Methods for standardization of antibiotics MCQs With Answer

Introduction: Methods for standardization of antibiotics MCQs With Answer is an essential resource for B. Pharm students focusing on antibiotic potency, bioassays, and analytical techniques. This guide covers key methods such as microbiological assays (agar diffusion, tube and plate methods), physicochemical assays (HPLC, UV spectrophotometry), MIC/MBC determination, and USP/CLSI standards. Emphasis is placed on assay validation parameters—accuracy, precision, linearity, LOD/LOQ—and use of reference standards, control strains, and calibration curves to ensure reproducible potency results. Practical principles, sample preparation, and interpretation of results are highlighted to build exam-ready competence. Now let’s test your knowledge with 50 MCQs on this topic.

Q1. Which method measures antibiotic potency by comparing inhibition zones produced by sample and reference on an agar plate?

• Agar diffusion bioassay
• HPLC assay
• UV spectrophotometry
• Mass spectrometry

Correct Answer: Agar diffusion bioassay

Q2. In broth microdilution, the lowest concentration of antibiotic that prevents visible growth is called:

• Minimum bactericidal concentration
• Minimum inhibitory concentration
• Minimum selective concentration
• Maximum tolerated concentration

Correct Answer: Minimum inhibitory concentration

Q3. Which analytical method is most suitable for quantifying antibiotic components with high specificity and sensitivity?

• Plate count
• HPLC
• Kirby-Bauer
• Agar well diffusion

Correct Answer: HPLC

Q4. In microbiological assay validation, which parameter assesses agreement between measured and true values?

• Precision
• Specificity
• Accuracy
• Robustness

Correct Answer: Accuracy

Q5. The log-dose response relationship in antibiotic bioassays is used to calculate:

• Retention time
• Potency/slope ratio
• pH stability
• Solubility

Correct Answer: Potency/slope ratio

Q6. Which control organism is commonly used for validating assays of penicillin?

• Staphylococcus aureus
• Escherichia coli
• Bacillus subtilis
• Pseudomonas aeruginosa

Correct Answer: Bacillus subtilis

Q7. What does MBC represent in antibiotic testing?

• The concentration that inhibits growth
• The lowest concentration that kills 99.9% of organisms
• The time to achieve bacteriostasis
• The concentration causing growth enhancement

Correct Answer: The lowest concentration that kills 99.9% of organisms

Q8. Which guideline is widely referenced for antimicrobial susceptibility testing standards?

• ICH Q2
• CLSI
• ISO 9001
• EMA GMP Annex 1

Correct Answer: CLSI

Q9. In HPLC method validation, which parameter assesses instrument response reproducibility?

• Linearity
• Precision
• Specificity
• Stability

Correct Answer: Precision

Q10. The agar well diffusion assay measures antibiotic activity by:

• Counting surviving colonies
• Measuring zone diameters around wells
• Recording UV absorbance
• Calculating peak area in HPLC

Correct Answer: Measuring zone diameters around wells

Q11. What is the main disadvantage of microbiological assays compared to HPLC?

• Lower specificity and higher variability
• Higher cost and complexity
• Cannot detect activity
• Requires mass spectrometer

Correct Answer: Lower specificity and higher variability

Q12. In a cylinder plate assay, uniform diffusion is ensured by:

• Using gradient elution
• Applying equal volumes and consistent agar depth
• Heating the agar to 100°C
• Varying inoculum size across plates

Correct Answer: Applying equal volumes and consistent agar depth

Q13. Potency of an antibiotic in international units (IU) is determined using:

• Physicochemical purity only
• Biological reference standards
• Only HPLC peak areas
• Gram staining

Correct Answer: Biological reference standards

Q14. Which factor most affects zone sizes in agar diffusion assays?

• Mobile phase composition
• Diffusion coefficient and inoculum density
• Column temperature
• Detector wavelength

Correct Answer: Diffusion coefficient and inoculum density

Q15. The purpose of standard curve in antibiotic bioassay is to:

• Determine molecular weight
• Relate concentration to biological response for potency estimation
• Measure pH stability
• Estimate color change

Correct Answer: Relate concentration to biological response for potency estimation

Q16. Which dilution method is preferred for determining MIC in a high-throughput format?

• Tube macrodilution
• Broth microdilution
• Agar streak method
• Disk diffusion

Correct Answer: Broth microdilution

Q17. In HPLC assay of antibiotics, a C18 column is often chosen because it provides:

• Strong ion exchange
• Reversed-phase separation for moderately nonpolar compounds
• Chiral separation
• Gas-phase separation

Correct Answer: Reversed-phase separation for moderately nonpolar compounds

Q18. Which validation parameter ensures the method measures the intended analyte in presence of excipients?

• Accuracy
• Specificity
• Precision
• Robustness

Correct Answer: Specificity

Q19. A 3×3 parallel line assay in microbiological standardization is used to:

• Determine MIC only
• Compare potency of sample and standard across concentrations
• Measure HPLC retention time reproducibility
• Assess sterility

Correct Answer: Compare potency of sample and standard across concentrations

Q20. When interpreting MIC results, a bacteriostatic antibiotic endpoint is usually:

• Complete sterilization
• Prevention of visible growth
• Increase in turbidity
• Colorimetric change to red

Correct Answer: Prevention of visible growth

Q21. Which preservative or excipient can interfere with microbiological assays?

• Sodium chloride at isotonic levels
• Residual disinfectants like chlorhexidine
• Pure water
• Sterile agar

Correct Answer: Residual disinfectants like chlorhexidine

Q22. Limit of detection (LOD) in an analytical method indicates:

• The highest concentration that can be measured accurately
• The smallest concentration that can be reliably distinguished from blank
• Amount of solvent required
• Time for complete degradation

Correct Answer: The smallest concentration that can be reliably distinguished from blank

Q23. Which antibiotic class commonly requires acid labile handling during assay preparation?

• Penicillins
• Macrolides
• Aminoglycosides
• Fluoroquinolones

Correct Answer: Penicillins

Q24. In HPLC assay, an internal standard is used to:

• Improve peak symmetry only
• Correct for injection and detector variability
• Change retention time
• Denature analyte

Correct Answer: Correct for injection and detector variability

Q25. Which microbiological assay format is most appropriate to determine bactericidal activity (MBC)?

• Plate subculturing from MIC wells
• HPLC area comparison
• UV absorbance at 600 nm only
• Disk diffusion diameter measurement

Correct Answer: Plate subculturing from MIC wells

Q26. During bioassay, the term “parallelism” refers to:

• Two dose-response lines having similar slopes indicating comparable potency
• Running two HPLC columns side by side
• Using two different detectors
• Simultaneous sterilization methods

Correct Answer: Two dose-response lines having similar slopes indicating comparable potency

Q27. Which mobile phase component is commonly used for reversed-phase HPLC of antibiotics?

• Hexane
• Water and acetonitrile or methanol
• Pure chloroform
• Formalin

Correct Answer: Water and acetonitrile or methanol

Q28. A robustness study evaluates:

• Effect of small deliberate changes in method parameters on results
• Long-term stability of reference standard
• Microbial contamination rate
• Molecular weight distribution

Correct Answer: Effect of small deliberate changes in method parameters on results

Q29. Which detection mode in HPLC is most common for antibiotics with chromophores?

• Refractive index detector
• UV-Visible detector
• Flame ionization detector
• Electrochemical detector

Correct Answer: UV-Visible detector

Q30. In agar diffusion, increasing agar depth generally causes:

• Larger zones of inhibition
• Smaller zones of inhibition due to reduced diffusion
• No effect on zones
• Color change of medium

Correct Answer: Smaller zones of inhibition due to reduced diffusion

Q31. For antibiotics prone to oxidation, which storage measure improves assay stability?

• Storing at elevated temperature
• Protecting from light and using inert atmosphere
• Adding strong oxidizers
• Frequent freeze-thaw cycles

Correct Answer: Protecting from light and using inert atmosphere

Q32. Which statistical test is commonly used to compare potency estimates between sample and standard in bioassays?

• ANOVA and parallel-line analysis
• Chi-square for proportions
• Mann-Whitney U only
• Fisher’s exact test

Correct Answer: ANOVA and parallel-line analysis

Q33. In antibiotic standardization, the reference standard should be:

• Uncharacterized crude extract
• Certified with known potency and expiry
• Prepared freshly without documentation
• Any commercially available tablet

Correct Answer: Certified with known potency and expiry

Q34. Disk diffusion (Kirby-Bauer) results are interpreted using:

• Retention time charts
• Zone diameter breakpoints provided by guidelines
• HPLC calibration curves
• Gram stain morphology

Correct Answer: Zone diameter breakpoints provided by guidelines

Q35. Which parameter indicates linearity in an HPLC method?

• Correlation coefficient (r) close to 1 over working range
• Single peak only
• Retention time variability
• Detector noise level

Correct Answer: Correlation coefficient (r) close to 1 over working range

Q36. What is the effect of high inoculum size on MIC determination?

• May increase observed MIC values (inoculum effect)
• Always decreases MIC values
• Has no impact on MIC
• Makes HPLC peaks broader

Correct Answer: May increase observed MIC values (inoculum effect)

Q37. Which antibiotic assay requires sterile technique and aseptic handling throughout?

• HPLC analysis in non-sterile lab only
• Microbiological bioassays (broth and agar methods)
• UV spectrophotometry of non-biological standards
• Mass spectrometry with dried samples

Correct Answer: Microbiological bioassays (broth and agar methods)

Q38. In potency calculation using slope ratio method, identical slopes indicate:

• Non-parallelism and invalid assay
• Parallelism and comparability, allowing potency estimation
• Need for different standards
• Failure of standard stability

Correct Answer: Parallelism and comparability, allowing potency estimation

Q39. Which factor is critical when selecting the indicator organism for an antibiotic bioassay?

• Organism nutritional requirements only
• Sensitivity and reproducible response to the antibiotic class
• Color of colonies only
• Ability to grow at 4°C only

Correct Answer: Sensitivity and reproducible response to the antibiotic class

Q40. An assay specificity study should demonstrate that:

• Other substances do not interfere with analyte measurement
• Method is only precise
• All excipients produce similar signals
• Reference standard is unnecessary

Correct Answer: Other substances do not interfere with analyte measurement

Q41. Which technique can separate antibiotic degradation products from the parent compound?

• Disk diffusion
• HPLC with appropriate chromatographic conditions
• Simple dilution in water
• Surface tension measurement

Correct Answer: HPLC with appropriate chromatographic conditions

Q42. What is the primary advantage of broth microdilution over tube macrodilution?

• Lower throughput
• Higher reagent and sample economy with high throughput capability
• Requires larger volumes
• Less sensitive endpoint detection

Correct Answer: Higher reagent and sample economy with high throughput capability

Q43. During assay validation, repeatability refers to:

• Variation under different labs and analysts
• Variation under same conditions over short time by same analyst
• Stability under shipping conditions
• Long-term storage degradation

Correct Answer: Variation under same conditions over short time by same analyst

Q44. Which solvent property is important when preparing antibiotic solutions for HPLC?

• Evaporation rate only
• Solubility of analyte and compatibility with mobile phase
• Color alone
• Magnetic susceptibility

Correct Answer: Solubility of analyte and compatibility with mobile phase

Q45. Stability-indicating assay should be able to:

• Detect and quantify analyte only when intact
• Separate and quantify degradation products and unchanged drug
• Only measure pH changes
• Only measure potency in IU

Correct Answer: Separate and quantify degradation products and unchanged drug

Q46. Which parameter is assessed by forced degradation studies?

• Method specificity under stress conditions and degradation pathways
• Only microbial contamination
• Routine precision
• Color of formulation

Correct Answer: Method specificity under stress conditions and degradation pathways

Q47. Which is NOT a microbiological method for antibiotic standardization?

• Agar diffusion
• Tube dilution
• HPLC peak area comparison
• Broth microdilution

Correct Answer: HPLC peak area comparison

Q48. When calculating final potency from a bioassay, you must account for:

• Only sample color
• Dilution factors and reference standard potency
• Detector wavelength only
• Column length only

Correct Answer: Dilution factors and reference standard potency

Q49. Which of the following best describes the role of USP monographs in antibiotic standardization?

• Provide unrelated clinical guidelines
• Specify official assays, reference standards, and acceptance criteria
• Only list brand names
• Mandate hospital dosing schedules

Correct Answer: Specify official assays, reference standards, and acceptance criteria

Q50. In designing a bioassay, why is it important to include multiple concentrations of standard and sample?

• To assess only color change
• To establish dose-response relationship and ensure parallelism for potency estimation
• To increase agar turbidity
• To avoid statistical analysis

Correct Answer: To establish dose-response relationship and ensure parallelism for potency estimation

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

Mail- Sachin@pharmacyfreak.com