for the study of water diffusible enzymes [2]
Acetone
phosphatase, lipases
2 Fixatives involved for brain tissues→ rabies diagnosis
+nissi granules:
+negri bodies:
Camoy’s fluid
Acetone
Acetone as solvent includes certain ______ → in
_____________ techniques for _____ _____.
metallic salts
freeze substitution
tissue blocks
– Evaporates rapidly
– POOR: dissolves fat + preserves glycogen
Acetone
– this procedure involves thermal coagulation of tissue proteins for RAPID DIAGNOSIS
– for frozen tissue section + preparation of [bacteriologic/ parasitology smears]
Heat fixation
bacteriologic
works as physical agent similar to vacuum, oven, agitation
– Optimum temp: 45-55C
microwave technique
process of placing an already fixed tissue in a 2nd fixative.
SECONDARY FIXATION
Post-Chromatization
– Mordant: __________ for [#] hrs→ better staining & aid in _____ preservation of tissues.
2.5-3% potassium dichromate
24 hrs
cytologic
FIXATION ARTEFACTS [2]
Formalin pigment
Crush Artefact
Formalin Pigment
– Known ______ produced under [alkaline/acid] conditions
– [-] by fixation in ________
– Under microscope: [color] stain
artefact
acid
phenol-formalin
black/brown
– in surgical specimen (liver biopsies)
Crush artefact
Crush artefact
– intense [basophilic/eosinophilic]staining
– Due to [full/partial] coagulation of _____ by ______.
– Incomplete: _______.
eosinophilic
partial
protein
ethanol
wax impregnation
failure to arrest early autolysis of cells is caused by ?
failure to fix immediately
insufficient fixative
CAUSE OF:
removal of substances SOLUBLE in fixing agent
LOSS or inactivation of enzymes needed for study
Wrong choice of fixative
CAUSE OF:
[+] artefact pigments on tissue sections
incomplete washing of fixative
CAUSE OF:
Tissues are soft & feather-like in consistency
incomple fixation
CAUSE OF:
Shrinkage & Swelling of cells and tissue structures
Over fixation
CAUSE OF:
Tissue blocks are brittle/hard
Prolonged fixation
