Bioautographic techniques MCQs With Answer

Introduction: Bioautographic techniques combine chromatographic separation with biological assays to detect bioactive compounds directly on chromatographic media. For M.Pharm students, mastering these methods is essential for natural product screening, antimicrobial discovery, and enzyme inhibitor identification. This blog presents focused multiple-choice questions that probe the principles, practical setup, selection of indicator organisms, detection chemistries, variants of bioautography (direct/contact/agar-overlay/HPTLC-bioautography), and interpretation of results. Emphasis is placed on experimental variables—plate types, solvents, incubation conditions, controls, and limitations—to build competence in designing, troubleshooting, and critically evaluating bioautographic experiments commonly used in bioprocess and pharmaceutical research.

Q1. Which principle best describes bioautography?

• Separation of compounds by electrophoresis followed by mass spectrometry
• Chromatographic separation followed by in situ biological detection on the stationary phase
• Direct microbial culture without chromatographic separation
• Quantitative HPLC analysis using UV detection

Correct Answer: Chromatographic separation followed by in situ biological detection on the stationary phase

Q2. Which of the following is NOT a common variant of bioautography?

• Direct contact bioautography
• Agar-overlay bioautography
• Fluorescent plate bioautography
• High-performance thin-layer chromatographic (HPTLC) bioautography

Correct Answer: Fluorescent plate bioautography

Q3. In agar-overlay bioautography, why is a thin agar layer used to cover the chromatogram?

• To solubilize all separated compounds for extraction into solvent
• To allow diffusion of bioactive compounds from the stationary phase into the agar for microbial contact
• To prevent solvent evaporation during chromatography
• To provide nutrients for column stationary phase regeneration

Correct Answer: To allow diffusion of bioactive compounds from the stationary phase into the agar for microbial contact

Q4. Which indicator is commonly used to reveal bacterial viability in bioautography by changing color upon metabolic activity?

• Resazurin
• Silver nitrate
• Phenolphthalein
• Coomassie Blue

Correct Answer: Resazurin

Q5. Which stationary phase is most frequently used for TLC-based bioautography?

• Silica gel
• Ion-exchange resin
• Reverse osmosis membrane
• Polystyrene plate

Correct Answer: Silica gel

Q6. What is the main advantage of HPTLC-bioautography over conventional TLC-bioautography?

• HPTLC uses thicker plates that prevent diffusion
• Higher resolution and better reproducibility allowing finer separation and localized bioactivity detection
• HPTLC eliminates the need for indicator organisms
• HPTLC plates are disposable and cheaper than TLC plates

Correct Answer: Higher resolution and better reproducibility allowing finer separation and localized bioactivity detection

Q7. When performing contact bioautography, what is a critical procedural step to ensure transfer of compounds to the agar?

• Heating the chromatogram to 100°C before contact
• Applying a cold press for immediate freezing
• Ensuring close, uniform contact between TLC plate and agar surface for a defined time
• Immersing both plate and agar in organic solvent

Correct Answer: Ensuring close, uniform contact between TLC plate and agar surface for a defined time

Q8. Which factor is most likely to cause false-negative results in bioautography assays?

• Use of an indicator strain that is highly sensitive to the analyte
• Poor diffusion of nonpolar compounds into the aqueous agar medium
• Applying too thin an agar overlay
• Using resazurin as a metabolic indicator

Correct Answer: Poor diffusion of nonpolar compounds into the aqueous agar medium

Q9. For detecting enzyme inhibitors by bioautography, what modification helps visualize inhibition zones?

• Incorporation of a chromogenic or fluorogenic substrate in the overlay to reveal enzyme activity
• Pre-staining the plate with silver stain
• Heating the plate to denature the enzyme
• Adding antibiotics to the overlay medium

Correct Answer: Incorporation of a chromogenic or fluorogenic substrate in the overlay to reveal enzyme activity

Q10. Which control is essential when interpreting antimicrobial bioautography results?

• An overlay lacking agar
• A plate developed with an inert solvent and overlaid with the same microorganism to check for solvent effects
• A chromatogram developed with pure water and left untested
• A plate incubated without any microorganism as an enzyme control

Correct Answer: A plate developed with an inert solvent and overlaid with the same microorganism to check for solvent effects

Q11. Which detection approach is appropriate when bioautography uses viable bacteria but no visible inhibition zones are produced?

• Use of metabolic viability stains (e.g., tetrazolium salts) to reveal localized reduced metabolism
• Switching to gas chromatography for direct bioactivity detection
• Measuring fluorescence of silica gel under UV only
• Applying heat to induce visible zones

Correct Answer: Use of metabolic viability stains (e.g., tetrazolium salts) to reveal localized reduced metabolism

Q12. When correlating an inhibition zone on a bioautogram to the separated compound, what analytical step is commonly performed next?

• Quantitative PCR of the inhibition zone
• Scraping the silica at the zone, eluting the compound, and analyzing by LC-MS or NMR
• Direct titration of the agar with strong acid
• Measuring the zone diameter and correlating to MIC values without further analysis

Correct Answer: Scraping the silica at the zone, eluting the compound, and analyzing by LC-MS or NMR

Q13. Which solvent property is most critical when selecting a developing solvent for bioautography of natural products?

• Ability to fluoresce at 254 nm
• Compatibility with both silica and the biological overlay to minimize toxic residues and ensure compound transfer
• High UV absorption to aid visualization
• Viscosity greater than 10 cP

Correct Answer: Compatibility with both silica and the biological overlay to minimize toxic residues and ensure compound transfer

Q14. In agar-overlay bioautography, what is the typical rationale for using a low agar concentration (e.g., 0.75–1.0%)?

• To solidify the agar faster
• To enhance diffusion of compounds from the chromatogram into the overlay
• To increase nutrient content for the microorganisms
• To prevent microbial growth entirely

Correct Answer: To enhance diffusion of compounds from the chromatogram into the overlay

Q15. Which microbial strain selection principle improves the detection of a target antimicrobial class in bioautography?

• Choosing a strain with intrinsic resistance to the target class
• Using an indicator strain with known susceptibility profile matched to the expected mode of action
• Selecting any fast-growing environmental isolate without characterization
• Using fungal strains exclusively for antibacterial screening

Correct Answer: Using an indicator strain with known susceptibility profile matched to the expected mode of action

Q16. What is a common limitation of direct bioautography compared with agar-overlay methods?

• Direct bioautography provides higher sensitivity for nonpolar compounds
• It may require harsh staining that kills indicator organisms, reducing viability assessment
• Direct bioautography always uses radioactive detection
• It eliminates the need for chromatographic separation

Correct Answer: It may require harsh staining that kills indicator organisms, reducing viability assessment

Q17. Why might a researcher choose to perform bioautography at two different incubation temperatures?

• To ensure agar solidification at both temperatures
• Different microorganisms or enzyme activities have optimal temperatures, revealing broader activity profiles or thermolabile activity
• To change solvent polarity during incubation
• To accelerate elution of compounds from silica

Correct Answer: Different microorganisms or enzyme activities have optimal temperatures, revealing broader activity profiles or thermolabile activity

Q18. Which of the following improves semi-quantitative estimation of activity from bioautograms?

• Measuring Rf values only
• Creating calibration curves by spotting known concentrations of active standard and comparing inhibition zone intensity or area
• Using thicker silica layers to trap more compound
• Applying more solvent to the chromatogram after overlay

Correct Answer: Creating calibration curves by spotting known concentrations of active standard and comparing inhibition zone intensity or area

Q19. Which post-bioautography staining reveals bacterial zones of inhibition by producing a white zone against a colored background through tetrazolium reduction?

• Vanillin-sulfuric acid
• MTT or TTC (tetrazolium salts) staining
• Dragendorff’s reagent
• Ninhydrin spray

Correct Answer: MTT or TTC (tetrazolium salts) staining

Q20. When designing a bioautographic screen for lipophilic antibiotics, which adaptation increases detection sensitivity?

• Using a purely aqueous overlay without emulsifiers
• Incorporating a small percentage of a non-toxic surfactant or solvent in the overlay to aid diffusion of lipophilic compounds
• Drying the developed plate at high temperature to fix compounds
• Covering the plate with plastic wrap to prevent contamination

Correct Answer: Incorporating a small percentage of a non-toxic surfactant or solvent in the overlay to aid diffusion of lipophilic compounds

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