Microbiology Lab 2 Quiz

What is the purpose of each lab procedure in the multi-lab ferment project up to agarose gel electrophoresis (AGE)?

To isolate, identify, and analyze microorganisms from fermented samples using staining, DNA extraction, PCR, and gel electrophoresis.

How is each lab procedure in the multi-lab ferment project dependent on the previous one?

Each step builds on the last: cultures → Gram stain → DNA extraction → PCR → gel electrophoresis. Each provides information or material for the next.

What are the steps in performing a Gram stain and the purpose of each?

1. Crystal violet – primary stain

2. Iodine – mordant, binds dye

3. Alcohol – decolorizer (removes dye from Gram-negative)

4. Safranin – counterstain (colors Gram-negative pink)

How do you interpret Gram-stained specimens?

• Purple: Gram-positive (thick peptidoglycan)

• Pink: Gram-negative (thin wall + outer membrane)

• Observe shape (cocci/rods) and arrangement (chains/clusters

What is the difference between differential and selective media?

• Differential: distinguishes bacteria by biochemical traits (e.g., color change).

• Selective: supports some microbes but inhibits others.

What are the thermal cycling steps of PCR and their purpose?

1. Denaturation (94–95°C): DNA strands separate.

2. Annealing (50–65°C): Primers bind to target sequence.

3. Extension (72°C): Taq polymerase builds new DNA strands.

What are the necessary ingredients/reagents for PCR and their purpose?

Template DNA, primers, nucleotides (dNTPs), buffer, Taq polymerase, and Mg²⁺ ions — all needed for DNA amplification.

Why is the 16S rRNA gene used as the target for PCR amplification?

It’s highly conserved among bacteria but has variable regions that help identify species.

Why do primers bind to conserved regions of the 16S rRNA gene instead of variable regions?

Conserved regions allow binding across many bacteria; variable regions create unique sequences for identification.

What is the purpose of positive and negative controls in PCR?

• Positive control: ensures reagents and setup work correctly.

• Negative control: ensures no contamination or false positives.

What components are needed for PCR positive and negative controls?

• Positive: known bacterial DNA template.

• Negative: reaction without DNA (water instead).

What are the steps of agarose gel electrophoresis (AGE)?

1. Prepare gel with agarose and buffer.

2. Load DNA samples and ladder.

3. Apply electric current.

4. DNA moves through gel — smaller fragments move faster.

5. Stain and view under UV light

How does AGE separate DNA?

By size — smaller DNA fragments travel farther through the gel pores.

How is DNA visualized after AGE?

With a fluorescent stain (like ethidium bromide or GelRed) under UV light.

How do you interpret an AGE gel image after it’s run?

Compare DNA bands to a ladder (marker) to estimate size in base pairs; verify if expected bands appear.

What are the basic differences (advantages & limitations) between the four modern clinical microbiology techniques?

• Microscopy: fast, shows morphology but limited ID accuracy.

• Culture: confirms growth and drug sensitivity, but slow.

• Biochemical testing: identifies metabolic traits; needs pure culture.

• Molecular methods (PCR, sequencing): rapid and precise; more expensive and technical.