LAB PRACTICAL 2

What is the process by which bacteria take up free, extracellular DNA from their environment?

Bacterial transformation.

What is a competent bacterium?

A bacterium that is in a physiological state capable of taking up DNA.

What common reagent is used to prepare competent cells for transformation?

CaCl₂.

What is the purpose of heat shock in bacterial transformation?

It creates a thermal imbalance that drives DNA into the cell.

What key plasmid is often used in transformation experiments?

pGLO.

What does the gene ampR on the pGLO plasmid provide?

Ampicillin resistance.

What is the role of GFP in the pGLO plasmid?

It serves as a reporter gene.

What is special about the E. coli MM294 strain?

It is highly transformable and lacks restriction enzymes.

How does CaCl₂ facilitate transformation?

It provides Ca²⁺ ions that neutralize charges and allow DNA binding.

Why is ampicillin included in the medium during transformation?

To select for cells that have taken up the plasmid containing ampR.

What does a positive result look like in the experimental control plates of a transformation lab?

Growth in the +DNA LB/Amp plate.

What are the characteristics of UV radiation?

UV is nonionizing radiation.

What type of DNA damage does UV radiation cause?

Pyrimidine dimers, particularly thymine dimers.

What UV wavelength is most damaging to DNA?

UV-C, specifically between 200–280 nm, with maximum absorption at 260 nm.

What is one mechanism that repairs UV-induced DNA damage?

Nucleotide excision repair.

Which two bacterial species are commonly used in UV sensitivity experiments?

Bacillus subtilis and Staphylococcus epidermidis.

What is the effect of keeping petri dish lids on during UV exposure?

It blocks UV radiation, preventing lethal effects.

What are standard conditions used in Kirby-Bauer testing?

Mueller-Hinton agar and 0.5 McFarland inoculum, incubated at 35°C for 16–18 hours.

What is the purpose of inoculum in the Kirby-Bauer test?

To create a uniform bacterial lawn.

How is the zone of inhibition defined?

The clear area around an antibiotic disk where bacteria cannot grow.

What factors can affect the size of the zone of inhibition?

Antibiotic diffusion rate, potency, bacterial susceptibility, agar composition, and inoculum density.

How do you measure the zone of inhibition?

By measuring the diameter in millimeters (mm).

How are the results of the Kirby-Bauer test interpreted?

By comparing the measured diameter to CLSI interpretive tables.

What does 'S', 'I', and 'R' stand for in antibiotic susceptibility testing?

'S' stands for susceptible, 'I' for intermediate, and 'R' for resistant.

How do you determine effective antimicrobials for treatment?

By choosing antibiotics where the organism is designated as susceptible (S) according to the chart.

What does PCR stand for?

Polymerase Chain Reaction.

What is the purpose of PCR?

To amplify a specific DNA sequence.

Name the three main events of PCR.

Denaturation, annealing, and extension.

What temperature is denaturation performed at during PCR?

Approximately 95°C.

What role does Taq polymerase play in PCR?

It synthesizes new DNA during the extension phase.

What is the function of primers in PCR?

To define the target region and provide a free 3' OH for DNA synthesis.

How can mecA be identified on a gel?

By looking for a band at approximately 310 bp compared to a DNA ladder.

What is the size of the mecA PCR product band?

Approximately 310 bp.

What are two advantages of PCR over the Kirby-Bauer method for MRSA detection?

PCR is fast and sensitive.

What is one disadvantage of the Kirby-Bauer method compared to PCR?

It is slower and less specific.

What are the characteristics of Staphylococci?

Gram-positive cocci that appear in clusters and are non-motile.

What type of broth is m-Staphylococcus Broth?

Selective due to high salt concentration.

What is mannitol salt agar (MSA) used for?

It is selective for Staphylococci and differentiates based on mannitol fermentation.

What type of hemolysis is indicated by a green color on blood agar?

Alpha (α) hemolysis.

Which toxin primarily causes beta hemolysis in S. aureus?

Alpha-toxin (α-hemolysin).

What does a positive coagulase test indicate?

Clot formation in the test sample.

What is staphyloxanthin?

A golden carotenoid pigment in S. aureus that protects against oxidative damage.

What appearance distinguishes S. aureus on MSA?

Yellow colonies with a yellow halo.

What coagulase result is expected for S. aureus?

Positive for coagulase.

Which tests are major for distinguishing Staphylococci?

Coagulase test, mannitol fermentation, and novobiocin susceptibility.

What are the characteristics of Streptococci?

Gram-positive cocci that appear in chains or pairs and are catalase-negative.

What is blood agar categorized as?

Enriched and differential due to hemolysis patterns.

What differentiates Staph from Strep?

The catalase test; Staph is positive, Strep is negative.

What tests are used for identifying Streptococci and Enterococci?

Hemolysis pattern, bacitracin sensitivity, optochin sensitivity, bile esculin, and salt tolerance.

How does a positive bile esculin test appear?

The medium turns black.

What does a positive salt tolerance test look like?

Turbidity in the broth.

What is the general purpose of biochemical tests in microbiology?

To determine metabolic capability.

What is the purpose of ELISA?

To detect antigens or antibodies through antigen-antibody binding.

What is the primary difference between direct and indirect ELISA?

Direct ELISA uses an enzyme-linked primary antibody while indirect uses a secondary antibody.

What is the role of known antigen in indirect ELISA?

To capture specific antibodies from the patient serum.

What does a positive indirect ELISA result look like?

A strong color change.

What is the follow-up action after a positive ELISA?

Perform a confirmatory test like Western blot or PCR.