Table 1- practical questions

1. Describe 3 important steps in the preparation of a smear used in staining technique - from BROTH.

Use 1 loopful of broth on the slide, spread into a thin smear, air-dry completely, then heat-fix. Do not add water because broth is already liquid.

2. Describe 3 important steps in the preparation of a smear used in staining technique - from agar.

Add 1–2 drops of water to the slide, gently touch a colony, mix it into the water and spread thinly, air-dry completely, then heat-fix.

3. What will happen if you take too many cells during preparation of smear for gram staining?

The smear becomes too thick, so individual cells are hard to see and the Gram stain result may be inaccurate.

4. What would happen if you don’t heat-fix while smearing from bacteria on plate?

The bacteria may wash off the slide during staining/rinsing because they were not fixed to the glass.

5. What would happen if you didn’t air-dry completely while smearing from bacteria on plate?

The smear can steam, splatter, or distort the bacterial shape when heat-fixed.

6A. Classify the organism based on the result shown in tube “A”.

Look at growth in the FTM tube: growth only top = obligate aerobe; growth only bottom = obligate anaerobe; growth throughout but more at top = facultative anaerobe; even growth throughout = aerotolerant anaerobe; band below top = microaerophile.

6B. Classify the organism based on the result shown in tube “B”.

Use the FTM growth pattern: top only = obligate aerobe; bottom only = obligate anaerobe; throughout but heavier top = facultative anaerobe; even throughout = aerotolerant anaerobe; band just below top = microaerophile.

6C. Classify the organism based on the result shown in tube “C”.

Use the FTM growth pattern: top only = obligate aerobe; bottom only = obligate anaerobe; throughout but heavier top = facultative anaerobe; even throughout = aerotolerant anaerobe; band just below top = microaerophile.

7A. Give the name of the media used in 2042 in the oxygen tolerance test.

Fluid thioglycollate medium, or FTM.

7B. Give the name of the indicator present in the media used in 2042 in the oxygen tolerance test.

Resazurin, which indicates oxygen presence.

8A. What is the name of the indicator and inhibitor in media “MSA”?

Indicator: phenol red. Inhibitor: 7.5% sodium chloride.

8B. What type of bacteria will grow in media “MSA”?

Staphylococcus species grow because they tolerate high salt.

9A. What is the name of the indicator and inhibitor in media “MacConkey”?

Indicator: neutral red. Inhibitors: bile salts and crystal violet.

9B. What type of bacteria will grow in media “MacConkey”?

Gram-negative bacteria grow; Gram-positive bacteria are inhibited.

10A. What is the difference between alpha, beta and gamma hemolysis?

Alpha = partial hemolysis/greenish zone. Beta = complete hemolysis/clear zone. Gamma = no hemolysis/no change.

10B. What type of hemolysis pattern is shown in media “D” by an arrow?

Identify what the arrow points to: clear zone = beta hemolysis; greenish zone = alpha hemolysis; no change = gamma hemolysis.

11. What is the name of the inhibitor in media “CNA” and what type of bacteria will grow in here?

Inhibitors: colistin and nalidixic acid. Gram-positive bacteria grow; Gram-negative bacteria are inhibited.

12. Name the indicator of glucose/lactose fermentation.

Phenol red.

13A. Identify the result for glucose test in tube E. What is the principle behind this result?

If the tube is yellow, glucose fermentation is positive because acid was produced. If red/orange, glucose fermentation is negative. If pink/fuchsia, alkaline reaction/no fermentation.

13B. Identify the result for lactose test in tube F. What is the principle behind this result?

If the tube is yellow, lactose fermentation is positive because acid was produced. If red/orange, lactose fermentation is negative. If pink/fuchsia, alkaline reaction/no fermentation.

13C. What is the name of the small tube in tube E and F? What is the function of it?

The small tube is a Durham tube. It traps gas from fermentation; a bubble means gas positive.

An organism with an optimal growth temperature of 60°C is considered a...

Thermophile

An organism with an optimal growth temperature of 35°C would be considered to be a...

Mesophile

Turbidity is an example of what kind of measurement for determining bacterial counts in a culture?

Indirect measurement

True or False: A direct microscopic count cannot reliably distinguish between living and dead cells.

True

In a serial dilution, the number of colonies that grow on the plates will [blank] with each consecutive dilution step.

Decrease

In which phase do bacteria grow slowly as they adapt to a new growth medium?

Lag phase

During which growth phase do nutrients completely deplete and toxic waste products accumulate, causing the number of dying cells to exceed the number of new cells?

Death (Decline) phase

A complex aggregation of microbes attached to and growing on a surface is called a...

Biofilm

What term describes free-floating or free-swimming microbial cells that live suspended in aquatic environments?

Planktonic

What is an example of a reactive oxygen species (ROS)?

Superoxide anion (O₂•⁻), Hydrogen peroxide (H₂O₂), Hydroxyl radical (•OH), or Singlet oxygen (¹O₂)

An organism that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation or anaerobic respiration if oxygen is absent, is a [blank] anaerobe.

Facultative