Jawaharlal Nehru Technological University Hyderabad B. Pharmacy II Year Pharmaceutical Microbiology Previous Question Paper (November/December - 2020 - R17)

Bacteria are prokaryotic microorganisms characterized by a simple cellular organization lacking a membrane-bound nucleus and organelles. Their ultra-structure comprises several essential components:

• Cell Envelope: Consists of the glycocalyx (capsule or slime layer), the cell wall (composed of peptidoglycan), and the cytoplasmic membrane, which regulates transport.
• Cytoplasm: Contains the nucleoid (circular DNA), ribosomes (70S type for protein synthesis), plasmids (extrachromosomal DNA), and inclusion bodies (storage granules).
• Appendages: Flagella provide motility, while pili and fimbriae facilitate attachment and genetic exchange.
• Endospores: Highly resistant, dormant structures formed by certain bacteria (e.g., Bacillus) to survive adverse environmental conditions.

Bacteria are classified morphologically based on their shape into the following categories:

• Cocci: Spherical or oval cells. Variations include diplococci (pairs), streptococci (chains), staphylococci (grape-like clusters), and tetrads (groups of four).
• Bacilli: Rod-shaped bacteria. These may appear as single rods, diplobacilli (pairs), or streptobacilli (chains).
• Spiral forms: Includes vibrios (comma-shaped), spirilla (rigid, helical shapes), and spirochetes (flexible, corkscrew-like organisms).
• Pleomorphic bacteria: Organisms that lack a constant shape and can alter their morphology based on environmental conditions.

A pure culture contains only one type of microorganism. Isolation is the process of separating a specific microbe from a mixed population, while preservation maintains the viability and genetic stability of the culture over time.

Isolation Methods:

• Streak Plate Method: A loopful of mixed culture is streaked over the surface of an agar plate in a pattern that dilutes the cells until individual colonies form.
• Pour Plate Method: The sample is serially diluted in molten agar (cooled to 45°C) and poured into petri dishes. Colonies develop both on the surface and within the medium.
• Spread Plate Method: A small volume of diluted sample is spread evenly over the surface of a pre-solidified agar plate using a sterile L-shaped glass rod.
• Serial Dilution Method: The sample is diluted in a series of sterile broth tubes until a single cell is statistically expected in the final dilution, which is then inoculated onto a medium.

Preservation Methods:

• Refrigeration (4°C): Cultures are stored on agar slants in a refrigerator. This slows metabolic activity, extending viability for weeks or months.
• Paraffin Method: Sterile liquid paraffin is poured over the culture slant to exclude oxygen and prevent dehydration, allowing storage for up to a year.
• Cryopreservation (-20°C to -80°C): Cultures are suspended in a cryoprotectant like glycerol or skim milk to prevent ice crystal damage and stored in deep freezers.
• Lyophilization (Freeze-drying): The culture is rapidly frozen and then dehydrated under a high vacuum. This removes water by sublimation, resulting in a stable powder that can be stored for years.
• Liquid Nitrogen Storage (-196°C): The most effective method for long-term storage, where cultures are kept in ampoules submerged in liquid nitrogen, maintaining viability for decades.