PREPARATION OF BIOLOGICAL SPECIMENSFOR LIGHT MICROSCOPY

Dr. Ratheesh Chandra P.

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

Reagents of Fixatives

3. Acetic Acid Water miscible Rapid penetration No hardening effect Makes tissues soft

4. Chromic acid Water miscible Oxidiser Slow Penetration

Reagents of Fixatives

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

Killing and Fixing Fluids (Fixatives)

Acetic Acid

Alcohol Mixtures

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

Killing and Fixing Fluids (Fixatives)

1. Chromo acetic acid ( Weak)Chromic acid 1% - 50mlAcetic acid 1% - 50 ml

2. Chromo acetic (Medium)Chromic acid 1% - 70mlAcetic acid 1% - 20 mlWater- 10 ml

3. Chromoic acetic : StrongChromic acid 1% - 97 mlAcetic acid 1% - 3 ml

Chromo Acetic Acid

Mixtures

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

1. Navaschin’s FormulaSol. A: Chromic acid (1%) - 15 mlGlacial acetic acid - 10 mlDistilled water - 90 ml

Sol. B:Formalin - 40 mlDistilled 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)

Craf I Chromic acid 1% 20 ml Acetic acid 1% 75 ml Formalin 5

ml

Craf IIChromic acid 1 %

20 mlAcetic acid 10% 10 mlFormalin 5 mlDistilled water 65 ml

Chromo Acetic Acid

Formalin Mixtures

Killing and Fixing Fluids (Fixatives)

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)

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

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.

20253030

2520150

10152540

557085

100

70604530

201000

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)

123456789

10

9080706050403020100

102030405060708090

100

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 SeriesParaffin 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

Para

ffin

wax

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 involved1. 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

5. Microtomy

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 - classificationPrinciple 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 : NucleusCytoplasmic: Cytoplasm

Microtechnical purposes

Histological: defines tissues (xylem, phloem etc.)Cytological : Define cell components (nucleus, chromosomes etc.)

StainsNatural 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 StainingProgressive 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

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