Practice Questions For Microbiology

List of (9) Agar mediums and what they reveal:

• TSA (Tryptic Soy Agar): A general-purpose medium used to grow many types of bacteria.

• BHI (Brain Heart Infusion): A nutrient-rich medium used to grow bacteria that need extra nutrients.

• PDA (Potato Dextrose Agar): Used mainly to grow fungi, including molds and yeasts.

• LB (Luria-Bertani): Commonly used to grow bacteria such as E. coli.

• Blood agar: Used to grow many bacteria and determine whether they break down red blood cells.

• EMB (Eosin Methylene Blue): Selects mainly for Gram-negative bacteria and helps distinguish lactose fermenters.

• MacConkey agar: Selects mainly for Gram-negative bacteria and distinguishes lactose fermenters, which appear pink. (Lactose Fermentation is an anaerobic process)

• Mueller-Hinton agar: Used for standardized antibiotic susceptibility testing.

• Hektoen Enteric (HE) agar: Gram-negative enteric bacteria (naturally live inside the intestinal tract)

What are 2-3 common mistakes that could make a gram stain less reliable?

1) Incorrect Decolorization - too much decolorizer (ethanol) is used, causing the Gram-positive cells to look pink; or too little can leave the Gram-negative cells purple
2) Smear is too thick - clumped cell stain and uneven decolorization
3) Damaged or Old culture - cells with damaged walls can lose crystal violet and can appear falstely gram-negative.

Phenol red turns yellow below pH 6.8 and fuchsia above pH 7.4. Carbohydrate utilization creates acid and turns the medium yellow; peptone utilization can produce ammonia and turn it fuchsia. T/F

False;
Phenol red is yellow below pH 6.8
Red/Pink (Fuchsia) above 8.4
Neutral Red-orange at 7.4

carbohydrate fermentation: Sugar → fermentation → acidic products → release H+ → lower pH → yellow

Peptone utilization → peptones → amino acid breakdown → ammonia → use up H → higher pH → pink

What is the purpose of the Durham tube in the broth tube?

A Durham tube is a small inverted tube that traps gas released during microbial metabolism (usually Carbohydrate Fermentation). The results are recorded separately and help with further distinction.

 What are the primary stain and counterstain for the acid-fast stain?

The primary stain is carbon fuchsin, while the counterstain is malachite green. Acid-Alcohol is the decolorizer, not the counterstain.
The purpose is to see if the cells retain the carbon fuchsin after decolorization. If they retain the primary stain, this means that their cell walls contain thick, waxy mycolic acids that block acid-alcohol from stripping out the color.

What is the difference between a simple stain and an endospore stain?

A simple stain uses one dye to show overall cell morphology, while an endospore stain uses a differential stain to distinguish resistant endospores from vegetative cells. (Spore-forming vs. non-spore-forming → spores are single-celled units adapted for asexual reproduction or survival.)

Why do redox indicator agars tell us?

Redox indicators change color to help show whether oxygen is present in the medium. This is useful when growing anaerobes, which may need conditions without oxygen.

• Pink: Indicates exposure to oxygen.

• Colorless: Indicates reduced conditions, usually with little or no oxygen.

T/F: The aerobic plate count indicates the level of microorganisms in a product. Spiral plate counting is used for milk, foods, and cosmetics, with a mechanical plater inoculating a rotating agar plate with liquid sample.

True.

• Aerobic plate count: It tells you roughly how many living bacteria (and other microbes that grow with oxygen) are in a product. A higher count means more microbes, which signals poorer sanitation or quality.

• Spiral plating: A machine spreads a liquid sample onto a spinning agar plate in a spiral, using less and less sample as it moves outward. That means one plate holds a range from heavy to light concentration, so you don't need to dilute the sample multiple times. You then count the colonies that grow.

1. After an acid-fast stain using malachite green as the counterstain, a student observes red bacterial cells against a green background. What does this result indicate?

   1. The bacteria lost the primary stain during decolorization.

   2. The bacteria retained carbol fuchsin after treatment with acid alcohol.

   3. The bacteria are definitively identified as Mycobacterium tuberculosis.

   4. The bacteria took up the malachite green counterstain.

2. The bacteria retained carbol fuchsin after treatment with acid alcohol: The red cells are acid-fast-positive because they retained the primary stain despite decolorization. Material that takes up the green counterstain provides contrast. This reaction is consistent with an acid-fast envelope, but it does not definitively identify Mycobacterium tuberculosis; species identification requires additional testing.

1. An oxidase test produces no color change within the specified reading time, but turns purple several minutes later. What should the student conclude?

   1. The organism is oxidase-positive because any purple color indicates a positive result.

   2. The organism cannot perform cellular respiration.

   3. The test is negative within the valid reading window; the delayed color change should not be counted as positive.

   4. The organism is definitively identified as Escherichia coli.

3. The test is negative within the valid reading window; the delayed color change should not be counted as positive: Oxidase reagent can oxidize after prolonged exposure, producing a misleading late purple color. The kit or laboratory protocol determines the valid reading interval. A negative result does not mean that the organism cannot respire; it may use respiratory components that this test does not detect. The result alone also does not identify E. coli.

What is a catalase test?

The catalase test detects the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen: 2 H₂O₂ → 2 H₂O + O₂. Immediate bubbling after hydrogen peroxide is added is a positive result because oxygen gas is released. No visible bubbling is negative under the test conditions

3. A bacterial species is inoculated on EMB agar. 

(a) The bacteria do not grow. Why?
(b) If the bacteria ferment lactose, what would you expect to see?
(c) The bacteria produce clear colonies. Why?

(a) The bacteria do not grow. Why? EMB agar is selective: the dyes eosin Y and methylene blue inhibit the growth of Gram-positive bacteria. So the species is most likely a Gram-positive organism (e.g., Staphylococcus, Streptococcus, Bacillus) and is inhibited by the dyes.

(b) If the bacteria ferment lactose, what would you expect to see? If the bacteria ferment lactose, they produce acid, which lowers the pH of the medium and causes the eosin and methylene blue dyes to leak in the colonies, so the colonies appear dark purple to black. Strong, rapid fermenters such as E. coli produce enough acid to give the colonies a green metallic sheen, while weaker fermenters such as Klebsiella and Enterobacter form pink to purple, mucoid colonies without the sheen.

(c) The bacteria produce clear colonies. Why? Clear (colorless or translucent) colonies indicate the bacteria are non-lactose fermenters (e.g., Salmonella, Shigella, Proteus, Pseudomonas). They grow on the medium, so they are Gram-negative and tolerate the dyes, but they cannot ferment lactose. They use the peptones in the medium instead, which produces alkaline byproducts (ammonia). The pH does not drop, so the dyes do not precipitate, and the colonies stay colorless (the agar may look pale or slightly amber/pink).

E. coli grows from 20,000 to 160,000 cells in 2 hours. How long would 15 doubling cycles take? 

First find the growth ratio: 160,000/20,000 = 8 = 2³, so the population underwent 3 doubling cycles in 2 hours. Generation time = 120 minutes/3 = 40 minutes per doubling. Fifteen doublings therefore require 15 × 40 = 600 minutes = 10 hours.

Bacteria are distributed throughout a broth tube rather than primarily at the top or bottom. Which listed types is present: facultative anaerobic, obligate anaerobic, or obligate aerobic?

Facultative anaerobic. Growth throughout the broth means the bacteria can grow both with and without oxygen. Obligate aerobes need oxygen and would cluster at the top, where oxygen is highest, while obligate anaerobes are killed by oxygen and would collect at the bottom, where it is lowest. Facultative anaerobes grow wherever they like, though they actually grow best near the top, since aerobic respiration yields more energy, and still grow throughout the tube by switching to fermentation or anaerobic respiration where oxygen is absent.

What type of stain is used in a capsule stain?

A capsule stain uses a negative stain (typically India ink or nigrosin) combined with a simple counterstain such as crystal violet or safranin. The negative stain darkens the background, and the simple stain colors the bacterial cell, while the capsule, which does not take up either stain, shows up as a clear halo around the cell.

what is a phage?

a virus that infects bacteria. It carries a nucleic acid genome inside a protein coat

T/F: Biotin is responsible for biosynthetic reactions involving carbon dioxide fixation and release, as well as fatty acid synthesis.

True in the intended cofactor sense. Biotin, vitamin B7, acts as a cofactor for enzymes that transfer carboxyl groups.

T/F: Many cyanobacteria grow with mineral salts and light, synthesize their own vitamins, and can nevertheless be challenging to culture because of sensitivity to contaminants. 

True.

Many cyanobacteria are photoautotrophs. They need only light, CO2, and a mineral salts medium (like BG-11), and they make their own vitamins and organic compounds, so they don’t need rich nutrients.

 ______ is used to monitor water quality and wastewater discharges. It uses microbial growth to determine the concentration of an essential growth compound. 

Bioassay (microbiological assay)

It uses the growth of a test microorganism to measure the concentration of an essential growth compound, such as a vitamin or amino acid, in a sample. Because the organism’s growth is proportional to the amount of that nutrient available, comparing its growth in the sample against a standard curve gives the concentration of that particular nutrient

Plaques are:
A. clear areas caused by viral infection
B. stained infected areas
C. virus colonies on agar
D. bacterial colonies on agar

A. Clear areas in a lawn of cultured cells caused by virus infection. A phage infects susceptible bacteria and spreads locally, reducing or destroying growth in that area. Plaques may be completely clear or turbid depending on the phage-host interaction.

How do viruses cause cell death?

1. Entry: The virus attaches to a receptor and gets its genome (DNA or RNA) into the cell.

2. Takeover: The viral genome uses the host’s ribosomes, enzymes, nucleotides, and energy to make viral proteins and copy itself. Many viruses also shut down the host’s own DNA, RNA, and protein synthesis, so the cell can’t maintain itself.

3. Assembly: New virus particles are built inside the cell, using up its resources and often wrecking its internal structure.

4. Release: The cell dies when the viruses exit. In the lytic cycle, the cell bursts (lysis), releasing hundreds or thousands of new viruses. Enveloped viruses can instead bud out through the membrane, which damages it and can also kill the cell.

Other ways viruses kill cells:

• Apoptosis (programmed cell death): Infected cells often detect the invasion and trigger their own death to stop the spread. Some viruses also push this process themselves.

• Immune attack: Cytotoxic T cells and NK cells destroy infected cells, so some of the damage you see in disease is from the immune system.

• Toxic viral proteins: Some viral proteins directly poison the cell or disrupt its membrane.

What is flora?

microorganisms that live in humans but do not cause disease

Five benefits of normal flora:

1. Competitive exclusion: They take up attachment sites and nutrients, making it hard for pathogens to colonize.

2. Antimicrobial production: They make bacteriocins, fatty acids, and acids that inhibit or kill competing microbes
   Vitamin synthesis: Gut bacteria such as E. coli make vitamin K and some B vitamins that the host absorbs.

3. Aid in digestion: Intestinal flora break down fibers and complex carbohydrates humans cannot digest, producing short-chain fatty acids that nourish colon cells

4. Immune system stimulation: They help the immune system develop and stay primed, for example by promoting IgA production and lymphoid tissue development.

What is the main function of a bacterial capsule? 

The capsule primarily provides protection. Capsules can also help cells retain water, adhere to surfaces, and participate in biofilms. In pathogenic bacteria, it reduces recognition and engulfment by phagocytic immune cells (increase virulence)

Which stain is used primarily to help identify Mycobacterium:
a. flagellar; b. Gram; c. acid-fast; d. spore; e. negative? 

c. Acid-fast stain. The mycolic-acid-rich envelope of many mycobacteria retains carbol fuchsin after acid-alcohol decolorization, producing red or pink acid-fast cells.

What is the purpose of a Simmons citrate slant, and what result would you expect with E. coli?

The Simmons citrate slant tests whether a bacterium can use citrate as its sole carbon source (and ammonium salts as its nitrogen source). It is used to help identify and differentiate members of the Enterobacteriaceae (Gram-negative, rod-shaped bacteria that function as facultative anaerobes).

If a bacterium uses citrate (via the enzyme citrate permease), it produces alkaline byproducts, raising the pH, so the medium changes from green to blue and growth is visible on the slant.

Expected result with E. coli: E. coli is citrate-negative. It cannot use citrate as its only carbon source, so there is no growth and no color change (the slant stays green).

4. What is your final serial dilution concentration if the starting culture contains 1 mL broth and 0.1 mL microbial inoculum and you made 9 plates, including a control plate? 

Final = (10^-1)^n = (10^-1)^8 = 10^(-1 × 8) = 10^-8
(take out one for control)

Trend between gram stain and oxidase?

Oxidase-positive organisms are mostly Gram-negative, and most Gram-positive bacteria are oxidase-negative.
(Exception: Many Gram-negatives are oxidase-negative if part of the Enterobacteriaceae family)

Why do we measure the zone of inhibition? 

The zone diameter measures how far visible bacterial growth is inhibited around an antimicrobial disk.

How do you calculate growth (PFU/mL)? 

 CFU/mL = colony count ÷ (dilution plated × volume plated in mL).

What are the two life cycle of a bacteriophage?

Lytic: The phage takes over, makes new phages, and bursts (lyses) the cell to release them.

Lysogenic: The phage DNA integrates into the bacterial chromosome as a prophage and gets copied along with the host’s DNA until a trigger switches it to the lytic cycle.

Explain the Gram Stain Step to Step (think CIDS)

A Gram stain is a differential stain that separates bacteria into two groups based on their cell wall

1. Prepare a thin bacterial smear on a slide: Spread a small amount of bacterial sample onto a clean glass slide. Use a drop of water to spread it thinly and evenly.
2. Allow the smear to air-dry completely, then heat-fix with a Bunsen burner
3. The primary stain (crystal violet) covers the smear for about 1 minute, before it is gently rinsed with water
4. Apply Gram’s Iodine (the mordant) for about 1 minute to ensure the stain is retained more strongly, then rinse with water
5. Apply the decolorizer (95% ethanol) for a few seconds until the agent on the slide runs clear
6. Cover the smear with safranin (the counterstain) for about 30 s- 1 min; wash with water
7. Put under the microscope and observe results

What is the acronym for the bacteria?

ESRAMBB

Give an example of a Lactose Fermenter and a non-fermenter and how it appears on MacConkey plate”

E.coli: Pink means lactose fermenter
Salmonella: Colorless means non-fermenter

Here are some practice problems. Unless stated otherwise, assume 25 mL per plate, 28 g agar per 1,000 mL water, and 5% (vol/vol) blood.
Problem 1
How much DI water, agar and blood (in µL) do you need for 6 plates?

Problem 2
How much DI water, agar and blood (in µL) do you need for 15 plates?

Problem 3
Your plates hold 20 mL each. How much DI water, agar and blood (in µL) do you need for 10 plates?

Problem 4
You prepare 2 litres of blood agar.
a) How many grams of agar do you need?
b) How many µL of blood do you need?
c) How many 25 mL plates can you pour?

Problem 5
You need 8 plates, but you want to make 10% extra to allow for losses. How much DI water, agar and blood (in µL) do you need?

Problem 6 (challenge)
You only have 7.0 g of agar powder left.
a) How much DI water can you use with it?
b) How many 25 mL plates will that make?
c) How much blood (in µL) do you need?

Problem 1: Total volume = 6 × 25 = 150 mL

• Water: 150 mL

• Agar: 28 × 150/1000 = 4.2 g

• Blood: 0.05 × 150 = 7.5 mL = 7,500 µL

Problem 2: Total volume = 15 × 25 = 375 mL

• Water: 375 mL

• Agar: 28 × 375/1000 = 10.5 g

• Blood: 0.05 × 375 = 18.75 mL = 18,750 µL

Problem 3: Total volume = 10 × 20 = 200 mL

• Water: 200 mL

• Agar: 28 × 200/1000 = 5.6 g

• Blood: 0.05 × 200 = 10 mL = 10,000 µL

Problem 4: 2 L = 2,000 mL

• a) Agar: 28 × 2 = 56 g

• b) Blood: 0.05 × 2,000 = 100 mL = 100,000 µL

• c) Plates: 2,000 / 25 = 80 plates

Problem 5: Base volume = 8 × 25 = 200 mL, and 200 × 1.10 = 220 mL

• Water: 220 mL

• Agar: 28 × 220/1000 = 6.16 g

• Blood: 0.05 × 220 = 11 mL = 11,000 µL

Problem 6:

• a) Water: 7.0 g ÷ 28 g × 1,000 mL = 250 mL

• b) Plates: 250 / 25 = 10 plates

• c) Blood: 0.05 × 250 = 12.5 mL = 12,500 µL

Bacteria Phage and Antibiotic relationship

Bacteriophages are viruses that infect and kill specific bacteria. Bacteria evolve resistance to phages quickly, which sounds like a problem. But resistance to a phage often requires changing the very structure the phage uses to latch on, and sometimes that structure is also part of the bacterium’s antibiotic defense.

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