Microbiology Lab: Midterm 2

What is a smear?

A thin layer of bacteria placed on a slide.

What happens if a smear prepared is not a thin layer of cells?

It can lead to poor staining, cell overlap or clumping, obscured cell morphology, or blocking light transmission under the microscope.

Why do we air dry a slide?

To prevent bacteria to be aerosolized and to prevent splatter.

Describe smear preparation.

1. Smear culture in a thin film across the slide

2. Let slide air dry

3. Pass slide through fire to heat fix

What is heat-fixing?

Pass slide through fire to kill bacteria and fix it to the slide.

Describe a simple stain.

Use a basic dye, meaning positively charged, to stain the slide for 1 minute. Choose 1 of 3 basic dyes: Safranin, Methylene Blue, or Crystal Violet. Then rinse with water.

What is the goal of a Gram Stain?

To see 2 types of bacteria: Gram-Positive and Gram-Negative bacteria.

Describe the Gram-Staining technique.

1. Primary Stain

2. Mordant

3. Decolorizing Agent

4. Counterstain

What is the most crucial step of a Gram Stain?

Using acetone alcohol so Gram-Negative bacteria lose their color. Otherwise, they will stay purple and indistinguishable from Gram-Positive bacteria.

What is Spore Staining mainly used for?

It is used mainly for the Bacillus genus.

Why is Bacillus a virulent bacteria?

1. Before these bacteria die, they can form endospores, which can stay dormant for months to years before they become active again.

2. They are highly resistant to environmental conditions, like heat, cold, radiation, dehydration, and detergents.

Why is the Spore Staining method used?

Bacillus have thick spore coats, so they are difficult to stain with traditional methods. Steam is used as mordant so the primary stain can penetrate the endospore.

Which dyes are used for Spore Staining?

Malachite Green stains the spore and Safranin stains the cell pink.

Describe Capsule Staining.

Use an acidic dye (Congo Red or Nigrosin) and a basic dye. The capsule will appear as a halo around the bacterial cell.

Why do capsules add to a bacteria’s virulence?

It makes it hard for the immune system to eliminate the bacteria.

Why is no heat-fixing required for Capsule Staining?

Capsules can shrink, collapse, or dislodge with heat, which would make them invisible to us.

Describe cardinal temperatures.

1. Minimum Temperature: The lowest temperature at which growth occurs.

2. Optimal Temperature: The temperature at which growth is fastest.

3. Maximum Temperature: The highest temperature at which growth is still possible before heat causes damage or denaturation.

Define psychrophile.

A microorganism that thrives in cold environments (0-20°C)

Define mesophile.

A microorganism that grows best in moderate temperatures (20-45°C)

Define thermophile.

A microorganism that thrives at high temperatures (45-80°C)

Define hyperthermophile.

A microorganism that thrives in extremely high temperatures (above 80°C)

Define acidophile.

Bacteria that like acidic environments (pH lower than 7)

Define neutrophile.

Bacteria that like neutral environments (pH of 7)

Define alkiliphile.

Bacteria that like basic environments (pH greater than 7)

Antiseptic vs. Disinfectant

Antiseptics, like alcohol, are safe for human use, versus disinfectants, which are used for objects.

Transient vs. Resident Bacteria

Transient bacteria are temporary bacteria acquired from the environment that can be washed off, while resident bacteria are part of your natural flora and are non-pathogenic and usually beneficial to you.

How does UV light inhibit the growth of microorganisms?

UV light damages the bacteria’s DNA causing mutations that eventually affect protein production and kill the bacteria.

What is the most lethal UV wavelength?

260 nm

Describe the Kirby-Bauer method.

Antibiotic disks are applied on a bacterial lawn to see whether the bacteria is susceptible to an antibiotic. A clear zone of inhibition forms around the disk if it is.

Why do we use MH agar plates?

MH agar minimizes variability, allowing for reliable comparisons of antiobiotics.

Define antibiotics.

Antibiotics are chemical substances, naturally produced by microorganisms or synthetically made, that kill or inhibit the growth of bacteria. They are a subset of antimicrobials that specifically target bacteria. Antibiotics are ineffective against viruses, fungi, and parasites.

Define antimicrobials.

Antimicrobials are agents that kill or inhibit the growth of microorganisms, including bacteria, viruses, fungi, and parasites.

Define bactericidal.

An antimicrobial agent that kills bacteria rather than just inhibiting their growth.

Define bacteriostatic.

An antimicrobial agent that inhibits the growth and reproduction of bacteria, but does not directly kill them.

Describe broad versus narrow spectrum antibiotics.

Broad-spectrum antibiotics target a wide range of bacteria (both Gram-positive and Gram-negative species). Narrow-spectrum antibiotics target a specific group of bacteria, usually either Gram-positive or Gram-negative, or even a single bacterial species.

Describe serial dilution.

A technique used to dilute a bacterial culture step-by-step in a series of defined concentrations.

Describe the pour plate technique.

A method used to isolate and enumerate microorganisms in a sample by embedding the sample within agar medium.

What do dilution factors mean?

Dilution factors are a way of expressing the degree to which a sample has been diluted in a series of dilutions, often to achieve a desired concentration or to make measurements more manageable.

How do you calculate dilution factor?

Formula:

Dilution Factor = Volume of the sample / Total volume after dilution

How do you calculate bacterial concentration?

Formula:

Bacterial Concentration (CFU/mL) = (# of colonies / Volume plated) x Dilution Factor