BIO221 Lab Material Review: Antimicrobial Methods, Biofilms, and Bacterial Genetics

Experiment 11: Antibiotic Disc Diffusion (Kirby-Bauer Method)

Principles of the Kirby-Bauer Method

The Kirby-Bauer disc diffusion method is a standardized technique used to evaluate the effectiveness of antibiotics against specific bacteria. It is widely used in clinical microbiology to determine bacterial susceptibility to antimicrobial agents.

• Purpose: To assess whether a bacterium is susceptible, intermediate, or resistant to various antibiotics by measuring zones of inhibition around antibiotic discs placed on an agar plate inoculated with the test organism.

• Parameters for Standardization:

  • Type and depth of agar medium (usually Mueller-Hinton agar, 4 mm deep)

  • Inoculum density (standardized to 0.5 McFarland turbidity standard)

  • Incubation time and temperature (typically 16-18 hours at 35°C)

  • Antibiotic disc potency

• Bacteriostatic vs. Bactericidal:

  • Bacteriostatic: Inhibits bacterial growth but does not kill the organism.

  • Bactericidal: Kills the bacteria directly.

• Determining Resistance: If the zone of inhibition is below the standard cutoff for a given antibiotic, the bacterium is considered resistant.

• Synergy and Antagonism:

  • Synergy: Two antibiotics work better together than alone (zones of inhibition may merge or increase).

  • Antagonism: One antibiotic interferes with the action of another (zones may flatten or decrease).

  • Therapeutic Use: Synergistic combinations may be preferred for treatment; antagonistic combinations should be avoided.

Example: Testing Staphylococcus aureus against penicillin and vancomycin to determine the best treatment option.

Experiment 11: Antibiotic MIC and MBC

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

MIC and MBC are quantitative measures of antibiotic effectiveness. MIC is the lowest concentration of an antibiotic that inhibits visible growth, while MBC is the lowest concentration that kills 99.9% of the original inoculum.

• MIC (Minimum Inhibitory Concentration): The smallest amount of antibiotic that prevents visible bacterial growth after overnight incubation.

• MBC (Minimum Bactericidal Concentration): The lowest concentration of antibiotic that results in microbial death (no growth when subcultured onto antibiotic-free media).

• Steps for Calculating MIC:

  1. Prepare serial dilutions of the antibiotic in broth.

  2. Inoculate each tube with a standardized bacterial suspension.

  3. Incubate overnight at 35°C.

  4. Observe for turbidity (growth) or clarity (no growth).

  5. The lowest concentration with no visible growth is the MIC.

• Units for MIC: Typically expressed in micrograms per milliliter (μg/mL).

• Determining MBC: Subculture from tubes with no visible growth onto antibiotic-free agar. The lowest concentration with no colony growth is the MBC.

• Mechanisms of Antibiotics: Antibiotics may target cell wall synthesis (e.g., penicillins), protein synthesis (e.g., tetracyclines), nucleic acid synthesis (e.g., fluoroquinolones), or metabolic pathways (e.g., sulfonamides).

Example: Determining the MIC and MBC of ampicillin against Escherichia coli using serial dilution and subculture methods.

Experiment 13: UV Radiation vs. Spore-formers

Effects of Ultraviolet (UV) Radiation on Bacteria

UV radiation, particularly at 260 nm, is effective at damaging bacterial DNA, leading to mutations and cell death. Spore-forming bacteria are more resistant to UV due to their protective spore coats.

• Spore-forming Bacteria: Commonly found in soil and environments with fluctuating conditions. Examples include Bacillus and Clostridium species.

• Differences from Other Bacteria: Spore-formers can survive harsh conditions, including UV exposure, due to their endospores.

• Mutations Caused by UV: UV induces thymine dimers in DNA, which can block replication and transcription.

• DNA Repair: Some bacteria can repair UV-induced damage via photoreactivation or nucleotide excision repair.

• Isolation in Lab: Heat or chemical treatments can be used to select for spore-formers, as spores survive conditions that kill vegetative cells.

Example: Exposing Bacillus subtilis and Escherichia coli to UV light and comparing survival rates.

Exercise 12: Biofilm Formation

Biofilm Development and Antimicrobial Resistance

Biofilms are structured communities of bacteria adhering to surfaces and embedded in a self-produced extracellular matrix. They are important in medical and environmental microbiology due to their resistance to antimicrobials.

• Steps of Biofilm Formation:

  1. Initial attachment of planktonic cells to a surface

  2. Irreversible attachment and microcolony formation

  3. Maturation of biofilm structure

  4. Production of extracellular polymeric substances (EPS)

  5. Dispersion of cells to new locations (sometimes considered part of maturation)

• Disinfectants vs. Antiseptics:

  • Disinfectants: Chemicals used to destroy microorganisms on non-living surfaces.

  • Antiseptics: Chemicals safe for use on living tissues to reduce microbial load.

• DNA Transfer in Biofilms: Horizontal gene transfer (e.g., conjugation, transformation) is facilitated within biofilms, contributing to antibiotic resistance.

• Bacteria in Biofilms: Examples include Pseudomonas aeruginosa, Staphylococcus epidermidis, and Streptococcus mutans.

• Persister Cells: Dormant variants of regular cells that are highly tolerant to antibiotics and contribute to chronic infections.

• Examples of Biofilms: Dental plaque, catheter-associated infections, and biofouling in water systems.

Example: Formation of dental plaque as a biofilm on teeth surfaces.

Experiment 14: Conjugation

Bacterial Conjugation and Horizontal Gene Transfer

Conjugation is a process of horizontal gene transfer in bacteria involving direct cell-to-cell contact, often mediated by a plasmid such as the F factor.

• Examples of Horizontal Gene Transfer: Conjugation, transformation, and transduction.

• F Factor: A plasmid (fertility factor) that carries genes for pilus formation and DNA transfer.

• Transconjugant Cell: A recipient cell that has acquired new genetic material via conjugation.

• Selective Media: McConkey agar or other selective media can be used to distinguish transconjugants from non-transconjugants based on antibiotic resistance or metabolic traits.

• Conjugation Procedure: Mix donor and recipient strains, incubate to allow mating, and plate on selective media to identify transconjugants.

Example: Transfer of antibiotic resistance genes from one Escherichia coli strain to another via conjugation.

Experiment 15: Transformation

Bacterial Transformation and Genetic Engineering

Transformation is the uptake of naked DNA from the environment by a bacterial cell, leading to genetic change. It is a key tool in molecular biology.

• Bacterial Transformation: The process by which bacteria take up foreign DNA from their surroundings.

• Plasmid Used: Commonly, the pGlo plasmid, which carries a gene for green fluorescent protein (GFP) and an antibiotic resistance marker.

• Making Competent Cells: Cells are treated with calcium chloride or other methods to make their membranes permeable to DNA.

• Induction of GFP: Arabinose in the growth medium activates the promoter for GFP expression in transformed cells.

Example: Transformation of E. coli with pGlo plasmid and selection on LB agar with ampicillin and arabinose.

Identifying Bacterial Unknowns

Microbiological Identification and Experimental Design

Identifying unknown bacteria is a fundamental skill in microbiology, requiring careful experimental design and aseptic technique.

• Hypothesis: A testable statement predicting the outcome of an experiment.

• Maintaining Pure Cultures: Use of aseptic technique, streak plating, and careful handling to avoid contamination.

• Consequences of Misidentification: Incorrect diagnosis and treatment in clinical settings, failure in industrial processes, or flawed research results.

• Strain Variation: Different strains of the same species may have varying biochemical or staining properties, complicating identification.

Example: Misidentifying Staphylococcus aureus as Staphylococcus epidermidis could lead to inappropriate antibiotic therapy.

Summary Table: Key Laboratory Methods and Their Purposes

Method	Main Purpose	Key Outcome
Kirby-Bauer Disc Diffusion	Test antibiotic susceptibility	Zone of inhibition indicates sensitivity/resistance
MIC/MBC Determination	Quantify antibiotic effectiveness	MIC: Inhibition; MBC: Bacterial death
UV Radiation	Assess bacterial survival and mutation	Spore-formers show higher resistance
Biofilm Formation	Study microbial communities	Biofilm structure and resistance
Conjugation	Horizontal gene transfer	Acquisition of new traits (e.g., resistance)
Transformation	Genetic engineering	Expression of new genes (e.g., GFP)
Unknown Identification	Determine bacterial species	Accurate diagnosis and research

Additional info: These topics correspond to core laboratory techniques and concepts in college-level microbiology, including antimicrobial testing, microbial genetics, and identification methods.