Pgtrb_Unit IV: Microtechniques: Microscopy – Principle and applications, Light

microscope: Bright field, Dark field, Phase contrast microscopy, Fluorescence Microscope,
Electron microscope (TEM & SEM). Microtome: Types, Principles and operating
mechanisms. Maceration, Squashes, Smears, Whole mount and clearing techniques. Fixation
and fixatives, dehydration, clearing, infiltration, Embedding, Block making and sectioning.
Stains and staining techniques.
ApTrb_ Unit IV : Micro techniques and histochemistry

Rotary Microtome

 It is most commonly used microtome.

 This device operates with a staged rotary action such that the actual cutting is part of
the rotary motion.

 In a rotary microtome, the knife is typically fixed in a horizontal position.

 A rotary action of the hand wheel actuate the cutting movement.

 Here the advantage over the rocking type is that it is heavier and there by more
stable. Hard tissues can be cut without vibration.

 Serial sections or ribbons of sections can easily be obtained.

 The block holder or block (depends upon the type of cassette) is mounted on the steel
carriage that moves up and down and is advanced by a micrometer screw.

 Auto-cut microtome has built in motor drive with foot and hand control. With suitable
accessories the machine can cut thin sections of paraffin wax blocks and 0.5 to 2.0
micrometer thin resin sections.

 Advantages 1. The machine is heavy, so it is stable and does not vibrate during
cutting.

 2. Serial sections can be obtained.

 3. Cutting angle and knife angle can be adjusted.

4. It may also be used for cutting celloidin embedded sections with the help of special holder
to set the knife

 The flywheel in microtomes can be operated by hand.

 This has the advantage that a clean cut can be made, as the relatively large mass of the
flywheel prevents the sample from being stopped during the sample cut.

 The flywheel in newer models is often integrated inside the microtome casing.

 The typical cut thickness for a rotary microtome is between 1 and 60 µm.
  For hard materials, such as a sample embedded in a synthetic resin, this design of
microtome can allow for good “Semi-thin” sections with a thickness of as low as 0.5
µm.

TYPES OF MICROTOME

 Sledge Microtome is a device where the sample is placed into a fixed holder (shuttle),
the sledge placed upon a linear bearing, a design that allows for the microtome to
readily cut many coarse sections. Applications for this design of microtome are of the
preparation of large samples, such as those embedded in paraffin for biological
preparations. Typical cut thickness achievable on a sledge microtome is between is 10
and 60 micron.

 Cryomicrotome For the cutting of frozen samples, many rotary microtomes can be
adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup. The
reduced temperature allows for the hardness of the sample to be increased, such as by
undergoing a glass transition, which allows for the preparation of semithin samples.
However the sample temperature and the knife temperature must be controlled in
order to optimise the resultant sample thickness

 Ultramicrotome A ribbon of ultrathin sections prepared by room temperature

ultramicrotomy, floating on water in the boat of a diamond knife used to cut the
sections. The knife blade is the edge at the upper end of the trough of water.

 Vibrating microtome The vibrating microtome operates by cutting using a vibrating

blade, allowing the resultant cut to be made with less pressure than would be required
for a stationary blade. The vibrating microtome is usually used for difficult biological
samples. The cut thickness is usually around 30-500 µm for live tissue and 10- 500
µm for fixed tissue.
  Saw microtome The saw microtome is especially for hard materials such as teeth or
bones. The microtome of this type has a recessed rotating saw, which slices through
the sample. The minimal cut thickness is approximately 30 µm, and can be made for
comparatively large samples.

 The laser microtome is an instrument for contact free slicing. Prior preparation of the
sample through embedding, freezing or chemical fixation is not required, thereby
minimizing the artefacts from preparation methods. Alternately this design of
microtome can also be used for very hard materials, such as bones or teeth as well as
some ceramics. Dependent upon the properties of the sample material, the thickness
achievable is between 10 and 100 µm.

Ultramicrotomy

 An ultramicrotome is a main tool of ultramicrotomy. It can allow for the preparation

of extremely thin sections, with the device functioning in the same manner as a
rotational microtome, but with very tight tolerances on the mechanical construction.

 As a result of the careful mechanical construction, the linear thermal expansion of the
mounting is used to provide very fine control of the thickness.

 These extremely thin cuts are important for use with transmission electron microscope
(TEM) and Serial Block-Face Scanning Electron Microscopy (SBFSEM), and are
sometimes also important for light-optical microscopy.

 The typical thickness of these cuts is between 40 and 100 nm for transmission
electron microscopy and often between 30 and 50 nm for SBFSEM. Thicker sections
up to 500 nm thick are also taken for specialized TEM applications or for light
microscopy survey sections to select an area for the final thin sections.

 Diamond knives (preferably) and glass knives are used with ultramicrotomes. To
collect the sections they are floated on top of a liquid as they are cut and are carefully
picked up onto grids suitable for TEM specimen viewing. The thickness of the section
can be estimated by the thin-film interference colors of reflected light that are seen as
a result of the extremely low sample thickness.

MICROTOME KNIVES

 It is the important instrument used to cut uniform thin serial sections of the tissue.

 Various types of knives are used with different microtomes. For routine purpose
wedge (C type) knife is used.

 It is plain on both sides. The size varies from 100 mm to 350 mm in length.

 Microtome knives are made of good quality of high carbon or steel which is tempered
at the tip. Hardness of knife is essential to obtain good tissue sections.
  Sharpening of microtome knife - To achieve good sections knife should be very sharp.
The knife is put in the knife back to sharpen.

 Knife can be sharpened manually or by the use of automatic machine.

 Honing - This is done to remove nicks and irregularity from the knife edge. Coarse
and fine honing is done using different abrasives.

 Stropping - The purpose of stropping is to remove the “burr” formed during honing
and to polish cutting edge. Other types of knives are diamond and glass knives. These
knives are very expensive and used for ultramicrotomy.

Squash Technique (for mitosis: root tips, young meristems)

Principle

Softens middle lamella so individual cells separate; stain highlights chromosomes; gentle
“squash” spreads cells into a single layer for crisp chromosome views.

Common Applications

 Mitotic stages (prophase → telophase)

 Chromosome counts & karyotyping
 Checking ploidy, mitotic index
 Studying effects of mutagens/colchicine

Fixatives (prepare fresh if possible)

 Farmer’s fixative (3:1 ethanol:glacial acetic acid)

 OR Carnoy’s II (6:3:1 ethanol:chloroform:acetic acid)

Pretreat (optional; arrests metaphase & spreads chromosomes)

 Colchicine 0.05% (30–120 min) or

 Saturated p-dichlorobenzene (PDB) (1–3 h), then rinse

Cell-wall softening / hydrolysis

 1 N HCl (room temp or 60 °C water bath)
 (Optional enzymatic mix) 2% pectinase + 2% cellulase in 0.01 M citrate buffer, pH
~4.5 (15–30 min)
Stains
 2% Aceto-orcein (in 45% acetic acid), or
 1–2% Aceto-carmine (in 45% acetic acid), or
 Feulgen (Schiff reagent) after HCl hydrolysis for DNA-specific staining
 Counterstain (optional): Fast Green 0.1% (brief)

Mountant
 45% acetic acid (temporary) or DPX/Canada balsam (permanent)
Feulgen Variant (DNA-specific)

1. Fix in Carnoy’s.
2. Hydrolyze in 1 N HCl at 60 °C for 8–10 min.
3. Rinse in cold HCl (brief), then distilled water.
4. Stain in Schiff reagent 30–60 min in dark; rinse in SO₂ water or running water.
5. Squash in 45% acetic acid; mount.

Smear Technique (for meiosis: pollen mother cells in anthers)

Principle
Thin “smear” of cell suspension from anther spreads PMCs in a single layer; acetic acid
softens; stain contrasts chromosomes/spindle/chiasmata.
Common Applications
 Meiosis I & II staging (leptotene → tetrads)
 Chiasma frequency, crossing-over studies
 Pollen fertility/abnormalities, meiotic index
 Cytotaxonomy, polyploidy screening
Reagents
 Fixative: Farmer’s (3:1) or Carnoy’s I (3:1 ethanol:acetic acid)
 Stain (choose one):
o 1–2% Aceto-carmine (classic for PMCs)
o 2% Aceto-orcein
o Acetocarmine with ferric chloride (1% FeCl₃ in stain; intensifies)
o Aceto-propionic orcein (for sharper plates)
 Mountant: 45% acetic acid (temporary) or DPX (permanent after dehydration)
Specimens
Young flower buds at appropriate stage (test a range of bud sizes to capture full meiosis).
Procedure (Acetocarmine Smear of PMCs)

1. Collect & Fix: Harvest young buds; fix whole buds 1–24 h in Farmer’s; store in
70% ethanol if needed. (Fresh unfixed buds also work for quick classroom demos.)
2. Dissect: On a clean slide, add a drop of 1–2% acetocarmine. Transfer 1–3 anthers
into the drop.
3. Release PMCs: Tear anthers with needles; express contents into the stain to form
a uniform suspension. Remove remaining wall fragments with forceps.
4. Acetic Spread: Add an extra drop of 45% acetic acid; mix gently to reduce
viscosity and help nuclear spreading.
5. Smear:
o Place cover slip; gently press or drag the edge of the cover slip across
the drop once to create a thin smear.
6. Permanent Mount (optional): Gently lift cover slip after partial drying, dehydrate
(70%→90%→100% ethanol), clear in xylene, mount in DPX.

Feature Squash Smear

Mitotic cells (root tips,
Target Meiotic PMCs (anthers)
meristems)
Spreading Vertical pressure under cover slip Horizontal drag to thin film
Wall HCl or enzymes Not always needed; acetic acid +
 Feature Squash Smear
softening mechanical release
Aceto-orcein / Aceto-carmine /
Typical stain Acetocarmine / Aceto-orcein
Feulgen
Chiasma counts, meiotic index, pollen
Best for Karyotyping, mitotic index
fertility

MACERATION OF PLANT MATERIALS

 Purpose: To separate plant tissues into individual cells (fibers, sclereids, tracheids,
vessels) for microscopic study.
 Principle: Lignified and thick-walled cells resist chemicals and remain intact,
while middle lamella and primary walls are dissolved, allowing separation.
1. Harlow’s Method
 Reagent: 10% Nitric Acid (HNO₃).
 Procedure:
1. Take small pieces of plant material (wood, stem, leaf midrib).
2. Boil gently in 10% HNO₃ for 5–10 minutes until tissue softens.
3. Wash thoroughly with distilled water to remove acid.
4. Transfer to 5% potassium chlorate solution for bleaching (optional step
for clearer cells).
5. Tease the tissue gently with a needle or shake in water to separate cells.
 Advantages: Quick method, gives clear lignified elements.
 Limitation: Can damage delicate thin-walled cells due to strong acid.
2. Schultze’s Method
 Reagent: Mixture of Potassium Chlorate (KClO₃) crystals + concentrated
Nitric Acid (HNO₃).
 Procedure:
1. Place tissue in a test tube.
2. Add a small amount of KClO₃ crystals.
3. Pour conc. HNO₃ over it (caution: violent reaction possible).
4. Heat carefully until tissue becomes soft and bleached.
5. Wash well with distilled water.
6. Tease the material – cells (especially fibers, vessels, sclereids) separate
easily.
 Advantages: Produces very clear, bleached cells useful for permanent slides.
 Limitation: Harsh and dangerous chemicals (toxic fumes, explosive reaction if
overheated). Needs great care.
3. Jeffrey’s Method
 Reagents:
o 10% Chromic Acid solution (oxidizing agent).
o 10% Nitric Acid solution.
 Procedure:
1. Boil tissue in 10% Chromic Acid for 2–3 minutes.
2. Transfer immediately to 10% Nitric Acid, boil again for 2–3 minutes.
3. Wash thoroughly in water.
4. Tease the softened tissue gently to isolate cells.
 Advantages: Safer compared to Schultze’s method; better preservation of delicate
elements.
Limitation: Takes longer than Harlow’s method.

Comparison Table
Method Reagents Used Duration Advantages Limitations
Quick (5–10 May damage delicate
Harlow’s 10% HNO₃ Simple, rapid
min) cells
KClO₃ + conc. Very clear, bleached Dangerous
Schultze’s Moderate
HNO₃ prep (toxic/explosive)
10% Chromic acid + Preserves delicate
Jeffrey’s Slower Time-consuming
10% HNO₃ tissues, safer

Histological Techniques in Plants: Killing, Fixation, Embedding & Block

Making
Plant histology involves preparing thin sections of plant organs (root, stem, leaf, flower, seed
etc.) for microscopic study. Since fresh plant tissues dry, collapse or disintegrate quickly,
they must be preserved and hardened by special processes before sectioning.
1. Killing
 Definition: Process of rapidly stopping the life activities of the cells to prevent
autolysis (self-digestion), enzymatic action, or bacterial decay.
 Importance: Prevents shrinkage, distortion, or plasmolysis of cells.
 Methods:
o Chemical killing: Strong fixatives (alcohol, FAA – Formalin-Acetic-
Alcohol) kill protoplasm instantly.
o Heat killing: Rarely used for plant histology (sometimes for algal or
fungal preparations).
2. Fixation
 Definition: Process of preserving tissues in as close to living condition as possible
by using fixatives that stabilize and harden cellular components.
 Objectives:
1. Arrest physiological activities (stop metabolism).
2. Prevent autolysis and putrefaction.
3. Coagulate and stabilize proteins and other cell constituents.
4. Enhance staining properties of tissues.
5. Maintain structural integrity of tissues.
 Common Fixatives for Plants:
o FAA (Formalin–Acetic acid–Alcohol)
 Formula: 5 ml Formalin + 5 ml Glacial Acetic Acid + 90 ml
70% Ethanol.
 Excellent for general plant fixation.
o Carnoy’s fixative (Ethanol + Glacial acetic acid; sometimes with
chloroform).
o Chromic acid mixtures (rare, for cytological studies).
o Bouin’s fluid (for delicate tissues).
Procedure:
0. Collect fresh plant material (small pieces, 2–5 mm thick).
1. Immerse immediately in excess fixative (at least 10x tissue volume).
 2. Keep for 12–48 hrs depending on tissue hardness.
3. Wash material in 70% ethanol to remove excess fixative (especially
acetic acid or formalin).
3. Embedding
After fixation, tissues are dehydrated and infiltrated with a medium that can solidify into a
block for section cutting.
Steps:
1. Dehydration
o Remove water from tissues using graded series of alcohol (e.g., 50%,
70%, 90%, absolute).
o Essential because embedding media (wax, resin) are hydrophobic.
o Time: 30 min to 1 hr per grade, longer for woody tissues.
2. Clearing
o Replace alcohol with a clearing agent miscible with wax.
o Common clearing agents: Xylene, chloroform, benzene, cedarwood oil.
o Tissue becomes transparent at this stage.
3. Infiltration
o Clearing agent is gradually replaced by melted paraffin wax (58–60°C
melting point for plants).
o Tissues are kept in molten wax baths (2–3 changes, 1–2 hrs each).
4. Embedding
o Tissue is placed in a mold filled with molten wax and properly oriented
(so that desired section plane is obtained).
o Rapid cooling (on ice or cold water) solidifies the block.
4. Block Making
 Process: Final stage of embedding in which tissue is enclosed in a solid block of
wax for sectioning.
 Steps:
1. Select a mold (L-shaped metal or paper boat).
2. Pour molten paraffin wax.
3. Place tissue in correct orientation.
4. Allow to cool quickly (prevents crystal formation in wax).
5. Remove block from mold and trim edges.
6. Block is now ready for microtome sectioning (thin slices 5–15 µm).
Applications
 Study of plant anatomy (root, stem, leaf).
 Research in cytology and histochemistry.
 Detection of pathological changes in plants.
 Teaching demonstrations in botany.

In summary:
 Killing stops life activities.
 Fixation preserves tissue structure.
 Embedding gives support for sectioning.
 Block making produces a stable block for thin microtome sections.
Sectioning & Staining of Paraffin-Embedded Plant Tissues
1. Sectioning of Plant Tissue Using Microtome
Equipment:
 Rotary or sliding microtome
 Sharp microtome knife (steel / disposable)
 Brush & forceps
 Warm water bath (40–45 °C)
 Microscope slides
Procedure:
1. Trimming the Block
o Trim excess paraffin around the tissue block into a trapezoid/pyramid
shape.
o This exposes tissue and gives support during cutting.
2. Clamping the Block
o Fix the paraffin block onto the microtome chuck with correct orientation.
3. Cutting Sections
o Adjust thickness to 5–15 µm (plants often require 8–12 µm due to rigid
cell walls).
o Turn the wheel steadily → thin slices of wax ribbon form.
o Sections adhere into a continuous paraffin ribbon due to adhesive
property of wax.
4. Handling the Ribbon
o Using a fine brush or forceps, transfer the ribbon onto warm water bath
(40–45 °C).
o The warm water helps flatten the sections and remove wrinkles.
5. Mounting on Slides
o Place flattened sections carefully on clean slides coated with adhesive
(e.g., Mayer’s albumin or poly-L-lysine).
o Drain excess water and dry slides on a slide warmer at 37–40 °C (few
hours to overnight).

2. Staining of Paraffin Ribbon

Since tissues are embedded in paraffin, staining requires deparaffinization &

rehydration before applying dyes.

A. Deparaffinization
1. Place slide in xylene (2–3 changes, 5–10 min each) → removes paraffin.
2. Transfer to absolute alcohol (2–3 min).

B. Rehydration
 Pass slides through descending alcohol grades (95% → 70% → 50%) and
finally distilled water.
 Tissue is now rehydrated and ready for staining.

C. Staining Methods (for Plant Tissues)

1. Double Staining (most common for plants)
o Safranin O (red dye) → stains lignified, cutinized, suberized cell walls
(xylem, sclerenchyma, fibers).
 o Fast Green or Light Green → counterstains cellulose walls, cytoplasm
(phloem, parenchyma, collenchyma).

Protocol:

o Stain slides in Safranin (0.5–1%) → 1–2 hrs (or overnight for woody
tissues).
o Wash in water → remove excess dye.
o Counterstain with Fast Green (0.1%) for few seconds to 1 min.
o Rinse quickly in 95% alcohol (Fast Green is alcohol-soluble, so don’t
overdo).
2. Other Stains (depending on purpose):
o Hematoxylin & Eosin (H&E) → general histology.
o Toluidine Blue O → metachromatic stain, excellent for primary
screening.
o PAS reaction (Periodic Acid-Schiff) → detects polysaccharides,
mucilage, cuticle.

D. Dehydration & Clearing

1. Pass slides through ascending alcohol series (70% → 90% → absolute).
2. Clear in xylene (2–3 changes, 5 min each).

E. Mounting
 Add a drop of mounting medium (e.g., DPX, Canada balsam).
 Place coverslip gently to avoid air bubbles.
 Dry slides → ready for microscopic observation.

3. Expected Results

 Xylem (wood elements, fibers, lignified tissues) → Red (Safranin).

 Phloem, parenchyma, collenchyma → Green (Fast Green).
 Cuticle / Suberin / Lignin → Red.

This gives excellent contrast to study tissue differentiation.

In summary:

 Microtome sectioning → thin, wrinkle-free ribbons.

 Deparaffinization → Rehydration → Staining → Dehydration → Clearing →
Mounting → permanent slides with clear tissue differentiation.

HISTOCHEMICAL TECHNIQUES

 Histochemistry is an important technique that is used for the visualization of

biological structures. As such, it is concerned with the identification and distribution
of various chemical components of tissues through the use of stains, indicators as well
as microscopy.
  Essentially, identification and distribution of chemical constituents of tissues is
achieved through the exploitation of unique chemical environments in cells,
heterologous expression techniques as well as enzymatic activities. T
 he various methods for histochemical techniques includes the staining of: • Proteins

Carbohydrates • Lipids • Enzymes

Lipids Staining: This technique is dependent on dyes that are soluble in lipids. Some of the
most common dyes used include: • Sudan VI • Sudan Black • Oil Red O •

 Nile Blue Lipid staining is a useful technique that is used for demonstrating
intracellular lipids in various tissue sections.

Protein and Amino Acids : Some of the methods used for specific amino acids include: •
Millon’s Reaction • Sakaguchi Reaction Tetrazotized Benzidine Reaction

Millon’s Reagent : Millon’s reagent is used for detecting amino acid tyrosine:

Principle : In this technique, the mercurous and mercuric nitrate (components of the reagent)
reacts with hydroxybenzene radicals to form a compound that is red in color. Tyrosine
contains the phenolic group, which forms the red coloration in the presence of Millon’s
reagent. The compound formed through this reaction is called mercuric fumarate.

Sakaguchi Test : The Sakaguchi reaction test involves the use of the Sakaguchi reagent.
This reagent is composed of 1-naphthol and sodium hypobromite and forms a reddish
compound when mixed with the sample containing arginine. The test is positive for any
amino acid that contains the guanidine group in Arginine. Therefore, the Guanidine group in
an amino acid will react with the α-Naphthol and alkaline hypobromite in the reagent to give
of a red-colored complex indicating the presence of such amino acids.

Tetrazotized Benzidine Reaction : Although it has been shown to be less effective,

Tetrazotized benzidine is used in histochemistry to detect non-collagen proteins. The
procedure involves the coupling of Tetrazotized benzidine with beta naphthol or Hyaluronic
acid. The method, commonly referred to as Tetrazotized benzidine helps in the detection of
such non-collagen proteins as tyrosine, histidine as well as tryptophan

Nucleic Acids and DNA

 Feulgen’s Reaction : This is a relatively new technique that is used for demonstrating
DNA in tissue sections. It is a sensitive means of detecting aldehydes, which makes it
the ideal method for detecting the presence of DNA. Here, the section is treated with
dilute hydrochloric acid in order to remove the bases.
 The sugar part that remains reacts as an aldehyde ultimately forming a visible color.
Therefore, this method can be said to be divided in to two main parts: 1. The first part
of the procedure is the hydrolysis phase that involves the use of 5N HCl, ambient
temperature for 40 minutes. This step is aimed at separately selecting 2 purine bases
(Adenine and Guanine) which are removed from the DNA molecule. 2. The second
 step is the staining phase. The reagent used is preferred because it is highly selective
for DNA rather than RNA. Here, RNA does not react because of the presence of
hydroxyl on carbon 2 of ribose, which prevents the acid (HCl) from hydrolyzing
sugar. The reaction is also precise for the localization of DNA given that deoxyribose
radicals are bound to phosphoric acid of the apurinic acid molecule following the
removal of purine bases.
 Some of the stains used for both DNA and RNA include, • Methyl Green Pyronin
Stain • Acridine Orange

PAS Reaction (Periodic Acid Schiff)

 This is one of the most popular histochemical techniques for the detection of
glycogen. It has been shown to be one of the best techniques for demonstrating
carbohydrates in tissue. In this technique, the periodic acid oxidizes tissue
carbohydrates to produce aldehyde groups. This group then condenses with the
reagent to form a bright red coloration to demonstrate the tissue component with
carbohydrate attachments. The diastase and a-amylase in the reagent act on the
glycogen and depolymerize it into smaller sugar units (maltose and glucose) which
are then washed out of the section.
 Some of the other stains used for staining sacchrides include, • Lectins • Ruthenium
Red • Alcian Blue

Histochemical localisation of common plant metabolites and chemical constituents

1. Lignin

 Reagent/Test: Phloroglucinol–HCl test (Wiesner reaction)

 Result: Lignified cell walls stain red to magenta.
 Other tests: Mäule reaction → brownish-red colour.

2. Pectin

 Reagent/Test: Ruthenium red

 Result: Pectic substances in middle lamella and primary wall stain pink to red.
 Other option: Coralline (meta-chromatic staining).

3. Cellulose

 Reagent/Test: Iodine–Zinc Chloride solution

 Result: Cellulose walls show blue-violet colour.
 Alternative: Chlor-Zinc Iodine → blue colour.

4. Starch

 Reagent/Test: Iodine–Potassium Iodide (IKI)

 Result: Starch grains stain blue to blue-black (amylose) or reddish-brown (amylopectin).
5. Lipids

 Reagent/Test: Sudan dyes (Sudan III, Sudan IV, Sudan Black B, Oil Red O).
 Result: Lipid bodies stain orange/red/black depending on dye used.
 Osmium tetroxide (OsO₄): Fixes and stains lipids black.

6. Proteins

 Reagent/Test: Mercuric bromophenol blue (or Xanthoproteic test).

 Result: Proteins stain blue (bromophenol) or yellow (xanthoproteic).

7. Nucleic acids

 DNA: Feulgen reaction → DNA stains red/purple.

 RNA: Pyronin-methyl green stain → RNA stains red, DNA green.

8. Mucilage

 Reagent/Test: Ruthenium red or Toluidine blue

 Result: Mucilage stains deep pink/red (Ruthenium) or violet/blue (Toluidine).