Lan practical 1 micro practice

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Broth Growth Patterns (Pellicle, Sediment, Turbidity, Flocculent)

16. Which tube shows a pellicle?
    A: The one with a film/skin at the top.

17. Which tube shows sediment?
    A: The one with growth settled at the bottom.

18. Which tube shows flocculent growth?
    A: Clumps suspended in broth.

19. Why does a pellicle indicate aerobic metabolism?
    A: Bacteria cluster at the oxygen-rich surface.

20. Trick Q: If growth is evenly turbid throughout, what two categories could it be?
    A: Facultative anaerobe or aerotolerant anaerobe.

21. Why would an obligate anaerobe only grow at the bottom?
    A: Lacks enzymes to detoxify oxygen radicals.

22. Fun fact Q: Why is flocculent growth sometimes compared to “snow in a snow globe”?
    A: Because the clumps swirl when shaken.

Oxygen Requirements (FTM)

23. Which tube shows obligate aerobes?
    A: Growth only at top.

24. Which tube shows obligate anaerobes?
    A: Growth only at bottom.

25. Which tube shows microaerophiles?
    A: Growth just below surface.

26. Which tube shows facultative anaerobes?
    A: Growth throughout but denser at top.

27. Which tube shows aerotolerant anaerobes?
    A: Growth evenly distributed throughout.

28. What chemical in FTM reduces oxygen?
    A: Sodium thioglycolate.

29. What dye indicates oxygen in FTM?
    A: Resazurin (pink when oxygen is present).

30. Trick Q: Why does oxygen kill obligate anaerobes?
    A: They lack SOD and catalase, so ROS build up.

31. If an organism grows best in low oxygen but not no oxygen, what is it?
    A: Microaerophile.

32. Why do facultative anaerobes grow faster at the top?
    A: They prefer aerobic respiration (more ATP yield) but can ferment if needed.

Antibiotic Sensitivity (Kirby-Bauer)

33. What type of media is used in Kirby Bauer?
    A: Mueller-Hinton agar.

34. What does a large zone of inhibition mean?
    A: Susceptible.

35. What does no zone of inhibition mean?
    A: Resistant.

36. If the zone diameter is in the “intermediate” range, what does that mean?
    A: The antibiotic may work at higher doses.

37. Trick Q: If two antibiotics have the same zone diameter, are they equally effective?
    A: Not necessarily — interpretation depends on standards for each drug.

38. Which organism is more likely to be resistant to penicillin: Gram-positive or Gram-negative?
    A: Gram-negative (outer membrane blocks entry). (same answer if it asks, why wouldnt a gram positive test be resistant to penicillin? because it doesnt have an outer membrane that blocks entry)

    Penicillin Resistance & Gram Reaction

    • Gram-positive bacteria

      • Penicillin usually works well because it targets peptidoglycan cross-linking.

      • They do not have an outer membrane, so the drug can easily reach the cell wall.

      • If they’re resistant, it’s usually due to enzymes or altered PBPs (penicillin-binding proteins), not an outer barrier.

    • Gram-negative bacteria

      • Often resistant to penicillin because their outer membrane blocks or slows entry of the drug.

      • Even if penicillin gets through, efflux pumps or the enzymes in the periplasmic space may inactivate it.

    ---

    Lab-Test Style Question on this:

    Q: Why would a Gram-negative bacterium be more resistant to penicillin than a Gram-positive bacterium?
    A: Gram-negatives have an outer membrane that blocks drug entry; Gram-positives lack this barrier.

    Q (trick style): Why wouldn’t a Gram-positive be resistant to penicillin just because of its structure?
    A: Because it lacks an outer membrane — penicillin can reach the peptidoglycan directly. Resistance in Gram-positives comes from enzymes (β-lactamase) or modified binding proteins, not structural exclusion.

39. Why is Mueller-Hinton agar used?
    A: It allows uniform diffusion of antibiotics.

40. Why is it important to standardize inoculum density in Kirby-Bauer?
    A: Too heavy = smaller zones, too light = larger zones.

Colony Morphology (Slants/Plates)

43. Define friable growth.
    A: Dry, brittle, crusty colonies.

44. Define spreading edge growth.
    A: Colonies with irregular, fuzzy edges that creep outward.

45. What does “mucoid” morphology look like?
    A: Slimy, sticky, shiny colonies.

46. Trick Q: What structural feature causes mucoid colonies?
    A: Capsule production.

47. Why might colonies appear pigmented?
    A: Bacteria produce pigments (e.g., Serratia marcescens = red).

48. What tool is used to streak an agar slant?
    A: Inoculating loop.

Serial Dilution/Colony Counts

49. Why do we dilute cultures before plating?
    A: To obtain countable colonies (30–300).

50. If you plate 0.1 mL of a 10⁻⁴ dilution and count 50 colonies, what was the CFU/mL of the original?
    A: 50 ÷ 0.1 mL × 10⁴ = 5 × 10⁶ CFU/mL.

51. Trick Q: Why don’t we count plates with <30 colonies?
    A: Not statistically reliable.

52. Why don’t we count plates with >300 colonies?
    A: Too crowded, inaccurate.

53. What’s the purpose of using sterile saline or broth in dilutions?
    A: To disperse cells evenly without nutrients.

Temperature Classification

54. Which group grows at refrigerator temps?
    A: Psychrophiles.

55. Which group grows at body temp (37 °C)?
    A: Mesophiles.

56. Which group grows at hot springs (60–80 °C)?
    A: Thermophiles.

57. Which group survives boiling temps (>80 °C)?
    A: Hyperthermophiles.

58. Trick Q: Which is more dangerous in food spoilage: psychrotrophs or psychrophiles?
    A: Psychrotrophs (can grow in fridge but also at body temp).

59. What is the growth range of mesophiles?
    A: 20–45 °C.

60. What is the optimum temperature for E. coli?
    A: ~37 °C.

Metabolism / Fermentation “Trick Questions”

61. What is the final electron acceptor in fermentation?
    A: An organic molecule (like pyruvate or acetaldehyde).

    Final Electron Acceptor Questions

    Q1. What is the final electron acceptor in aerobic respiration?
    A: Oxygen (O₂).

    Q2. What is the final electron acceptor in anaerobic respiration?
    A: Inorganic molecules other than oxygen (e.g., nitrate, sulfate, carbonate).

    Q3. What is the final electron acceptor in fermentation?
    A: An organic molecule, such as pyruvate or acetaldehyde. (pyruvate will come up alot later on in the semester)

62. What is the final electron acceptor in aerobic respiration?
    A: Oxygen.

63. What is the final electron acceptor in anaerobic respiration (non-fermenters)?
    A: Inorganic molecules like nitrate or sulfate.

64. Trick Q: Why does fermentation only make 2 ATP per glucose?
    A: Only glycolysis runs; no ETC or Krebs.

65. Why does facultative anaerobe growth look heavier at the top of broth?
    A: Because oxygen allows more ATP production.

66. In fermentation tubes, why does a color change to yellow indicate acid?
    A: Sugar was fermented → acid byproducts lower pH.

67. What is the gas bubble in a Durham tube?
    A: CO₂ or H₂ from fermentation.

68. Trick Q: If glucose is fermented to lactate, is oxygen used?
    A: No — fermentation is anaerobic.

General Trick/Conceptual Questions

69. Why does Gram-positive bacteria resist penicillin better once it produces β-lactamase?
    A: β-lactamase destroys penicillin’s β-lactam ring.

70. Why do Gram-negative bacteria often appear resistant?
    A: Outer membrane blocks many antibiotics.

71. Which oxygen requirement group produces catalase?
    A: Obligate aerobes and facultative anaerobes.

72. Which group does NOT produce catalase?
    A: Obligate anaerobes.

73. Trick Q: Why does aerobic respiration yield more ATP than fermentation?
    A: ETC + oxidative phosphorylation produce ~34 ATP more.

74. Why do obligate anaerobes sometimes survive briefly in oxygen?
    A: Some may have low levels of detox enzymes.

75. Why is Mueller-Hinton agar poured at a standardized depth?
    A: Depth affects diffusion rates of antibiotics.

76. Why do we stain bacteria instead of viewing them unstained?
    A: Increases contrast since bacteria are transparent.

77. Trick Q: What happens if you use too much oil on oil immersion?
    A: Image becomes blurry, lens can get contaminated.

78. Why are mesophiles the most common pathogens?
    A: Because their optimum is human body temperature.

79. Why are psychrophiles not usually pathogenic?
    A: They cannot grow at body temp.

80. What does the term “fastidious” mean for bacteria?
    A: Requires special growth factors/nutrients.

Broth Growth Patterns (TSB tubes, pellicle/flocculent/sediment)

Q1. These organisms were inoculated into TSB. Which tube shows a pellicle?
A1. The tube with a thick layer at the top (pellicle = floating mat on the surface).

Q2. Describe what flocculent growth looks like.
A2. Flocculent looks like clumps or cotton-ball-like masses floating in the broth.

Q3. Why is uniform turbidity throughout the broth not always “facultative anaerobe”?
A3. Because other organisms (like aerotolerant anaerobes) can also grow evenly throughout. Uniform turbidity just means they can tolerate oxygen, not necessarily use it.

Fun Fact: “Pellicle” comes from Latin pellicula meaning “little skin.” Easy way to memorize: pellicle = “skin on top.”

Oxygen Requirements (FTM tubes)

Q4. Which tube shows a microaerophile?
A4. The one with growth just below the surface (needs O₂ but at lower concentration than atmosphere).

Q5. What compound in fluid thioglycollate medium scavenges oxygen?
A5. Sodium thioglycolate.

Q6. What oxygen term applies to an organism growing only at the bottom of the tube?
A6. Obligate anaerobe.

Q7. Why can oxygen be toxic to some organisms?
A7. Because they lack enzymes like catalase and superoxide dismutase to detoxify reactive oxygen species (ROS).

Tip to memorize: Oxygen tolerance = enzyme protection. If no enzymes → O₂ is poison.

Antibiotic Sensitivity (Kirby-Bauer test)

Q8. Measure the zones of inhibition for penicillin and erythromycin. Which antibiotic was more effective against S. aureus?
A8. The larger clear zone = more effective (often penicillin against Gram-positives).

Q9. What media is used in the Kirby-Bauer test?
A9. Mueller-Hinton agar.

Q10. If E. coli shows no clear zone around penicillin, how is it classified?
A10. Resistant.

Memory trick: Bigger halo = stronger antibiotic effect. No halo = resistant.

Colony Morphology (Slants or Plates)

Q11. Which organism shows friable morphology?
A11. The dry, brittle, powdery colony.

Q12. Which organism shows spreading edge morphology?
A12. The one with a fuzzy, irregular edge that creeps outward.

Serial Dilution Plates

Q14. After performing a 1:1000 dilution, you count 45 colonies on the plate. How many CFU/mL were in the original culture?
A14. 45 × 1000 = 45,000 CFU/mL. -----> 4.5 × 10⁴ CFU/mL

Q15. Why do we perform serial dilutions before plating?
A15. To reduce bacterial numbers so colonies can be counted.

Temperature Growth

Q16. Which organism is a mesophile?
A16. The one growing best at ~37 °C.

Q17. Organism D grows at 60 °C. How would you classify it?
A17. Thermophile.

Q18. What is the normal growth range of mesophiles?
A18. About 20–45 °C.

Memory trick: “Meso = middle,” so mesophiles like middle/room-to-body temperatures.

Microscope Parts

Q19. Which part of the microscope adjusts light intensity and contrast?
A19. The condenser & iris diaphragm.

Q20. Which knob should only be used with the 40× and 100× objectives?
A20. Fine focus.

Q21. What is the total magnification if you are using the 10× ocular and 40× objective?
A21. 400×.

More Practice Questions:

1. Kirby-Bauer Media

Q: What medium is used to cultivate bacteria in the Kirby-Bauer test? Why?
A: Mueller-Hinton agar is used because it allows antibiotics to diffuse evenly and supports consistent bacterial growth, making results reliable and comparable.

2. Salt Tolerance

Q: Bacteria A grows in both 1% and 5% NaCl. Bacteria B grows in 1% NaCl but not 5%.
a. What term describes Bacteria A?
A: Halophile.

b. In the 5% NaCl plate, are Bacteria A’s cells in a hypotonic, isotonic, or hypertonic state?
A: Isotonic.

c. What about Bacteria B?
A: Hypertonic (water leaves the cells, inhibiting growth).

3. Oxygen Requirements (FTM Tube)

Q: Bacteria A grows only at the bottom of the FTM tube.
a. What is Bacteria A?
A: Obligate anaerobe.

b. Why only at the bottom?
A: It lacks enzymes (e.g., superoxide dismutase, catalase) needed to neutralize toxic oxygen byproducts.

4. Oxygen Requirements (TSB Tube)

Q: Bacteria B grows evenly throughout a TSB broth. A partner calls it “facultative anaerobe.” Do you agree?
A: No. TSB lacks an oxygen gradient, so the term doesn’t apply. Even growth is described as “uniform fine turbidity.”

5. Mesophiles

Q: What is the temperature range for mesophiles? Optimal temperature?
A: Grow between ~10 °C and 50 °C, with an optimum at 37 °C.

6. Colony Morphology

Q: Draw an “umbonate” colony.
A: (Raised with a knob-like center.)

7. Pure Culture

Q: What is a pure culture?
A: A culture containing only one type of microorganism, free of contaminants, with genetically identical cells.

8. Inoculating a Fresh TSB Tube

Steps:

• Wash hands, disinfect workspace, and wear PPE.

• Label the fresh TSB tube (name, date, inoculum).

• Flame the inoculating loop until red-hot, then cool.

• Swirl the pure TSB culture, open aseptically, collect a loopful.

• Open the fresh TSB tube aseptically, insert loop without touching sides, swirl gently, then recap loosely.

• Reflame the loop.

• Incubate tube at the proper time and temperature.

• After observation, discard properly, remove PPE, and wash hands.

9. Temperature Classification

Q: If an organism grows at 25 °C, is it a psychrophile or mesophile?
A: Insufficient data. If it only grows below 30 °C → psychrophile. If it also grows above 30 °C → mesophile.

10. Transfer Tools

Q: Which tool is used…

• Slant to slant: Inoculating needle.

• Broth to broth: Inoculating loop.

11. Aseptic Technique

Q: Why is aseptic technique used?
A: To prevent contamination of cultures, media, environment, and ourselves, ensuring reliable results and safety.

12. Cell Concentration Calculation

Q: 0.1 mL of a 1:10,000 dilution gives 30 colonies. What is the original concentration?
A: (30 × 10,000) ÷ 0.1 mL = 3,000,000 cells/mL.

13. Streak Plate

Q: Why use the streak plate technique? How does it work?
A: To isolate pure colonies from a mixed culture. Bacteria are spread across quadrants, diluting each time, until individual colonies appear.

14. Flocculant Growth

Q: Your partner describes Bacteria C as “flocculant.” What does this mean?
A: Bacteria clump together as visible flakes in broth cultures.

15. Spread Plate vs. Swab

Q: Why use a spread plate instead of a swab for standard plate counts?
A: The spread plate allows inoculation with a known, precise volume, which is essential for calculating concentration. A swab transfers an unknown amount.

The PHILES 

1. What organism(s) is a mesophile?

A mesophile is a microorganism that grows best at moderate temperatures, typically in the range found in the human body. Common mesophiles include Escherichia coli, Staphylococcus aureus, and many pathogens that infect humans.

From your lab setup, if one of the plates shows best growth at 25°C–37°C but not at high temperatures (55°C) or low (4°C), that’s the mesophile.

2. How would you classify a mesophile?

Mesophiles are classified based on their optimal temperature range for growth:

• Psychrophiles: cold-loving (−5°C to 15°C)

• Psychrotrophs: grow in cold but prefer moderate (0–30°C)

• Mesophiles: moderate temperature (20–45°C, optimum ~37°C)

• Thermophiles: heat-loving (45–80°C)

• Hyperthermophiles: extreme heat (80°C+)

3. What is the normal growth range of a mesophile?

• 20°C to 45°C

• Optimum around 37°C (the human body temperature, which explains why most human pathogens are mesophiles)

Mesophiles

• Growth range: ~20 °C to 45 °C

• Optimum temperature: ~37 °C (human body temperature)

• Behavior: They grow poorly or not at all at refrigerator temps (~4 °C).

• Examples: E. coli, Staphylococcus aureus

• Relevance: Most human pathogens are mesophiles because our body is 37 °C.

Psychrotrophs

• Growth range: ~0 °C to 30–35 °C (they can tolerate colder conditions than mesophiles)

• Optimum temperature: ~20–25 °C (room temperature), not body temperature

• Behavior: They grow at refrigerator temps (~4 °C), though slowly, and that’s what sets them apart.

• Examples: Listeria monocytogenes, Pseudomonas species (spoil food in the fridge)

• Relevance: Major in food spoilage; some are opportunistic pathogens.

How to Tell Them Apart in Lab

• If your culture does not grow at 4 °C but does well at 37 °C → mesophile.

• If your culture grows at 4 °C and also at 20–25 °C, but not much at 37 °C → psychrotroph.

Mnemonic

• “Meso = Middle” → thrive in middle, body-like temps (20–45 °C).

• “Psycho = Fridge” → these "crazy cold lovers" can grow in the refrigerator (0–4 °C).