Laboratory Techniques BBO6T16

Syllabus topics (RTMNU)

• pH and Buffers: Definition and measurement of pH, Buffers, Buffer capacity, common buffer compositions (Phosphate, Borate and Tris)
• Principles of microscopy: Magnification, resolution, and numerical aperture
• Advanced optical techniques: Phase Contrast Microscopy, Electron Microscopy – Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM)
• Sample Preparation: Fixation, dehydration, and staining techniques specific to plant tissues

pH — definition and measurement

pH = −log₁₀[H⁺] (Sørensen, 1909). Scale 0–14 at 25 °C: pH 7 neutral, below 7 acidic, above 7 alkaline. A change of 1 pH unit = 10-fold change in [H⁺]. Plant cells: cytoplasm about 7.2–7.5; vacuole acidic (about 5–5.5).

Measurement: (1) Indicators and pH paper — colour change, approximate (universal indicator). (2) pH meter — glass electrode + reference electrode (calomel or Ag/AgCl), often combined; the potential changes by about 59 mV per pH unit at 25 °C. The meter is calibrated with standard buffers (pH 4.0, 7.0, 9.2), with temperature compensation; the electrode is kept wet and rinsed between samples.

Buffers, buffer capacity and common buffer compositions

A buffer is a solution of a weak acid and its conjugate base (or weak base and its conjugate acid) that resists change in pH when small amounts of acid or alkali are added. Henderson–Hasselbalch equation: pH = pK_a + log([A^–]/[HA]). A buffer works best within pK_a ± 1.

Buffer capacity (β): the amount (moles) of strong acid or base needed to change the pH of 1 L of buffer by 1 unit. It is highest when [A^–] = [HA] (pH = pK_a) and increases with total buffer concentration.

Principles of microscopy — magnification, resolution, numerical aperture

• Magnification: how many times the image is larger than the object. Total magnification = objective × eyepiece (e.g. 100× × 10× = 1000×). Magnification beyond the resolving power is "empty magnification".
• Resolution (resolving power): the minimum distance between two points that can still be seen as separate. Abbe/Rayleigh: d = 0.61 λ / NA. Smaller d = better resolution. With visible light (λ ≈ 500 nm) and NA 1.4, d ≈ 0.2 µm — the limit of the light microscope.
• Numerical aperture (NA) = n sin θ, where n = refractive index of the medium between specimen and objective and θ = half the angle of the cone of light entering the objective. Air n = 1.0; immersion oil n = 1.515 (cedarwood oil 1.51) raises NA to about 1.25–1.4 and improves resolution.

Advanced optical techniques — phase contrast, SEM and TEM

Phase contrast microscopy (Frits Zernike, Nobel Prize 1953): living, unstained cells are transparent; their parts slow light differently (phase difference) but the eye sees only amplitude (brightness) differences. An annular diaphragm under the condenser and a phase plate in the objective convert phase differences into differences of brightness, so organelles appear dark/bright. Used to study living cells, cell division, cytoplasmic streaming.

Electron microscopy: uses a beam of electrons (very short wavelength) focused by electromagnetic lenses in a vacuum, giving much higher resolution.

Sample preparation for plant tissues — fixation, dehydration, staining

1. Fixation: kills cells quickly and preserves structure as in life. For light microscopy: FAA (formalin–acetic acid–alcohol), Carnoy's fluid (for chromosomes); for EM: glutaraldehyde followed by osmium tetroxide (post-fixation, also stains membranes).
2. Dehydration: water is removed gradually through a graded ethanol (or tertiary butyl alcohol) series — 30%, 50%, 70%, 90%, 100% — to avoid shrinkage; then clearing in xylene, infiltration and embedding in paraffin wax (or epoxy resin for EM).
3. Staining: gives contrast. Safranin–fast green double staining (lignified/cutinised walls red, cellulose walls and cytoplasm green); toluidine blue O (polychromatic); iodine (starch blue-black); acetocarmine or Feulgen (chromosomes, DNA); Sudan III/IV (lipids, suberin); phloroglucinol–HCl (lignin, pink-red). Mounting in DPX or glycerine.

Likely exam questions

1. (Long, 8 marks) Define pH. How is it measured? Explain buffers and buffer capacity with the composition of phosphate, borate and Tris buffers.
2. (Long, 8 marks) Explain the principle and working of the transmission and scanning electron microscopes. Compare TEM and SEM.
3. (Short, 4 marks) Define resolution and numerical aperture of a microscope.
4. (Short, 4 marks) Write the principle of phase contrast microscopy.
5. (Short, 4 marks) Describe fixation and dehydration of plant tissues.

Syllabus topics (RTMNU)

• Centrifugation – Basic Principles: Sedimentation coefficient, Relative Centrifugal Force (RCF) vs. Revolutions per minute (RPM)
• Types of Centrifuges: High-speed, and Ultracentrifuge
• Separation Methods: Differential Centrifugation; Density Gradient Centrifugation
• Safety and Maintenance: Balancing, vacuum requirements, and temperature control
• Spectrophotometry: Beer-Lambert Law, Absorption Spectra, Instrumentation for UV-Visible spectrophotometer

Centrifugation — basic principles

Centrifugation separates particles (cells, organelles, macromolecules) suspended in a liquid by spinning them at high speed; the centrifugal force makes denser/larger particles sediment faster.

• Sedimentation coefficient (s): the sedimentation velocity per unit centrifugal field: s = v/(ω²r), where v = rate of sedimentation, ω = angular velocity (rad s^–1), r = distance from the axis. Unit: Svedberg (S) = 10^–13 s. s depends on mass, density and shape of the particle and on the medium (e.g. prokaryotic ribosome 70S = 50S + 30S; S values are not additive).
• RPM vs RCF: RPM (revolutions per minute) is only the speed of the rotor; relative centrifugal force (RCF, in × g) is the actual force, which also depends on the radius. RCF = 1.118 × 10^–5 × r (cm) × (rpm)². Protocols should state RCF because the same rpm gives different forces in different rotors.
  Example: r = 10 cm, 10,000 rpm → RCF = 1.118 × 10^–5 × 10 × (10,000)² = 11,180 × g.

Types of centrifuges — high-speed and ultracentrifuge

Separation methods — differential and density gradient centrifugation

Differential centrifugation: the homogenate is spun at successively higher speeds/longer times; at each step the pellet (heavier particles) is collected and the supernatant is spun again. Typical sequence for a plant/animal homogenate: about 1,000 × g, 10 min → nuclei, cell debris; about 10,000–20,000 × g, 20 min → mitochondria, chloroplasts; about 100,000 × g, 60 min → microsomes (ER fragments, ribosomes); supernatant → cytosol. Simple, but fractions are not very pure.

Density gradient centrifugation: the sample is layered on (or mixed with) a solution whose density increases towards the bottom of the tube.

• Rate-zonal: pre-formed shallow gradient (e.g. 5–20% sucrose); particles separate by size/shape (s value) into bands; run is stopped before they reach the bottom. Used for ribosomal subunits, RNA, proteins.
• Isopycnic (equilibrium): steep gradient (e.g. CsCl, sucrose 20–70%); each particle moves until it reaches the point where its density equals the medium (buoyant density), regardless of time. Used for DNA (Meselson–Stahl experiment, 1958), plasmids, organelles. Bands are collected by puncturing the tube.

Safety and maintenance of centrifuges

• Balancing: tubes placed opposite each other must be of equal mass (weigh on a balance); imbalance causes vibration, damage to the drive and rotor failure.
• Use correct tubes/rotors within their rated speed; never exceed maximum rpm; check rotor for cracks/corrosion; keep a rotor log (rotors are de-rated with age).
• Vacuum requirements: ultracentrifuges need a vacuum before high speed to cut air friction and heating; do not open until the rotor stops and vacuum is released.
• Temperature control: refrigeration (often 4 °C) protects enzymes, organelles and nucleic acids; pre-cool the rotor.
• Close the lid before starting (lid lock), use sealed buckets for infectious samples, clean spills and dry the chamber, lubricate O-rings.

Spectrophotometry — Beer–Lambert law, absorption spectra, UV-Vis instrumentation

Beer–Lambert law: A = log(I₀/I) = εcl, where A = absorbance, ε = molar absorption coefficient (L mol^–1 cm^–1), c = concentration (mol L^–1), l = path length (cm). Absorbance is proportional to concentration (in dilute solutions), so unknowns are found from a standard (calibration) curve. Transmittance T = I/I₀; A = −log T.

Absorption spectrum: a plot of absorbance against wavelength; the peak is λ_max, used for measurement (maximum sensitivity). Examples: DNA/RNA 260 nm (A₂₆₀/A₂₈₀ ≈ 1.8 for pure DNA), proteins 280 nm (Trp, Tyr), chlorophyll a peaks near 430 and 662 nm, chlorophyll b near 453 and 642 nm (in acetone).

Instrumentation of a UV-Visible spectrophotometer:

1. Light source: deuterium lamp (UV, 190–350 nm) and tungsten–halogen lamp (visible, 350–900 nm).
2. Monochromator: entrance slit, prism or diffraction grating, exit slit — selects a narrow band of wavelengths.
3. Sample holder: cuvettes of 1 cm path — quartz/fused silica for UV, glass or plastic for visible; a blank (reference) cuvette with solvent.
4. Detector: phototube, photomultiplier tube or photodiode array — converts light to electric signal.
5. Read-out: amplifier and digital display/computer (A or %T, spectrum). Double-beam instruments compare sample and reference continuously.

Likely exam questions

1. (Long, 8 marks) Explain the principle of centrifugation. Describe differential and density gradient centrifugation.
2. (Long, 8 marks) State the Beer–Lambert law. Describe the instrumentation of a UV-Visible spectrophotometer with a block diagram.
3. (Short, 4 marks) Distinguish between RPM and RCF. Calculate RCF at 12,000 rpm for a rotor radius of 8 cm.
4. (Short, 4 marks) What is the sedimentation coefficient? Define a Svedberg unit.
5. (Short, 4 marks) Write the safety precautions in using a centrifuge.

Syllabus topics (RTMNU)

• Electrophoresis – General Principle: Electrophoretic mobility, Types of electrophoresis
• Agarose Gel Electrophoresis (AGE): Principle, Procedure
• Polyacrylamide Gel Electrophoresis (PAGE): SDS-PAGE, Native-PAGE
• Two-Dimensional (2D) Electrophoresis: Basic concept
• Visual and fluorescent dyes used for detection of Nucleic acids and Proteins
• Blotting Techniques: Southern, Northern, and Western blotting (Basic concepts)

Electrophoresis — general principle and electrophoretic mobility

Electrophoresis is the movement of charged molecules in an electric field through a medium (gel or paper). Negatively charged molecules move to the anode (+), positive to the cathode (−).

Electrophoretic mobility μ = v/E (velocity per unit field strength) = q/(6πηr) for a sphere — it increases with net charge q and decreases with size r and with viscosity η of the medium. It also depends on pH (which sets the charge), ionic strength, voltage, temperature and gel pore size.

Types: paper electrophoresis, cellulose acetate, gel electrophoresis (agarose, polyacrylamide), isoelectric focusing (IEF), pulsed-field gel electrophoresis (large DNA), capillary electrophoresis, immunoelectrophoresis, 2-D electrophoresis.

Agarose gel electrophoresis (AGE) — principle and procedure

Principle: agarose (a polysaccharide from red seaweed) forms a porous gel. DNA has a uniform negative charge per unit length (phosphate backbone), so all fragments move towards the anode and are separated by size — small fragments move faster through the pores (migration roughly proportional to log of size). Gel strength 0.7–2% (lower % for larger DNA).

Procedure:

1. Dissolve agarose in TAE or TBE buffer by heating; cool to about 55 °C, add stain (e.g. ethidium bromide or a safer dye), pour into a casting tray with a comb; let it set.
2. Remove comb, place gel in the tank, cover with the same buffer (wells at the cathode end).
3. Mix DNA samples with loading dye (glycerol to make it sink + tracking dyes bromophenol blue, xylene cyanol); load into wells along with a DNA ladder (marker of known sizes).
4. Run at about 5–8 V per cm until the tracking dye has moved about ¾ of the gel.
5. View on a UV/blue-light transilluminator and photograph (gel documentation); compare bands with the ladder to estimate sizes.

Polyacrylamide gel electrophoresis (PAGE) — SDS-PAGE and native PAGE

Polyacrylamide gel is made by polymerising acrylamide with the cross-linker N,N′-methylene-bis-acrylamide, initiated by ammonium persulphate (APS) and catalysed by TEMED. Pore size is set by acrylamide % (e.g. 8–15%). It gives higher resolution than agarose and is used for proteins and small DNA/RNA.

• SDS-PAGE (Laemmli system): proteins are boiled with SDS (sodium dodecyl sulphate, an anionic detergent) and a reducing agent (β-mercaptoethanol or DTT). SDS unfolds the protein and coats it with uniform negative charge (about 1.4 g SDS per g protein), and the reducing agent breaks S–S bonds. Hence separation is by molecular mass only. A discontinuous system has a stacking gel (low %, pH 6.8) that concentrates samples into sharp bands and a resolving gel (higher %, pH 8.8). Molecular mass is found by comparing with protein markers.
• Native PAGE: no SDS or reducing agent; proteins keep their natural shape, charge and activity, and separate by charge, size and shape together. Used to study enzyme activity (zymograms, isozymes), protein complexes.

Two-dimensional (2D) electrophoresis — basic concept

Introduced by O'Farrell (1975). Proteins are separated in two directions at right angles: first dimension — isoelectric focusing (IEF) in a pH gradient strip, where each protein stops at its isoelectric point (pI); second dimension — SDS-PAGE, separating by molecular mass. Result: a gel with hundreds to thousands of spots. Used in proteomics — comparing protein profiles of plants under stress, identifying spots by mass spectrometry.

Visual and fluorescent dyes for detection of nucleic acids and proteins

Blotting techniques — Southern, Northern and Western (basic concepts)

Blotting is the transfer of separated molecules from a gel to a membrane (nitrocellulose or nylon/PVDF), where they are fixed and detected with a specific probe.

Likely exam questions

1. (Long, 8 marks) Explain the principle and procedure of agarose gel electrophoresis of DNA.
2. (Long, 8 marks) Describe SDS-PAGE and native PAGE. Add a note on two-dimensional electrophoresis.
3. (Short, 4 marks) What is electrophoretic mobility? On what factors does it depend?
4. (Short, 4 marks) Distinguish between Southern, Northern and Western blotting.
5. (Short, 4 marks) Name the dyes used to detect nucleic acids and proteins in gels.

Syllabus topics (RTMNU)

• Chromatography – General Principle: Partition law
• Paper chromatography and Thin Layer Chromatography (TLC)
• Gas chromatography
• Introduction to HPLC and HPTLC for secondary metabolite profiling
• Microtomy: Introduction to microtomy, Types of microtomes – Rotary microtome, Rocking microtome, Cryostat; Procedure of microtomy
• Polymerase Chain Reaction (PCR): Principle and procedure, Real-Time PCR

Chromatography — general principle: partition law

Chromatography (Tswett, 1906, plant pigments) separates the components of a mixture by their different distribution between a stationary phase and a mobile phase that moves over it.

Partition law (Nernst distribution law): at equilibrium a solute distributes between two immiscible phases in a constant ratio — the partition (distribution) coefficient K = C_s/C_m (concentration in stationary phase / in mobile phase). Components with higher K are held back longer; those with lower K move faster. Other mechanisms: adsorption, ion exchange, size exclusion, affinity.

Paper chromatography and thin layer chromatography (TLC)

• Paper chromatography: stationary phase = water held in cellulose of filter paper (Whatman No. 1); mobile phase = solvent mixture (e.g. butanol : acetic acid : water 4:1:5). Spot sample on base line, develop by ascending/descending method in a closed chamber, dry, locate spots (ninhydrin for amino acids, iodine vapour, UV). R_f = distance moved by solute / distance moved by solvent front. Example: spot 3.6 cm, front 12 cm → R_f = 0.30.
• TLC: a thin layer (0.25 mm) of adsorbent (silica gel G, alumina, cellulose) on a glass/aluminium/plastic plate; mainly adsorption. Faster (minutes), sharper spots, more sensitive, corrosive sprays can be used. Used for plant pigments (chlorophylls, carotenoids), alkaloids, flavonoids, checking purity, monitoring reactions; preparative TLC for isolation.

Gas chromatography (GC)

Principle: gas–liquid partition. Mobile phase = inert carrier gas (N₂, He, H₂); stationary phase = a high-boiling liquid coated on a solid support in a packed column or on the inner wall of a long capillary column, kept in a temperature-controlled oven. Volatile components separate according to boiling point and affinity for the stationary phase and emerge at different retention times.

Parts: carrier gas cylinder and flow controller → injection port (heated) → column in oven → detector — flame ionisation detector (FID, for organics), thermal conductivity detector (TCD), electron capture detector (ECD, pesticides) or mass spectrometer (GC-MS) → recorder (chromatogram; peak area ∝ amount).

Uses in botany: essential oils (menthol, citral), fatty acid methyl esters, plant volatiles, pesticide residues.

Introduction to HPLC and HPTLC for secondary metabolite profiling

• HPLC (High Performance Liquid Chromatography): liquid mobile phase pumped at high pressure (up to several hundred bar) through a column packed with very fine particles (3–5 µm), usually reversed-phase C18 silica with water–methanol/acetonitrile mobile phase. Parts: solvent reservoirs → high-pressure pump → injector → column → detector (UV/PDA, fluorescence, mass spectrometer) → data system. Fast, high resolution, quantitative; works for non-volatile and heat-sensitive compounds — phenolics, flavonoids, alkaloids, terpenoids, vitamins in plant extracts.
• HPTLC (High Performance TLC): improved TLC using pre-coated plates with finer, uniform particles, automatic band application of exact volumes, controlled development chambers and densitometric scanning for quantification. Many samples run side by side on one plate; gives a chemical fingerprint — widely used for standardisation and authentication of herbal drugs (e.g. quantifying marker compounds).

Microtomy — introduction, types of microtomes and procedure

Microtomy is cutting very thin, uniform sections of tissue (usually 5–15 µm) with a microtome for microscopic study of internal structure (anatomy, histochemistry).

• Rotary microtome: most common; a hand wheel moves the paraffin block up and down against a fixed knife and advances it by a set thickness each turn; produces a continuous ribbon of serial sections.
• Rocking microtome (Cambridge type): simple and cheap; the block holder is on a rocking arm that swings in an arc against a fixed knife; sections are slightly curved; suitable for routine paraffin sections.
• Cryostat (freezing microtome): a microtome inside a refrigerated chamber (about −20 °C); fresh tissue is frozen and cut without embedding; quick (minutes); preserves enzymes, lipids and antigens — used for histochemistry and rapid diagnosis. (Sliding microtomes are used for hard woody tissues.)

Procedure (paraffin method): killing and fixation (FAA) → washing → dehydration (graded alcohol/TBA series) → clearing (xylene) → infiltration with molten paraffin wax (58–60 °C) → embedding in moulds to make blocks → trimming and mounting block on holder → section cutting (ribbon) → floating ribbons on warm water and fixing on slides with adhesive (Haupt's or Mayer's albumin) → drying → removal of wax with xylene → rehydration → staining (safranin–fast green) → dehydration → mounting in DPX → labelling.

Polymerase Chain Reaction (PCR) — principle and procedure; Real-Time PCR

PCR (Kary Mullis, 1983; Nobel Prize 1993) makes millions of copies of a specific DNA segment in vitro within a few hours.

Components: template DNA; two short primers (forward and reverse) flanking the target; heat-stable Taq DNA polymerase (from Thermus aquaticus); dNTPs (dATP, dGTP, dCTP, dTTP); Mg²⁺; buffer; thermal cycler.

Steps of one cycle:

1. Denaturation (94–95 °C, 20–30 s): double-stranded DNA separates into single strands.
2. Annealing (about 50–65 °C, depending on primer T_m): primers bind to complementary sequences.
3. Extension (72 °C): Taq polymerase adds nucleotides from the primers, making new strands (5′→3′).

25–35 cycles; ideally DNA doubles each cycle, so after n cycles about 2ⁿ copies (2³⁰ ≈ 10⁹). An initial denaturation and a final extension are added. Product checked by agarose gel electrophoresis.

Real-Time PCR (quantitative PCR, qPCR): amplification is measured during each cycle using fluorescence — a DNA-binding dye (SYBR Green) or sequence-specific probes (TaqMan). The Ct (threshold cycle) is the cycle at which fluorescence crosses a threshold; the more starting template, the lower the Ct. No gel needed; gives the amount of starting DNA. With reverse transcription (RT-qPCR) it measures gene expression and detects RNA viruses (e.g. COVID-19 test). Uses: GMO detection, pathogen detection in plants, gene expression studies.

Applications of PCR in botany: DNA barcoding, molecular markers (RAPD, SSR, ISSR), cloning genes, detection of plant pathogens and transgenes, phylogenetics.

Likely exam questions

1. (Long, 8 marks) Explain the principle of chromatography. Describe paper chromatography, TLC and gas chromatography.
2. (Long, 8 marks) Describe the principle and steps of PCR. Add a note on real-time PCR.
3. (Short, 4 marks) Write a note on HPTLC in herbal drug analysis.
4. (Short, 4 marks) Describe the rotary microtome.
5. (Short, 4 marks) What is a cryostat? Give its uses.