MICROTECHNIQUE Killing and fixation module

PREPARATION OF BIOLOGICAL SPECIMENS
FOR LIGHT MICROSCOPY
Dr. Abdussalam, A.K.
Assistant Professor,
Dept. of Post Graduate Studies and Research in Botany
Sir Syed College, Taliparamba, Kannur
Mob: 9847654285, salamkoduvally@gmail.com

 Sample collection
 Killing and Fixation
 Dehydration
 Clearing
 Paraffin Embedding
 Microtomy
 Staining
 Observation
Steps

1. Killing and Fixation
 Essential requirement
 Performed by fixative
 Killing – Sudden stoppage of life processes
 Fixation - Preservation of a “life-like” state
 Purposes –
 Preservation of natural form
 Modifying RI
 Making material resistant and hard
 Preparing material for improving staining

Reagents of Fixatives
 No single reagent for the purpose
 Combinations of reagents
 Principle – Keep balance between properties
 1. Ethyl alcohol
 Water soluble
 Reducing agent
 Rapid Penetrability
 Shrink tissues
 Hardening effect
 Makes tissues difficult to stain

2. Formalin
 Aqueous Formaldehyde
 Reducing agent
 Water miscible
 Slow Penetration
 Causes shrinkage
 Great Hardening effect
 Makes staining difficult

3. Acetic Acid
 Water miscible
 Rapid penetration
 No hardening effect
 Makes tissues soft
4. Chromic acid
 Water miscible
 Oxidiser
 Slow Penetration

Killing and Fixing Fluids (Fixatives)
 Many groups based on their ingredients
 Selection depends on specific requirement
 Some stable
 Some unstable
 Some known by their ingredients
 Some by their investigators

1. Farmer’ s Formula
 Glacial Acetic acid- 5m ml
 Absolute Alcohol - 15 ml
 Ideal for cytological preparations – Root tips, Anther
 Fixation time – Root tips – 15 m, Anthers – 1 h
 Washing and storage in 70% alcohol
2. Carnoy’s Formula
 Absolute alcohol - 10 ml
 Chloroform - 15 ml
 Glacial Acetic acid- 5 ml
 Ideal for cytological preparations
 Fixation time – 10- 15 m.
 Washing and storage in 85% alcohol
Acetic
Acid
Alcohol
Mixtures

1. Rawlin’ Formula
 95% Ethyl alcohol - 50 ml
 Glacial Acetic acid - 5 ml
 Formalin - 10 ml
 Water - 35 ml
 For delicate materials
 Good hardening action and
 Materials may be stored in this for years even
 For hard woody materials decrease acid and increased formalin
 Fixation time: 18 hrs
 Wash in alcohol and store in same
Formalin
Acetic
acid
Alcohol
(FAA)
Mixtures

1. Chromo acetic acid ( Weak)
Chromic acid 1% - 50ml
Acetic acid 1% - 50 ml
2. Chromo acetic (Medium)
Chromic acid 1% - 70ml
Water - 10 ml
3. Chromoic acetic : Strong
Chromic acid 1% - 97 ml
ChromoChromo
AceticAcetic
AcidAcid
MixturesMixtures

 Recommended for delicate objects like
filamentous and thalloid plants, root tips, Floral
organs and small sections of leaves or stems
 Fixation time: Few minutes for algae, 12 hours
for small leaf and root tips
 24 hours for larger pieces of tissue
 Wash well in running water for 24 hours and
then in distilled water for 12 hours
ChromoChromo
AceticAcetic
AcidAcid
MixturesMixtures

1. Navaschin’s Formula
Sol. A: Chromic acid (1%) - 15 ml
Glacial acetic acid - 10 ml
Distilled water - 90 ml
Sol. B: Formalin - 40 ml
Distilled water - 60 ml
 Mix equal quantities of A and B just before use
 Fixation time : 12 hours
 Washing in water not required
 Navashin’s original formula has been modified by
many investigators and the name CRAF has been
coined for such types
ChromoChromo
AceticAcetic
AcidAcid
FormalinFormalin
MixturesMixtures

 Craf I
 Chromic acid 1% 20 ml
 Acetic acid 1% 75 ml
 Formalin 5 ml
 Craf II
Chromic acid 1 % 20 ml
Acetic acid 10% 10 ml
Formalin 5 ml
Distilled water 65 ml
ChromoChromo
AceticAcetic
AcidAcid
FormalinFormalin
MixturesMixtures

ZIRKLE-ERLIKI FORMULA
Potassium bichromate 1.25 gm
Ammonium bichromate 1.25 gm
Cupric sulphate 1.00 gm
Distilled water 200 ml
Recommended for studies of mitochondria, nucleoplasm, nucleoli and vacuoles
Dissolves chromatin spindles
Fixation time 24-28 hours
Wash in water
Potassium
Chromate
Mixtures

Materials should be fixed as soon as possible after collection – if possible on
the spot
Bearing in mind the properties of the reagent used, decide upon the proper
fixative
Materials for anatomical studies should be cut into pieces of 1x1x0.5 cm
without injuring tissues
Place them in flat bottomed tubes with cork and use the fixative and material
in the proportion by volume of 100 to 1 respectively
If pieces of materials do not sink in the fluid at once, air should be removed by
using an aspirator in repeatedly until the pieces sink at lest under the surface of
the liquid
Wash thoroughly after fixing for the required time
TECHNIQUES
TECHNIQUES OF FIXING

Dehydration
 Chemical removal of water and fixative from the specimen
 Replace them with dehydrating fluid - dehydrant
 Many dehydrants are alcohols. Several are hydrophilic so attract water from tissue.
 Practiced in graded series
 Progressively decreasing concentration of water
 Progressively increasing concentration of dehydrant

Dehydrants – Reagents in dehydration
 Some merely removes water
 Some acts also as solvents of mounting media
 Common dehydrants are ethyl alcohol, acetone, normal butyl alcohol , tertiary
butyl alcohol Glycerine, Dioxan etc.
Ethyl Alcohol/Isopropyl alcohol
 Most common
 Progressively increasing concentrations – 10%, 20%, 30%, 40% …… 100%
 Begin with a grade same as the water content in the tissue
 Time required – soft tissues ~30 minutes – Hard/ large tissue- ~6-12 hrs.

Normal Butyl Alcohol
 Advantage – solvents of paraffin – directly followed to impregnation
 Grades are prepared in combination with ethyl alcohol
Series No. 95 % Ethyl
Alcohol (ml)
Normal butyl
alcohol (ml)
Distilled water
(ml)
1.
2.
3.
4.
5.
6.
7.
8.
20
25
30
30
25
20
15
0
10
15
25
40
55
70
85
100
70
60
45
30
20
10
0
0
1 hour
2 hour

Tertiary Butyl Alcohol (TBA)
Series No. Absolute Alcohol (ml) 95% Ethyl
Alcohol (ml)
TBA
(ml)
Dist. Water
(ml)
1.
2.
3.
4.
5.
0
0
0
0
25
50
50
50
50
0
10
20
35
50
75
100
40
30
15
0
0
Dehydrate first in ethyl alcohol upto 50%
Three changes in absolute TBA

3. Clearing (Dealcoholization)
 Removal of alcohol from the tissues
 Replacing the dehydrating fluid with a fluid that is totally miscible with
both the dehydrating fluid and the embedding medium- Paraffin
 Transition step between dehydration and infiltration
 Only needed when the dehydrants are not solvents of wax
 Clearing agents- Xylene, Toluene, Chloroform, Benzene, Petrol etc.

Reagents in Clearing - Xylene
 Xylene- Conventional reagent in dealcoholization
 Practiced in graded series (30 min 1hr in each)
Series No. Ethyl alcohol (ml) Xylene (ml)
1
2
3
4
5
6
7
8
9
10
90
80
70
60
50
40
30
20
10
0
10
20
30
40
50
60
70
80
90
100

MICROTECHNIQUE Killing and fixation module

Clearing
 Clearing is transition step between dehydration and infiltration with the embedding medium
 Many dehydrants are immiscible with paraffin wax
 a solvent imiscible with both the dehydrant and the embedding medium us used to facilitate the
transition between dehydratin and infiltration step
 Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the
embedding medium.
 Choice of a clearing agent depends upon the following:
- The type of tissues to be processed, and the type of processing to be undertaken.
- The processor system to be used.
- Intended processing conditions such as temperature, vacuum and pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .

- xylene.
- Toluene.
- Chloroform.
- Benzene.
- Petrol.
Some clearing agents

Mounting
 The final stage in the preparation of tissues for microscopy is
mounting
 For stained preparations, the mounting medium or mount ant should
have the same refractive index as the section or nearer to that
 To be effective, a mountant should possess certain characteristics.
These include the following

 It should be colourless and transparent
 It should be able to completely permeate and fill tissue spaces
 It should have no adverse effect on tissue components
 It should be resistant to contamination particularly by
microorganisms
 It should be completely miscible with dehydrant or clearing agent
 The mountant may be hydrophobic or hydrophilic

 Hydrophobic mountant
 Canada balsam
 This is an oleorosin obtained from the bark of the fir Abis balsamea of the
family Pinaceae
 The dried resin is freely soluble in xylene and other organic solvents DPX
(Distrene, Polystyrene Xylene)
 DPX is one of the most commonly used mountants
 It is a colouless, neutral medium in which most standard stains are well
preserved
 This fast drying mounting medium prevents moisture from developing
under the cover glass and the consequent clouding of the specimen

`
Hydrophilinc mountant
 Water
 Glycerol
 Used as temporary mountant
 Having higher refractive index (1.460)
 Having longer drying time that water
 Phosphate buffered glycerol is commonly used

4. Paraffin infiltration (Embedding)
 Most commonly used waxes for infiltration are
the commercial paraffin waxes
 It us solid at room temperature but melts at
temperatures up to about 65°C or 70°C.
 Available in melting points at different
temperatures
 Dehydrated material is gradually infiltrated
with wax
 Liquid wax is recommended for the initial
infiltration

Paraffin Series
Paraffin Series Interval
50% TBA + 50 % Liquid Paraffin
100 % Liquid paraffin
1- 6 hours
1- 6 hours
20 %
40 %
60 %
80 %
100 %
1- 6 hours
1- 6 hours
1- 6 hours
1- 6 hours
1- 6 hours
at 70⁰ C
Oven
Paraffinwax

Paraffin Embedding
 Three changes in 100 % wax
 Paraffin block-material preparation
 Attachment of the block into the holder of the
microtome
 Sectioning with microtome

Steps involved
1. Killing and fixation
2. Dehydration
3. Clearing
4. Paraffin infiltration
5. Casting of wax impregnated material into blocks
6. Attachment of the block into the holder of the microtome
7. Microtomy
8. Affixing paraffin ribbon on glass slides
9. Removal of wax
10.Staining and mounting

Sections
 Sectioning allows light pass through the material
 FREE HAND SECTIONS
 SERIAL SECTIONS
FREE HAND SECTIONS
 Can be done if the material is hard
 Thin sections - 10 µM can be taken
 Sectioning with razor

Serial sections
 Serial sections are produced by paraffin method
 Paraffin infiltrated material are affixed on
wooden blocks
 Objects are cut into a series of sections
 Serial sections are placed on adhesive smeared
glass slides
 Serial sections enables the reconstruction of
structure of organ
 Orientation of vasculature, cellular organization
etc. can be studied

Stains and Staining
 Staining - Use of dyes to provide color to various tissue constituents
 Different tissue constituents react differently to dyes – contrast
 Chromogen
 Chromophore
 Auxochrome – acid/ alkali radicals. Responsible for solubility

Stains - classification
Principle Chemical Nature
Chemical Nature Basic : Colored organic base+ uncolored acetate, chloride or sulphate radical
(safranin, methylene blue, crystal violet)
Acidic : Metallic base (Na, K) + Colored organic radical (Aniline Blue, Eosin,
Orange G )
Neutral : Combinations of acidic and basic dyes (Giesma stain, Sudan black B)
Affinity to different
plant parts
Nuclear : Nucleus
Cytoplasmic: Cytoplasm
Microtechnical
purposes
Histological: defines tissues (xylem, phloem etc.)
Cytological : Define cell components (nucleus, chromosomes etc.)

Stains
Natural Dyes – dyes obtained from plant/ animal (Brazilin, Hematoxylin, Carmine)
Synthetic dyes – made from Coal tar – (Orange G, Safranine, Fast Green)
 Brazilin (Timber of Caesalpinia crista, C. echinata)
 Hematoxylin Hematoxylon campechianum
 Carmine Insect Dactylopius coccus
Staining Methods
1. Progressive staining
2. Regressive (Retrogressive staining)
3. Counter staining
4. Double, triple and quadruple staining

Methods of Staining
Progressive Staining
 Useful for beginners
 Tissues are understained first
 Gradually more stain is added until the desired intensity attained
 Staining interval required is determined by trial
Regressive (Retrogressive) Staining
 Overstained first
 Then destained until the desired intensity is attained
 Destaining agent – 70% alcohol with 1% acetic acid
 Proper washing after differentiation

Counterstaining
 Staining certain part of cells/ tissues with one stain
 Other parts with a contrasting color
Double/ Triple/ Quadruple staining
 Use of 2, 3, 4 colors on same section
 Double staining - Safranin O and Fast Green
 Triple staining - Safranin O, Gentian Violet and Orange G
 Quadruple Staining - Safranin O, Methyl violet, Fast Green and Orange G
Methods of Staining

Whole Mounts
 Used to preserve and retain natural color, form and shape of
whole plants/ plant parts
 Microscopic museum materials preserved in ethyl alcohol,
formalin
Water – 72 ml
Formaldehyde – 5 ml
Glacial acetic acid – 3 ml
Glycerine - 20 ml
 Temporary whole mounts – small filamentous algae- in 10%
glycerine/ coverslip

Whole Mounts
 Permanent whole mounts – Microscopic Material
 Constant handling requires preparation of permanent nature
1. Killing and fixation
2. Washing in water
3. Staining with hematoxylin for 30 min -1 hour
4. Destaining in 0.1% HCl
5. Transfer to glass slide
6. Covering with DPX and cover slip

Cytological Methods
 Used to study the minute details of the cell structure – nucleus
 Smear and squash methods are the most common.
 Smear – Smearing material on glass slide (Acetocarmine
method, Feulgen method)
 Squashes - component parts separate and are not studied
intact.
MACERATION
 Separation of cells of fixed plant or animal material through
hydrolysis
 Useful to visualize the 3d nature of structural elements
 Reagents used depends – nature of middle lamella

Maceration
Middle lamella
 Herbaceous – Pectin (boiling in water)
 Woody - Lignin (alkali/ acid/ enzyme treatment)
 3 common methods in practice
1. Schultze’ s Method
 Treatment with con. H2SO4+KClO3 and warming
 After thorough bleaching washing in water
2. Jeffrey’ s Method
 Treatment in equal vol 10% HNO3+K2CrO4 at 30° C - 40° C for 1 -2 days
 Thorough washing

3. Harlow’ s Method
 Treatment in chorine water –2 hours
 Washing in running water
 Boiling in 3% Na2SO3 – 15 min
 Washing
 Staining in Safranin
 Washing in water
 Dehydration with hygrobutol
 Infiltration with Canada balsum
 After placing the material on glass slides tease with needles
 Mount with cover slip
Maceration

MICROTECHNIQUE Killing and fixation module

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