Biochemical and Analytical Techniques for Biological Analysis

Biochemical & Analytical Techniques

Unit I — General Principles of Biological/Biochemical Analysis

pH: The pH scale quantifies the concentration of hydrogen ions (H⁺) in a solution, indicating its acidity or basicity. It ranges from 0 to 14. A pH value below 7 signifies an acidic solution, characterized by a higher concentration of H⁺ ions. A pH above 7 indicates a basic (alkaline) solution, with a lower concentration of H⁺ ions and a higher concentration of hydroxide ions (OH⁻). A pH of exactly 7 represents a neutral solution, where the concentrations of H⁺ and OH⁻ are equal, as in pure water.

pH testing is commonly used in forensic science to examine fluids, soils, chemical traces, and bodily substances found at crime scenes.

Buffer Solution

A buffer is a solution that minimizes changes in pH upon the addition of small amounts of a strong acid or base. It typically consists of a weak acid and its conjugate base, or a weak base and its conjugate acid. This composition allows the buffer to neutralize added H⁺ or OH⁻ ions, maintaining the solution’s pH within a narrow range. A buffer system must consist of either a weak acid and its conjugate base or a weak base and its conjugate acid. The selection of the acid–base pair is based on the target pH of the buffer solution.

Physiological Solution

Physiological solution: A chemically prepared, isotonic solution designed to provide the essential ionic environment and nutritional support required to maintain the viability of isolated tissues or organs during experimental procedures. It mimics the inorganic composition of blood plasma, offering a balanced mixture of cations (e.g., Na⁺, K⁺, Ca²⁺, Mg²⁺), anions (e.g., Cl⁻, HCO₃⁻, SO₄²⁻, H₂PO₄⁻), and glucose dissolved in distilled water. It also incorporates a buffer system (often bicarbonate-based or phosphate-based) to help maintain a physiological pH (~7.4).

Cell Culture

Cell culture refers to the process of growing animal or plant cells in a controlled, artificial environment outside their natural biological context. Cells used for culture may be directly isolated from tissues and enzymatically or mechanically disaggregated, or they may originate from an established cell line or cell strain. The success of cell culture depends on several factors:

• A suitable culture vessel providing a sterile, supportive surface or suspension environment.
• A nutrient-rich medium containing essential components such as:
  • Amino acids, carbohydrates, vitamins, and minerals
  • Growth factors and hormones specific to the cell type
• Controlled physicochemical conditions, including:
  • Temperature (typically 37 °C for mammalian cells).
  • pH stability, often maintained by CO₂-dependent bicarbonate buffering.
  • Osmotic pressure compatible with intracellular conditions.
  • Adequate gas exchange (mainly O₂ and CO₂).

Tissue Culture

Tissue culture is the in‑vitro cultivation of plant cells, tissues, or organs on a specially formulated nutrient medium under sterile and controlled conditions. This technique enables the growth and development of plant or animal material outside the parent organism, and under appropriate conditions.

Cell Fractionation

Cell fractionation is a laboratory technique used to rupture cells and separate their internal components for detailed examination of their structure, chemical makeup, and function. It is commonly used in cell biology and biochemistry to isolate organelles such as the nucleus, mitochondria, lysosomes, and ribosomes. To prevent damage and maintain the integrity of these organelles, the entire process is carried out in a cold, isotonic medium, which helps preserve osmotic balance and enzyme activity. The method consists of three main steps:

• Extraction: Cells are collected and suspended in a cold, isotonic buffer solution that maintains the stability of organelles during processing.
• Homogenization: The suspended cells are physically broken open using mechanical methods such as a blender, tissue grinder, or sonicator. This releases the internal components into the buffer, forming a uniform mixture called the homogenate.
• Centrifugation: The homogenate is subjected to differential centrifugation, where it is spun at increasing speeds to separate organelles based on their size and density. Heavier components like nuclei sediment at lower speeds, while smaller organelles like mitochondria and ribosomes are pelleted at higher speeds.

Biological Variations

Biological variation refers to the natural differences that exist between individuals of the same species. These differences can occur in physical traits, behavior, or genetic makeup, and they are important for diversity within a population. One major type of biological variation is phenotypic variation, which is the observable differences in characteristics (such as height, eye color, or blood type) between individuals. Phenotypic variation can be influenced by genetic factors, environmental factors, or a combination of both. Phenotypic variation can be categorized into two main types:

• Continuous variation: This type involves a wide range of small differences in a particular trait. The variation is gradual and usually measurable on a scale, such as height, body mass, or finger length. There are many possible values, and individuals do not fall into clearly defined groups. These traits are often influenced by multiple genes (polygenic inheritance) and can be affected by environmental factors.
• Discontinuous variation: This type includes traits with distinct, separate categories and no intermediate values. For example, blood group (A, B, AB, or O), sex (male or female), and the ability to roll the tongue are all discontinuous traits. These characteristics are usually controlled by a single gene and are less influenced by the environment.