Microbiology Lab Practical 2 Study Guide: Antibiotic Resistance, Biofilms, DNA Damage & Repair, and Genetic Exchange

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Antibiotic Resistance and Susceptibility Testing

Antibiotic Resistance

Antibiotic resistance occurs when bacteria evolve mechanisms to withstand the effects of antibiotics, often due to misuse or overuse of these drugs.

Bacteriostatic: Inhibits bacterial growth without killing the organisms.
Bactericidal: Kills bacteria directly.
Example: Prescribing antibiotics for viral infections can promote resistance.

Minimum Inhibitory and Bactericidal Concentrations

MIC (Minimum Inhibitory Concentration): The lowest concentration of an antibiotic that inhibits visible growth of a microorganism.
MBC (Minimum Bactericidal Concentration): The lowest concentration of an antibiotic that kills 99.9% of the original inoculum.
If MBC is close to MIC, the agent is bactericidal; if much higher, the agent is bacteriostatic.

Antibiotic Dilution Example

Serial dilutions of antibiotics are used to determine MIC by observing the lowest concentration that prevents visible growth.

Disk Diffusion (Kirby-Bauer) Method

This method tests bacterial sensitivity to antibiotics by measuring the zone of inhibition around antibiotic disks on an agar plate.

Requires standardization (Mueller-Hinton agar and McFarland standard).
Cannot distinguish between bacteriostatic and bactericidal effects.
Only determines efficacy for the tested bacteria-antibiotic combination.

Standardization Factors

Sensitivity of the organism
Antimicrobial molecule size and concentration
Density of culture
Diffusion rate of agent
Incubation time and temperature
Size of inoculum
Interactions

Biofilm Formation

Biofilms

Biofilms are structured communities of bacteria encased in a self-produced matrix of polysaccharides, proteins, lipids, and DNA. They are common in environments with excess nutrients.

Planktonic cells: Free-floating bacteria.
Sessile cells: Bacteria attached to a surface.

Stages of Biofilm Development

Surface attachment: Planktonic cells attach to a surface.
EPS matrix production: Cells secrete extracellular polymeric substances (EPS) to anchor themselves.
Maturation: Biofilm grows and develops complex structures.
Dispersion: Cells leave the biofilm to colonize new surfaces.
Quorum sensing: Cell-to-cell communication regulates gene expression and biofilm behavior.

Disinfectants, Antiseptics, and DNA Damage

Disinfectants vs. Antiseptics

Disinfectants: Used on inanimate objects to destroy bacterial cell membranes and interfere with metabolism.
Antiseptics: Used on living tissue; must be less toxic and are often broad-spectrum.

Chemical-Induced DNA Damage

Deaminating agents: Remove amino groups from nucleotides, causing mutations (e.g., cytosine to uracil).
Cytosine deamination: Converts cytosine to uracil, leading to mutations in subsequent DNA replications.

Bacterial DNA Repair Mechanisms

Base Excision Repair (BER)

BER is a cellular mechanism that repairs damaged DNA throughout the cell cycle by removing small, non-helix-distorting base lesions from the genome.

UV Radiation and DNA Damage

UV Radiation

UV light (especially UVC, 100–280 nm) is used to kill microbes by inducing DNA damage.
Optimal germicidal wavelengths: 260–265 nm.

Mechanisms of UV-Induced DNA Damage

Formation of photoproducts such as thymine dimers and thymine 6-4 photoproducts.
These lesions block DNA replication and transcription.

Types of DNA Damage

Thymine dimer: Covalent bond between adjacent thymines on the same DNA strand, blocking DNA polymerase.
Thymine 6-4 photoproduct: Dimer between carbons 6 and 4 of adjacent thymines, distorting the DNA backbone.

Bacterial Protection Mechanisms

Formation of endospores (highly resistant structures in Gram-positive bacteria such as Bacillus and Clostridium).
DNA repair mechanisms (e.g., photoreactivation, excision repair).

Endospores

Formation and Structure

Endospores are dormant, tough, and non-reproductive structures produced by certain bacteria to survive extreme conditions.

Endospore Formation Cycle

Initiated by nutrient deprivation or environmental stress.
Involves asymmetric cell division, engulfment, cortex and coat formation, and maturation.
Endospore is released when the mother cell lyses.

Types of Endospores

Endospore Shape	Cell Deformation	Endospore Position
Spherical	None	Central
Oval	Swollen	Subterminal
Oval	None	Terminal
Additional info: …	…	…

Endospore Structure

Structure	Function
Spore Coat	High protein content; enzymatic and chemical resistance
Cortex	Peptidoglycan-rich; high-temperature resistance
Outer/Inner Membranes	Permeability barriers
Core	Contains DNA, ribosomes, and dipicolinic acid for heat resistance
Small acid-soluble proteins (SASP)	Bind DNA, protect from UV and desiccation

DNA Damage Repair Mechanisms

Photoreactivation: Uses visible light to activate photolyase, which breaks thymine dimers.
Nucleotide Excision Repair (NER): Removes damaged DNA segments and fills the gap using the undamaged strand as a template.

Bacterial Genetic Exchange

Conjugation

Conjugation is the direct transfer of DNA from one bacterium to another via cell-to-cell contact, typically mediated by a plasmid (e.g., F plasmid).

Vertical gene transfer: Parent to offspring.
Horizontal gene transfer: Between unrelated cells.
Plasmids: Small, circular DNA molecules that replicate independently of the chromosome.

Example: F Plasmid Transfer

F+ cell (donor) transfers F plasmid to F- cell (recipient), making it F+.
Plasmids may carry genes for antibiotic resistance, virulence, or metabolism.

Summary Table: Growth on Selective Media

Antibiotic	F-	F+	TC
Cat	Growth, pink	No growth	*Growth, pink
Kan	No growth	Growth, yellow	**Growth, yellow
Cat + Kan	No growth	No growth	**Growth, pink/yellow
Additional info: Growth mostly F+ cells, very few TC (all pink); Growth mostly F+ cells (yellow), very few TC (bits of pink); Growth: TC cells only (all pink)

Transformation

Transformation is the uptake of free DNA from the environment by a bacterium. Cells must be competent (naturally or chemically induced) to take up DNA.

Competence can be induced by CaCl2 treatment and heat shock.
Plasmids must provide a selective advantage (e.g., antibiotic resistance) to be maintained.

Example: pGlo Transformation

pGlo plasmid contains a gene for green fluorescent protein (GFP) under the control of the arabinose promoter.
Only cells that receive the plasmid and are grown with arabinose will glow under UV light.

Results Interpretation

Plates without plasmid: No growth in presence of ampicillin.
Plates with plasmid: Growth in ampicillin; glowing colonies only with arabinose present.

Additional info: These topics correspond to Ch. 7 (Control of Microbial Growth), Ch. 8 (Microbial Genetics), Ch. 9 (Biotechnology & DNA Technology), and Ch. 20 (Antimicrobial Drugs) in standard Microbiology curricula.