Unit 1: Microbes

Models of Virus (T Phage) and Replication Cycles

This practical involves observing photographs or models to understand virus structure and life cycles.

T Phage (Bacteriophage)

A bacteriophage is a virus that infects bacteria. The T-phages (e.g., T4 phage) are known for their complex structure.

[Image of T4 Phage structure labeled]

• Head (Capsid): A protein coat (icosahedral) that encloses the genetic material (DNA).
• Collar: Connects the head to the tail.
• Tail Sheath: A contractile sheath that contracts to inject the DNA into the host bacterium.
• Base Plate: A hexagonal plate at the bottom of the tail.
• Tail Fibers: Long fibers that recognize and attach to specific receptors on the bacterial cell wall.
• Tail Pins: Short spikes on the base plate that anchor the phage to the host cell.

Lytic Cycle (Virulent Cycle)

This cycle results in the death (lysis) of the host cell and the release of new viruses.

[Image of Lytic Cycle of a Bacteriophage]

1. Attachment (Adsorption): The phage uses its tail fibers to attach to a specific receptor on the host bacterial cell wall.
2. Penetration (Injection): The tail sheath contracts, and the phage injects its DNA into the host's cytoplasm. The empty capsid remains outside.
3. Biosynthesis: The host cell's metabolic machinery (ribosomes, enzymes) is "hijacked" by the viral DNA. It stops making bacterial components and starts making viral components (viral DNA, capsid proteins).
4. Maturation (Assembly): The new viral components are assembled into complete, new phages (virions).
5. Lysis (Release): The phage produces an enzyme (like lysozyme) that breaks down the bacterial cell wall. The cell bursts (lyses), releasing hundreds of new phages to infect other cells.

Lysogenic Cycle (Temperate Cycle)

In this cycle, the viral DNA integrates into the host's chromosome and is replicated along with it, without immediately killing the cell.

[Image of Lysogenic Cycle of a Bacteriophage]

1. Attachment & Penetration: Same as the lytic cycle.
2. Integration: The viral DNA integrates itself into a specific site on the host's circular chromosome. The integrated viral DNA is now called a prophage.
3. Replication: The host cell lives and reproduces normally (e.g., by binary fission). Every time it divides, it copies the prophage along with its own DNA, passing it to its daughter cells. The bacteria containing a prophage is called a lysogenic bacterium.
4. Induction: The prophage can remain dormant for many generations. However, if the host cell is stressed (e.g., by UV light or chemicals), the prophage can "excise" (cut itself out) from the host chromosome and enter the lytic cycle, leading to biosynthesis, assembly, and lysis.

Study of Bacterial Reproduction

This practical involves observing prepared slides or photographs of different modes of bacterial reproduction.

Binary Fission (Asexual)

[Image of Binary Fission in a bacterium]

This is the most common form of bacterial reproduction. It is a simple asexual process.

1. The single, circular bacterial chromosome replicates.
2. The cell elongates, and the two chromosomes move to opposite ends (poles) of the cell.
3. A new cell wall and membrane (a septum) forms down the middle.
4. The cell divides into two identical daughter cells. This process can be very fast (e.g., every 20 minutes for E. coli).

Conjugation (Sexual-like Recombination)

This is not true sexual reproduction but a method of horizontal gene transfer. It involves the transfer of genetic material (usually a plasmid) from one bacterium to another through direct contact.

• A donor cell (F+) produces a sex pilus, a thin protein tube.
• The pilus attaches to a recipient cell (F-) and draws the two cells together.
* A conjugation bridge forms.
• The F+ cell copies its F-plasmid (Fertility plasmid) and transfers one copy to the F- cell.
• The F- cell receives the plasmid and becomes an F+ cell.

Endospores

This is not a form of reproduction but a survival mechanism. Some bacteria (like *Bacillus* and *Clostridium*) form endospores when environmental conditions are unfavorable (e.g., no food, high heat, drought).

• The bacterium copies its chromosome and surrounds it with a tough, multi-layered protective coat.
• The outer cell disintegrates, releasing the endospore.
* The endospore is dormant and extremely resistant to heat, desiccation, chemicals, and radiation. * It can survive for many years. When conditions become favorable again, the endospore germinates back into a vegetative (active) bacterial cell.

Gram Staining Technique (Curd/Root Nodules)

Objective

To differentiate bacteria into two main groups, Gram-positive and Gram-negative, based on differences in their cell wall structure.

Principle

• Gram-positive bacteria: Have a thick peptidoglycan cell wall. This wall traps the Crystal Violet-Iodine complex, resisting decolorization, and the cells remain purple.
• Gram-negative bacteria: Have a thin peptidoglycan wall *and* an outer lipid membrane. The alcohol-acetone decolorizer dissolves this outer membrane and washes out the Crystal Violet-Iodine complex. The cells become colorless and then take up the pink/red counterstain (Safranin).

[Image of Gram positive vs Gram negative cell wall staining mechanism]

Materials

• Fresh curd (for *Lactobacillus*) or crushed root nodules (for *Rhizobium*).
• Glass slides, inoculating loop, spirit lamp.
• Microscope.
• Reagents: Crystal Violet (primary stain), Gram's Iodine (mordant), 95% Ethanol or Acetone-Alcohol (decolorizer), Safranin (counterstain).

Procedure

1. Prepare Smear: Place a small drop of water on a clean slide. Mix a tiny amount of curd or crushed nodule material into the water. Spread it thinly.
2. Heat Fix: Allow the smear to air dry. Pass the slide (smear side up) quickly through a flame 2-3 times. This kills the bacteria and sticks them to the slide.
3. Primary Stain: Flood the smear with Crystal Violet. Let it stand for 1 minute. Wash with water. (All cells are purple).
4. Mordant: Flood the smear with Gram's Iodine. Let it stand for 1 minute. Wash with water. (Iodine forms a complex with Crystal Violet; all cells are still purple).
5. Decolorization: This is the critical step. Add the Alcohol/Acetone drop by drop until the purple color stops running off (usually 10-20 seconds). Immediately wash with water. (Gram+ cells stay purple; Gram- cells become colorless).
6. Counterstain: Flood the smear with Safranin. Let it stand for 30-60 seconds. Wash with water.
7. Dry and Observe: Blot the slide dry (do not wipe). Observe under the microscope, starting with low power and moving to the 100x oil immersion lens.

Observations

• Gram-positive (*Lactobacillus* from curd): Cells appear purple or violet. They are typically rod-shaped.
• Gram-negative (*Rhizobium* from root nodules): Cells appear pink or red. They are rod-shaped.

Preparation of Media (Nutrient Agar and Broth)

Nutrient Broth

A liquid medium used for growing general, non-fastidious bacteria.

Typical Composition (per 1000 mL):

• Peptone (protein digest): 5.0 g
• Beef Extract (source of vitamins, minerals): 3.0 g
• NaCl (for osmotic balance): 5.0 g
• Distilled Water: 1000 mL

Nutrient Agar

A solid medium used for growing bacteria as colonies. It is simply Nutrient Broth with a solidifying agent added.

Typical Composition (per 1000 mL):

• Nutrient Broth components (as above)
• Agar: 15.0 g

Procedure for Preparation

1. Weigh: Accurately weigh all the components (e.g., for 100 mL, use 1/10th of the amounts).
2. Dissolve: Add the components to a flask containing the distilled water. For agar, you must heat gently (e.g., on a hot plate or in a water bath) while stirring until the agar is completely dissolved. The solution will become clear.
3. Adjust pH: Cool the medium slightly and check the pH. Adjust to a neutral pH (~7.0-7.2) using 0.1M HCl (to lower) or 0.1M NaOH (to raise).
4. Dispense: Pour the medium into final containers (e.g., test tubes for broths or slants, or keep in the flask for pouring plates).
5. Sterilize: Plug the flasks/tubes with non-absorbent cotton. Sterilize in an autoclave at 121°C and 15 psi (pounds per square inch) pressure for 15-20 minutes. This kills all bacteria and endospores.
6. Cooling & Storage:
   • Broth: Can be stored after sterilization.
   • Agar Plates: After autoclaving, let the flask cool to about 45-50°C (warm to the touch) and pour into sterile Petri dishes. Let them solidify.