Biotechnology Talks by Anu Abhimannue

  Light Microscopy for Observing Bacterial Morphology Using Permanent Slides   Objective: To observe and identify the morphological characteristics of bacteria (such as shape and arrangement) using a compound light microscope and permanent stained slides .   Principle: Light microscopy uses visible light to magnify images of small specimens. In bacterial studies, staining techniques (e.g., Gram staining) enhance contrast, allowing for better observation of morphology —such as coccus , bacillus , spirillum , and their arrangements (e.g., diplo-, strepto-, staphylo-).   Materials Required: ·          Compound light microscope ·          Permanent bacterial slides ·          Immersion oil (for 100× objective) ·          Lens cleaning tissue ·        ...

  SEPARATION OF COMPOUNDS FROM A MIXTURE USING TLC Objective: To separate and identify individual compounds from a given mixture using Thin Layer Chromatography (TLC) technique. Principle: Thin Layer Chromatography is based on the differential adsorption of compounds on a stationary phase and their differential solubility in a mobile phase. ·          The stationary phase is a thin layer of adsorbent (e.g., silica gel or alumina) on a glass or plastic plate. ·          The mobile phase is a solvent or solvent mixture that moves up the plate via capillary action. ·          Compounds move at different rates depending on their polarity and affinity to the stationary and mobile phases. Materials Required: ·          TLC plate (silica gel-coated) ·          Developi...

  Spectrophotometry – Basic Instrumentation Objective: To understand the basic principle of spectrophotometry and the working of a UV-Vis spectrophotometer by measuring absorbance of a colored solution at a specific wavelength.   Principle: Spectrophotometry is based on the principle that molecules absorb light at specific wavelengths. The amount of light absorbed is proportional to the concentration of the solution (Beer-Lambert Law). Beer-Lambert Law : A = ε c l Where:   A = Absorbance ,  ε =  Molar absorptivity (L·mol⁻¹·cm⁻¹),  c  = Concentration (mol·L⁻¹),  l  =  Path length (cm)   Materials Required: ·          UV-Visible Spectrophotometer ·          Cuvettes (quartz or plastic, depending on wavelength) ·          Distilled water ·          Colored solution (...

  PREPARATION OF BUFFER & pH ANALYSIS   A buffer solution resists changes in pH upon the addition of small amounts of acid or base. It typically consists of a weak acid and its conjugate base or a weak base and its conjugate acid. Common buffer systems       §   Acetic acid/acetate (pH ~ 4.76) – acidic buffer §   Phosphate buffer (pH ~ 7.2) – neutral buffer §   Ammonium/ammonia (pH ~ 9.25) – basic buffer Henderson-Hasselbalch Equation is used to calculate the ratio of acid and base: pH=pKa+log⁡((A−)/(HA)), where: [A−] = concentration of conjugate base [HA] = concentration of weak acid   1.       PREPARATION OF 0.1 M TRIS-HCL BUFFER Objective: To prepare 0.1 M Tris-HCl buffer at pH 7.4 and verify its pH Theory: Tris is a weak base. When mixed with HCl, it forms the conjugate acid (Tris-HCl), creating a buffer. The pKa of Tris is ~8.1 at 25°C, making it ideal for buffers in the pH 7–9 r...