LAB PRACTICAL 2

✅ MICROBIOLOGY STUDY GUIDE — COMPLETE ANSWERS TO ALL 60 QUESTIONS

1. What is the theory behind bacterial transformation?

Bacterial transformation is the process by which bacteria take up free, extracellular DNA from their environment and incorporate it into their own cells. When the DNA is a plasmid, the new genes can be expressed immediately, causing a change in phenotype.

2. What does it mean for a bacterium to be competent?

Competence is the physiological state in which a bacterial cell is capable of taking up DNA. Some bacteria are naturally competent; others must be made competent using chemical or physical methods.

3. Procedure and reagents for bacterial transformation

  • Prepare competent cells (commonly with CaCl₂).

  • Mix plasmid DNA with competent cells on ice.

  • Perform a heat shock to induce uptake.

  • Allow recovery in nutrient broth.

  • Plate cells on selective media.
    Reagents: competent cells, CaCl₂, plasmid DNA, LB broth, selective agar (e.g., LB/Amp).

4. Name of plasmid used, genes on it, reporter and selective marker

A common plasmid is pGLO, which contains:

  • ampR = ampicillin resistance (selective marker)

  • GFP = green fluorescent protein (reporter gene) under an arabinose-inducible promoter (PBAD).

5. What is special about the E. coli MM294 strain?

MM294 is a safe, non-pathogenic laboratory strain engineered to be:

  • highly transformable,

  • lacking restriction enzymes that degrade foreign DNA,

  • genetically stable.

6. Role of CaCl₂ in transformation

CaCl₂ provides Ca²⁺ ions that neutralize negative charges on both the DNA and bacterial membrane, reducing repulsion and allowing DNA to bind to the cell surface.

7. Purpose of heat shock and recovery

  • Heat shock: creates a thermal imbalance that drives DNA into the cell.

  • Recovery: allows cells to repair membranes, begin expressing plasmid genes, and prepare for antibiotic exposure.

8. Why ampicillin is included in the medium

Ampicillin selects for only those cells that took up the plasmid containing the ampR gene. Non-transformed cells are killed.

9. Identify control and experimental plates (typical pGLO lab)

  1. +DNA LB/Amp — experimental
    → Growth = transformation occurred

  2. +DNA LB/Amp/Ara — experimental
    → Growth + fluorescence = plasmid present and GFP expressed

  3. –DNA LB/Amp — negative control
    → No growth = ampicillin selection is working

  4. –DNA LB — positive control
    → Heavy growth = bacteria were healthy

10. What kind of radiation is UV?

UV is nonionizing radiation. It possesses enough energy to excite molecules but not enough to ionize atoms.

11. How UV damages cells

UV causes pyrimidine dimers, especially thymine dimers, which distort DNA and block replication and transcription.

12. Which UV wavelength maximizes damage?

UV-C (200–280 nm)
Maximum absorption by DNA occurs at 260 nm, making it the most lethal.

13. Repair mechanisms for UV damage

  • Photoreactivation (light repair): photolyase breaks thymine dimers.

  • Nucleotide excision repair (dark repair): damaged DNA is removed and replaced.

14. Two bacteria typically used in UV lethal effects labs

Usually:

  • Bacillus subtilis (spore-forming; UV-resistant)

  • Staphylococcus epidermidis or E. coli (non–spore-forming; UV-sensitive)

15. Experimental design questions

a. Why 260 nm? DNA absorbs UV maximally → greatest damage.
b. Control? Plate exposed to 0 minutes UV.
c. Why keep the lid on? Plastic blocks UV → demonstrates shielding effect.

16. Typical UV sensitivity results

Non–spore-formers are highly sensitive; growth decreases quickly.
Spore-formers survive significantly longer.

17. Why the bacteria differ in UV sensitivity

Bacillus species form endospores, which contain:

  • thick protective layers

  • DNA-binding proteins

  • dehydration
    All of which help resist UV.

18. Effect of leaving petri lids on

No lethal effect because the lid blocks UV radiation.

19. Standard conditions for Kirby-Bauer

  • Mueller-Hinton agar

  • Standard 0.5 McFarland inoculum

  • Incubate 35°C for 16–18 hours

  • Standard antibiotic disk concentrations

20. Steps of Kirby-Bauer procedure

  1. Prepare standardized inoculum.

  2. Swab to create a uniform bacterial lawn.

  3. Place antibiotic disks.

  4. Incubate.

  5. Measure zones of inhibition.

  6. Interpret using CLSI charts.

21. What is a bacterial lawn?

A dense, confluent layer of bacterial growth across the entire plate; created by swabbing in multiple directions.

22. Zone of inhibition and factors affecting size

Zone of inhibition = clear area around disk where bacteria cannot grow.
Affected by:

  • antibiotic diffusion rate

  • potency

  • bacterial susceptibility

  • agar composition

  • inoculum density

23. How zones are measured

Measure diameter of the clear zone in millimeters (mm).

24. How zones are evaluated

Compare measured diameter to CLSI interpretive tables for each antibiotic.

25. How to assign R, I, S

  • S (Susceptible): zone ≥ susceptible breakpoint

  • I (Intermediate): zone between breakpoints

  • R (Resistant): zone ≤ resistant breakpoint

26. Meaning of R, I, S

  • R: resistant

  • I: intermediate

  • S: susceptible

27. Determining effective antimicrobials

Choose antibiotics where the organism is Susceptible (S) according to the chart.

28. What does PCR stand for?

Polymerase Chain Reaction.

29. Theory, procedure, and purpose of PCR

PCR amplifies a specific DNA sequence using:

  • template DNA

  • primers

  • Taq polymerase

  • nucleotides

Repeated temperature cycles exponentially increase target DNA.

30. Events of PCR

  • Denaturation: DNA strands separate (~95°C).

  • Annealing: primers bind (~50–65°C).

  • Extension: Taq polymerase extends DNA (~72°C).

31. Purpose of Taq polymerase

A heat-stable enzyme that synthesizes new DNA during extension without denaturing.

32. Purpose of primers

Primers define the target region and provide a free 3′ OH for DNA synthesis.

33. How mecA is located on a gel

Look for a band at the known bp size of the mecA PCR product, compared to the DNA ladder.

34. Size of mecA band

Typically ≈310 bp.

35. PCR vs Kirby-Bauer for MRSA detection

PCR advantages:

  • fast

  • sensitive

  • detects specific genes (e.g., mecA)

PCR disadvantages:

  • requires equipment

  • cannot detect phenotypic expression

  • more expensive

Kirby-Bauer advantages:

  • simple

  • detects actual resistance phenotype

Kirby-Bauer disadvantages:

  • slower

  • less specific

36. Characteristics of Staphylococci

  • Gram-positive cocci

  • Clusters

  • Non-motile

  • Catalase-positive

37. m-Staphylococcus Broth

  • Selective: high salt

  • Differential: mannitol + phenol red indicator

38. MSA & SM110

Both selective for Staphylococci due to 7.5% NaCl.

MSA:

  • mannitol + phenol red → yellow = positive

SM110:

  • encourages pigment production

  • shows colony morphology; no pH indicator

39. Hemolysis patterns on Blood Agar

  • Alpha (α): green partial hemolysis

  • Beta (β): clear zone

  • Gamma (γ): no hemolysis

40. Toxin causing beta hemolysis in S. aureus

Primarily alpha-toxin (α-hemolysin).

41. Coagulase test

Detects coagulase, which clots plasma.
Positive: clot formation
Negative: remains liquid

42. What is staphyloxanthin?

A golden carotenoid pigment produced by S. aureus; protects against oxidative damage.

43. Appearance of S. aureus

  • MSA: yellow colonies with yellow halo

  • SM110: golden-yellow colonies

  • Blood Agar: beta hemolysis

44. Coagulase result for S. aureus

Positive—forms a clot.

45. Major tests to distinguish Staphylococci

  • Coagulase test

  • Mannitol fermentation

  • Novobiocin susceptibility

46. Characteristics of Streptococci

  • Gram-positive cocci

  • Chains or pairs

  • Non-motile

  • Catalase-negative

47. Blood agar category

  • Enriched: 5% sheep blood

  • Differential: hemolysis patterns

48. Test that differentiates Staph from Strep

Catalase test.
Staph = positive
Strep = negative

49. Tests for Streptococci and Enterococci

  • Hemolysis pattern

  • Bacitracin (A disc)

  • Optochin (P disc)

  • Bile esculin

  • 6.5% NaCl tolerance

50. What positive and negative results look like

  • Bile esculin positive: blackening

  • Salt tolerance positive: turbidity

  • Optochin susceptible: clear zone

  • Bacitracin susceptible: zone

  • Hemolysis: alpha, beta, gamma as described

51–54. Test name, purpose, description, and interpretation

These refer generally to interpreting any biochemical test:

  • Name: e.g., Citrate test, Urease test, etc.

  • Purpose: determine metabolic capability

  • Description: includes substrate, enzyme, product, pH indicator, inoculation, incubation, reagents

  • Interpretation: color change or other visible reaction
    (This section is instructor-specific; answers provided match typical expectations.)

55. Theory and procedure for ELISA

ELISA detects antigens or antibodies using antigen–antibody binding and an enzyme that produces a color reaction.

General steps:

  1. Coat wells with antigen.

  2. Block unbound sites.

  3. Add sample.

  4. Add enzyme-linked antibody.

  5. Add substrate.

  6. Read color.

56. Direct vs indirect ELISA

  • Direct: enzyme-linked primary antibody binds antigen.

  • Indirect: antigen binds primary antibody (from patient serum), then enzyme-linked secondary antibody binds the primary.

57. Steps of indirect ELISA

  1. Coat plate with known antigen.

  2. Block.

  3. Add patient serum.

  4. Wash.

  5. Add enzyme-linked secondary antibody.

  6. Wash.

  7. Add substrate.

  8. Read color.

58. Role of materials in indirect ELISA

a. Known antigen: captures specific antibodies.
b. Test serum antibody: binds the antigen if present.
c. Enzyme-linked anti-human antibody: binds patient antibody.
d. Chromogen substrate: produces color when cleaved by enzyme.

59. Positive and negative indirect ELISA

  • Positive: strong color change

  • Negative: no color change

60. What to do after a positive ELISA

Perform a confirmatory test, such as:

  • Western blot

  • Nucleic acid test (PCR)

  • Second ELISA type
    Used to eliminate false positives.