This document outlines the detailed procedures for the preparation of biological specimens for light microscopy, including sample collection, fixation, dehydration, clearing, and embedding. It discusses various fixatives and their properties, as well as staining methods and equipment used in microscopy, emphasizing the importance of each step in preserving and preparing specimens for observation. Specific formulations for fixatives and dehydrants are also provided, alongside techniques for sectioning and staining specimens.

1. PREPARATION OF BIOLOGICAL SPECIMENS
FOR LIGHT MICROSCOPY
Dr. Ratheesh Chandra P.

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

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

22. 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

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

27. 5. Microtomy

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

38. ?