3. 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
4. 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
5. 2. Formalin
Aqueous Formaldehyde
Reducing agent
Water miscible
Slow Penetration
Causes shrinkage
Great Hardening effect
Makes staining difficult
Reagents of Fixatives
6. 3. Acetic Acid
Water miscible
Rapid penetration
No hardening effect
Makes tissues soft
4. Chromic acid
Water miscible
Oxidiser
Slow Penetration
Reagents of Fixatives
7. 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
8. 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
Killing and Fixing Fluids (Fixatives)
Acetic
Acid
Alcohol
Mixtures
9. Killing and Fixing Fluids (Fixatives)
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
10. Killing and Fixing Fluids (Fixatives)
1. Chromo acetic acid ( Weak)
Chromic acid 1% - 50ml
Acetic acid 1% - 50 ml
2. Chromo acetic (Medium)
Chromic acid 1% - 70ml
Acetic acid 1% - 20 ml
Water - 10 ml
3. Chromoic acetic : Strong
Chromic acid 1% - 97 ml
Acetic acid 1% - 3 ml
Chromo
Acetic
Acid
Mixtures
11. Killing and Fixing Fluids (Fixatives)
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
Chromo
Acetic
Acid
Mixtures
12. 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
Chromo
Acetic
Acid
Formalin
Mixtures
Killing and Fixing Fluids (Fixatives)
13. 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
Chromo
Acetic
Acid
Formalin
Mixtures
Killing and Fixing Fluids (Fixatives)
14. CRAF
In all these formalin should be added just before use
A few hours after the addition of formalin a color change
takes place.
Subsequently the color become olive green
This fluid now acts as a preservative
Fixation time 12 hours
No washing in water
Chromo
Acetic
Acid
Formalin
Mixtures
Killing and Fixing Fluids (Fixatives)
15. 2. 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
16. 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.
17. 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
18. 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
19. 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.
20. 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
21. 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
23. Paraffin Embedding
Three changes in 100 % wax
Paraffin block-material preparation
Attachment of the block into the holder of the
microtome
Sectioning with microtome
24. 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
25. 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
26. 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
28. 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
29. 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.)
30. 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
31. 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
32. 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
33. 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
34. 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
35. 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
36. 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
37. 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