BIOL 271 Microbiology w/ Lab Module 3 Exam Review
Advanced Comprehensive Question Bank v2.0
SECTION 1: MICROSCOPY THEORY & ADVANCED OPTICS
🟢 1. The resolving power of a microscope is mathematically expressed by the Abbe equation. Which of the following modifications would most
effectively increase the resolution of a light microscope?
A) Decreasing the numerical aperture of the objective lens from 1.4 to 0.25
B) Increasing the wavelength of illumination from 450 nm to 650 nm
🟢 C) Decreasing the wavelength of illumination from 550 nm to 380 nm while using an objective with NA 1.4
D) Decreasing the condenser numerical aperture from 1.25 to 0.90
🔴 Correct Answer: C – The Abbe equation (d = λ / 2NA) shows that resolution improves (smaller d) with shorter wavelength (λ) and higher numerical
aperture (NA). Decreasing wavelength to 380 nm (violet/UV range) significantly improves resolution when combined with high NA.
🟢 2. A microbiologist is examining a specimen under a 100x oil immersion objective with a numerical aperture of 1.4. The microscope has an ocular
lens magnification of 10x. If the working distance of this objective is 0.12 mm, what is the total magnification and approximate resolving power using
green light (550 nm)?
A) 1,000x total magnification; 0.196 μm resolution
B) 1,000x total magnification; 0.275 μm resolution
🟢 C) 1,000x total magnification; 0.196 μm resolution
D) 1,400x total magnification; 0.196 μm resolution
🔴 Correct Answer: C – Total magnification = 100 × 10 = 1,000x. Resolution (d) = λ / 2NA = 550 nm / (2 × 1.4) = .8 = 196.4 nm ≈ 0.196 μm.
🟢 3. The refractive index of immersion oil is approximately 1.515. What is the primary reason this specific refractive index is required for oil immersion
microscopy?
🟢 A) It matches the refractive index of glass (≈1.52), eliminating light refraction at the air-glass interface
B) It increases the wavelength of light reaching the specimen
C) It decreases the numerical aperture of the objective
D) It allows the use of lower intensity illumination
🔴 Correct Answer: A – Oil with refractive index matching glass prevents light scattering at the air-glass interface. Without oil, light refracts away from the
objective, reducing the numerical aperture and resolution.
🟢 4. A student is using a bright-field microscope and notices a halo artifact around the specimen when using the 100x objective. This is most likely
due to:
A) The condenser being fully closed
B) The objective being dirty
🟢 C) Improper use of immersion oil (air bubbles or incorrect amount)
D) The eyepiece being contaminated
🔴 Correct Answer: C – Air bubbles or incorrect oil application cause light scattering and halo artifacts around the specimen.
🟢 5. Which of the following adjustments would NOT improve the image quality when viewing an unstained, transparent bacterial specimen in a
bright-field microscope?
A) Closing the iris diaphragm to increase contrast
B) Reducing the light intensity
C) Using a higher numerical aperture objective
🟢 D) Using a lower numerical aperture condenser
,🔴 Correct Answer: D – Using a lower NA condenser would decrease resolution and image quality. Closing the iris, reducing light, and using high NA
objectives all improve contrast and resolution for unstained specimens.
🟢 6. The depth of field in a microscope is inversely proportional to the numerical aperture. Which of the following correctly describes the relationship
between NA and depth of field?
A) Higher NA = greater depth of field
🟢 B) Higher NA = shallower depth of field (thinner optical section)
C) Lower NA = shallower depth of field
D) NA does not affect depth of field
🔴 Correct Answer: B – Higher numerical aperture results in a shallower depth of field, which is why high power objectives require fine focusing
adjustments.
🟢 7. Chromatic aberration occurs when:
A) The specimen is not properly stained
B) The condenser is not centered
🟢 C) Different wavelengths of light are refracted to different degrees, causing color fringing
D) The objective lens is scratched
🔴 Correct Answer: C – Chromatic aberration is a lens defect where different wavelengths of light focus at different points, creating color fringing. This is
corrected in achromatic and apochromatic lenses.
🟢 8. Which of the following microscope configurations would provide the BEST resolution, assuming all else is equal?
A) Objective: 40x, NA 0.65, light source: 550 nm green
B) Objective: 60x, NA 0.85, light source: 550 nm green
C) Objective: 100x, NA 1.25, light source: 550 nm green
🟢 D) Objective: 100x, NA 1.40, light source: 450 nm blue
🔴 Correct Answer: D – Resolution improves with higher NA and shorter wavelength. The 100x NA 1.4 objective with 450 nm blue light provides the best
resolving power.
🟢 9. A researcher is using a fluorescence microscope to visualize GFP-tagged proteins in live bacterial cells. The excitation wavelength for GFP is
approximately 488 nm with emission at 509 nm. Which of the following filter cube configurations is appropriate?
🟢 A) Excitation filter: 488 nm, dichroic mirror: 505 nm, emission filter: 509 nm (bandpass)
B) Excitation filter: 509 nm, dichroic mirror: 488 nm, emission filter: 505 nm
C) Excitation filter: 505 nm, dichroic mirror: 488 nm, emission filter: 509 nm
D) Excitation filter: 509 nm, dichroic mirror: 505 nm, emission filter: 488 nm
🔴 Correct Answer: A – The excitation filter must pass 488 nm (excitation wavelength), the dichroic mirror must reflect excitation light and pass longer
wavelengths, and the emission filter must pass 509 nm (emission wavelength).
🟢 10. Photo-bleaching is a significant limitation in fluorescence microscopy. This phenomenon occurs when:
A) The fluorophore absorbs too much excitation light and degrades
B) The specimen is not properly fixed
C) The immersion oil contains fluorescent contaminants
🟢 D) The fluorophore is irreversibly photobleached by prolonged excitation, losing fluorescence
🔴 Correct Answer: D – Photo-bleaching is the irreversible destruction of fluorophores due to prolonged light exposure, reducing fluorescence signal.
🟢 11. Confocal microscopy offers improved resolution over conventional fluorescence microscopy primarily because:
🟢 A) It uses a pinhole to eliminate out-of-focus light, providing optical sectioning
B) It uses higher intensity lasers to saturate the fluorophores
C) It uses longer wavelength light to penetrate deeper
D) It uses digital image enhancement to improve resolution
, 🔴 Correct Answer: A – The pinhole in confocal microscopy blocks out-of-focus light, allowing optical sectioning and improved resolution.
🟢 12. Which of the following correctly describes the relationship between the pinhole size in confocal microscopy and the optical section thickness?
A) Larger pinhole = thinner optical section
🟢 B) Smaller pinhole = thinner optical section (better resolution)
C) Pinhole size does not affect optical section thickness
D) Pinhole size only affects brightness, not resolution
🔴 Correct Answer: B – Smaller pinholes block more out-of-focus light, resulting in thinner optical sections and better axial resolution, though at the cost
of reduced signal brightness.
🟢 13. Deconvolution microscopy is a computational technique that:
🟢 A) Uses mathematical algorithms to remove out-of-focus light from images
B) Uses physical pinholes to eliminate out-of-focus light
C) Uses laser beams to excite fluorophores
D) Uses electron beams to image specimens
🔴 Correct Answer: A – Deconvolution microscopy uses mathematical algorithms to computationally remove out-of-focus blur and improve image
resolution.
🟢 14. Super-resolution microscopy techniques such as STED, PALM, and STORM overcome the diffraction limit of light microscopy (≈200 nm) by:
A) Using longer wavelength light
B) Using higher numerical aperture objectives
🟢 C) Using specialized techniques that precisely control fluorophore emission
D) Using digital zoom to enlarge images
🔴 Correct Answer: C – Super-resolution techniques use specialized methods to control fluorophore emission (e.g., stimulated emission depletion,
photoactivation, stochastic switching) to achieve resolution below the diffraction limit.
🟢 15. The point spread function (PSF) of a microscope describes:
A) The total magnification of the system
B) The numerical aperture of the objective
🟢 C) The three-dimensional blurring pattern of a point source of light
D) The resolution limit of the human eye
🔴 Correct Answer: C – The PSF describes how a point source of light is spread out by the microscope optics, creating a three-dimensional diffraction
pattern.
SECTION 2: ADVANCED STAINING MECHANISMS & CHEMISTRY
🟢 16. The basic dyes used in simple staining (e.g., crystal violet, methylene blue) are cationic (positively charged). These dyes bind to bacterial cells
because:
A) Bacterial cell walls contain positively charged proteins
🟢 B) Bacterial cell walls contain negatively charged components (teichoic acids, LPS, and phospholipids)
C) Bacterial cells are electrically neutral but have hydrophobic properties
D) Bacterial cells are positively charged due to their high sodium content
🔴 Correct Answer: B – Basic dyes bind to negatively charged bacterial cell surface components, including teichoic acids, lipopolysaccharides, and
phospholipid phosphate groups.
🟢 17. An acidic dye such as nigrosin or India ink is used in negative staining. These dyes are anionic and:
Advanced Comprehensive Question Bank v2.0
SECTION 1: MICROSCOPY THEORY & ADVANCED OPTICS
🟢 1. The resolving power of a microscope is mathematically expressed by the Abbe equation. Which of the following modifications would most
effectively increase the resolution of a light microscope?
A) Decreasing the numerical aperture of the objective lens from 1.4 to 0.25
B) Increasing the wavelength of illumination from 450 nm to 650 nm
🟢 C) Decreasing the wavelength of illumination from 550 nm to 380 nm while using an objective with NA 1.4
D) Decreasing the condenser numerical aperture from 1.25 to 0.90
🔴 Correct Answer: C – The Abbe equation (d = λ / 2NA) shows that resolution improves (smaller d) with shorter wavelength (λ) and higher numerical
aperture (NA). Decreasing wavelength to 380 nm (violet/UV range) significantly improves resolution when combined with high NA.
🟢 2. A microbiologist is examining a specimen under a 100x oil immersion objective with a numerical aperture of 1.4. The microscope has an ocular
lens magnification of 10x. If the working distance of this objective is 0.12 mm, what is the total magnification and approximate resolving power using
green light (550 nm)?
A) 1,000x total magnification; 0.196 μm resolution
B) 1,000x total magnification; 0.275 μm resolution
🟢 C) 1,000x total magnification; 0.196 μm resolution
D) 1,400x total magnification; 0.196 μm resolution
🔴 Correct Answer: C – Total magnification = 100 × 10 = 1,000x. Resolution (d) = λ / 2NA = 550 nm / (2 × 1.4) = .8 = 196.4 nm ≈ 0.196 μm.
🟢 3. The refractive index of immersion oil is approximately 1.515. What is the primary reason this specific refractive index is required for oil immersion
microscopy?
🟢 A) It matches the refractive index of glass (≈1.52), eliminating light refraction at the air-glass interface
B) It increases the wavelength of light reaching the specimen
C) It decreases the numerical aperture of the objective
D) It allows the use of lower intensity illumination
🔴 Correct Answer: A – Oil with refractive index matching glass prevents light scattering at the air-glass interface. Without oil, light refracts away from the
objective, reducing the numerical aperture and resolution.
🟢 4. A student is using a bright-field microscope and notices a halo artifact around the specimen when using the 100x objective. This is most likely
due to:
A) The condenser being fully closed
B) The objective being dirty
🟢 C) Improper use of immersion oil (air bubbles or incorrect amount)
D) The eyepiece being contaminated
🔴 Correct Answer: C – Air bubbles or incorrect oil application cause light scattering and halo artifacts around the specimen.
🟢 5. Which of the following adjustments would NOT improve the image quality when viewing an unstained, transparent bacterial specimen in a
bright-field microscope?
A) Closing the iris diaphragm to increase contrast
B) Reducing the light intensity
C) Using a higher numerical aperture objective
🟢 D) Using a lower numerical aperture condenser
,🔴 Correct Answer: D – Using a lower NA condenser would decrease resolution and image quality. Closing the iris, reducing light, and using high NA
objectives all improve contrast and resolution for unstained specimens.
🟢 6. The depth of field in a microscope is inversely proportional to the numerical aperture. Which of the following correctly describes the relationship
between NA and depth of field?
A) Higher NA = greater depth of field
🟢 B) Higher NA = shallower depth of field (thinner optical section)
C) Lower NA = shallower depth of field
D) NA does not affect depth of field
🔴 Correct Answer: B – Higher numerical aperture results in a shallower depth of field, which is why high power objectives require fine focusing
adjustments.
🟢 7. Chromatic aberration occurs when:
A) The specimen is not properly stained
B) The condenser is not centered
🟢 C) Different wavelengths of light are refracted to different degrees, causing color fringing
D) The objective lens is scratched
🔴 Correct Answer: C – Chromatic aberration is a lens defect where different wavelengths of light focus at different points, creating color fringing. This is
corrected in achromatic and apochromatic lenses.
🟢 8. Which of the following microscope configurations would provide the BEST resolution, assuming all else is equal?
A) Objective: 40x, NA 0.65, light source: 550 nm green
B) Objective: 60x, NA 0.85, light source: 550 nm green
C) Objective: 100x, NA 1.25, light source: 550 nm green
🟢 D) Objective: 100x, NA 1.40, light source: 450 nm blue
🔴 Correct Answer: D – Resolution improves with higher NA and shorter wavelength. The 100x NA 1.4 objective with 450 nm blue light provides the best
resolving power.
🟢 9. A researcher is using a fluorescence microscope to visualize GFP-tagged proteins in live bacterial cells. The excitation wavelength for GFP is
approximately 488 nm with emission at 509 nm. Which of the following filter cube configurations is appropriate?
🟢 A) Excitation filter: 488 nm, dichroic mirror: 505 nm, emission filter: 509 nm (bandpass)
B) Excitation filter: 509 nm, dichroic mirror: 488 nm, emission filter: 505 nm
C) Excitation filter: 505 nm, dichroic mirror: 488 nm, emission filter: 509 nm
D) Excitation filter: 509 nm, dichroic mirror: 505 nm, emission filter: 488 nm
🔴 Correct Answer: A – The excitation filter must pass 488 nm (excitation wavelength), the dichroic mirror must reflect excitation light and pass longer
wavelengths, and the emission filter must pass 509 nm (emission wavelength).
🟢 10. Photo-bleaching is a significant limitation in fluorescence microscopy. This phenomenon occurs when:
A) The fluorophore absorbs too much excitation light and degrades
B) The specimen is not properly fixed
C) The immersion oil contains fluorescent contaminants
🟢 D) The fluorophore is irreversibly photobleached by prolonged excitation, losing fluorescence
🔴 Correct Answer: D – Photo-bleaching is the irreversible destruction of fluorophores due to prolonged light exposure, reducing fluorescence signal.
🟢 11. Confocal microscopy offers improved resolution over conventional fluorescence microscopy primarily because:
🟢 A) It uses a pinhole to eliminate out-of-focus light, providing optical sectioning
B) It uses higher intensity lasers to saturate the fluorophores
C) It uses longer wavelength light to penetrate deeper
D) It uses digital image enhancement to improve resolution
, 🔴 Correct Answer: A – The pinhole in confocal microscopy blocks out-of-focus light, allowing optical sectioning and improved resolution.
🟢 12. Which of the following correctly describes the relationship between the pinhole size in confocal microscopy and the optical section thickness?
A) Larger pinhole = thinner optical section
🟢 B) Smaller pinhole = thinner optical section (better resolution)
C) Pinhole size does not affect optical section thickness
D) Pinhole size only affects brightness, not resolution
🔴 Correct Answer: B – Smaller pinholes block more out-of-focus light, resulting in thinner optical sections and better axial resolution, though at the cost
of reduced signal brightness.
🟢 13. Deconvolution microscopy is a computational technique that:
🟢 A) Uses mathematical algorithms to remove out-of-focus light from images
B) Uses physical pinholes to eliminate out-of-focus light
C) Uses laser beams to excite fluorophores
D) Uses electron beams to image specimens
🔴 Correct Answer: A – Deconvolution microscopy uses mathematical algorithms to computationally remove out-of-focus blur and improve image
resolution.
🟢 14. Super-resolution microscopy techniques such as STED, PALM, and STORM overcome the diffraction limit of light microscopy (≈200 nm) by:
A) Using longer wavelength light
B) Using higher numerical aperture objectives
🟢 C) Using specialized techniques that precisely control fluorophore emission
D) Using digital zoom to enlarge images
🔴 Correct Answer: C – Super-resolution techniques use specialized methods to control fluorophore emission (e.g., stimulated emission depletion,
photoactivation, stochastic switching) to achieve resolution below the diffraction limit.
🟢 15. The point spread function (PSF) of a microscope describes:
A) The total magnification of the system
B) The numerical aperture of the objective
🟢 C) The three-dimensional blurring pattern of a point source of light
D) The resolution limit of the human eye
🔴 Correct Answer: C – The PSF describes how a point source of light is spread out by the microscope optics, creating a three-dimensional diffraction
pattern.
SECTION 2: ADVANCED STAINING MECHANISMS & CHEMISTRY
🟢 16. The basic dyes used in simple staining (e.g., crystal violet, methylene blue) are cationic (positively charged). These dyes bind to bacterial cells
because:
A) Bacterial cell walls contain positively charged proteins
🟢 B) Bacterial cell walls contain negatively charged components (teichoic acids, LPS, and phospholipids)
C) Bacterial cells are electrically neutral but have hydrophobic properties
D) Bacterial cells are positively charged due to their high sodium content
🔴 Correct Answer: B – Basic dyes bind to negatively charged bacterial cell surface components, including teichoic acids, lipopolysaccharides, and
phospholipid phosphate groups.
🟢 17. An acidic dye such as nigrosin or India ink is used in negative staining. These dyes are anionic and:
