Microbiological assays – principles MCQs With Answer

Microbiological assays – principles MCQs With Answer

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Introduction: Microbiological assays are essential bioassay techniques in B. Pharm curriculum, teaching principles of microbial enumeration, potency estimation, sterility testing, and antimicrobial activity measurement. Understanding assay design, controls, standard curves, statistical analysis and validation ensures reliable results in quality control, formulation testing and research. Key concepts include colony-forming units (CFU), most probable number (MPN), turbidimetric and plate-count methods, minimum inhibitory concentration (MIC), diffusion assays, endotoxin detection and preservative efficacy. This focused, keyword-rich overview helps B. Pharm students master microbiological assays — principles, calculations and interpretation — for practical exams and industrial applications. Now let’s test your knowledge with 50 MCQs on this topic.

Q1. Which principle best describes a turbidimetric microbiological assay?

  • Measurement of optical density proportional to microbial biomass
  • Counting colonies on solid medium after incubation
  • Measuring zones of inhibition around discs
  • Determining endotoxin levels using limulus amoebocyte lysate

Correct Answer: Measurement of optical density proportional to microbial biomass

Q2. In a plate count method, one colony typically represents:

  • A single viable cell or a clump of cells capable of forming a colony
  • A dead cell
  • A viral particle
  • If incubated, a chemical precipitate

Correct Answer: A single viable cell or a clump of cells capable of forming a colony

Q3. The Most Probable Number (MPN) method is most useful when:

  • Microbes are present in very low numbers or growth cannot be readily plated
  • Rapid quantification within minutes is required
  • Measuring endotoxin contamination in parenterals
  • Assessing antimicrobial potency by zone sizes

Correct Answer: Microbes are present in very low numbers or growth cannot be readily plated

Q4. In a diffusion assay (disk diffusion), the primary endpoint is:

  • Diameter of zone of inhibition indicating antimicrobial activity
  • Optical density change over time
  • CFU counted on a selective agar
  • Endotoxin-induced gelation time

Correct Answer: Diameter of zone of inhibition indicating antimicrobial activity

Q5. Which factor does NOT significantly influence plate count results?

  • Incubation time and temperature
  • Dilution accuracy and plating technique
  • Type of agar medium used
  • Color of the incubator walls

Correct Answer: Color of the incubator walls

Q6. The purpose of using a reference standard in biological assays is to:

  • Provide a basis for comparing potency and ensure assay validity
  • Contaminate the sample intentionally
  • Reduce incubation time
  • Increase turbidity without biological relevance

Correct Answer: Provide a basis for comparing potency and ensure assay validity

Q7. Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of an antimicrobial that:

  • Prevents visible growth of a microorganism after incubation
  • Kills 100% of cells within 1 hour
  • Produces a zone of inhibition of exactly 10 mm
  • Enhances microbial growth

Correct Answer: Prevents visible growth of a microorganism after incubation

Q8. A parallel-line assay is used in microbiological potency estimation to:

  • Compare dose-response curves of sample and standard assuming linearity
  • Measure endotoxin units directly
  • Detect presence of gram-negative bacteria only
  • Determine turbidity without standards

Correct Answer: Compare dose-response curves of sample and standard assuming linearity

Q9. Which statistical method is commonly used to calculate potency from quantal bioassay data?

  • Probit or logit analysis
  • Student’s t-test only
  • ANOVA without further modeling
  • Simple subtraction

Correct Answer: Probit or logit analysis

Q10. Sterility testing for pharmaceutical products primarily aims to detect:

  • Viable contaminating microorganisms in aseptic or terminally sterilized products
  • Chemical impurities like heavy metals
  • Endotoxin concentration in IU/ml accurately
  • Particle size distribution

Correct Answer: Viable contaminating microorganisms in aseptic or terminally sterilized products

Q11. In preservative efficacy testing (PET), the main criterion is:

  • Reduction in microbial counts over specified time points according to pharmacopeial limits
  • Increase in turbidity after preservative addition
  • Zone of inhibition size for preservatives
  • Change in pH only

Correct Answer: Reduction in microbial counts over specified time points according to pharmacopeial limits

Q12. Which medium is considered non-selective and supports a wide range of bacterial growth for total aerobic counts?

  • Nutrient agar
  • MacConkey agar
  • Sabouraud dextrose agar
  • Mannitol salt agar

Correct Answer: Nutrient agar

Q13. Endotoxin detection by LAL (Limulus Amebocyte Lysate) assay is based on:

  • Activation of clotting cascade in horseshoe crab blood components by lipopolysaccharide
  • Color change due to bacterial metabolism
  • Growth inhibition of indicator organisms
  • Precipitation of proteins in sample

Correct Answer: Activation of clotting cascade in horseshoe crab blood components by lipopolysaccharide

Q14. What is the main advantage of using a quantitative plate count over turbidity measurement?

  • Provides viable count (CFU) allowing direct estimation of viable organisms
  • Faster results within seconds
  • Unaffected by particulate matter
  • Measures endotoxin levels directly

Correct Answer: Provides viable count (CFU) allowing direct estimation of viable organisms

Q15. A bacteriostatic agent will:

  • Inhibit growth but not necessarily kill bacteria
  • Instantly lyse bacterial cells
  • Enhance bacterial replication
  • Neutralize endotoxins

Correct Answer: Inhibit growth but not necessarily kill bacteria

Q16. When validating a microbiological assay, which parameter assesses the ability to get the same result under identical conditions?

  • Repeatability (precision)
  • Linearity
  • Specificity
  • Robustness

Correct Answer: Repeatability (precision)

Q17. In an agar diffusion assay, a larger zone of inhibition typically indicates:

  • Greater antimicrobial potency or diffusion rate for that agent
  • Higher endotoxin content
  • Increased turbidity of the medium
  • Lower microbial susceptibility

Correct Answer: Greater antimicrobial potency or diffusion rate for that agent

Q18. Which method is most appropriate to distinguish bactericidal from bacteriostatic activity?

  • Minimum bactericidal concentration (MBC) determination following MIC
  • Disk diffusion only
  • Turbidimetric measurement alone
  • Endotoxin assay

Correct Answer: Minimum bactericidal concentration (MBC) determination following MIC

Q19. A parallel-line bioassay requires which of the following assumptions?

  • Sample and standard dose-response curves are linear and parallel over the tested range
  • Responses are binary only
  • Only a single concentration is tested
  • No replicates are needed

Correct Answer: Sample and standard dose-response curves are linear and parallel over the tested range

Q20. Selective media are used primarily to:

  • Suppress growth of unwanted organisms and favor target microorganism growth
  • Provide maximal growth for all microbes
  • Measure endotoxin by colorimetric change
  • Indiscriminately count all microbes

Correct Answer: Suppress growth of unwanted organisms and favor target microorganism growth

Q21. In microbiological assays, “specificity” refers to:

  • The ability of the assay to measure the analyte of interest without interference
  • The repeatability of measurements
  • The slope of the standard curve
  • Time required for incubation

Correct Answer: The ability of the assay to measure the analyte of interest without interference

Q22. What is a critical control in sterility testing to detect inhibition of microbial growth by the sample matrix?

  • Growth promotion test (positive control)
  • Negative control only
  • Using heat-killed organism only
  • Blank medium without inoculum

Correct Answer: Growth promotion test (positive control)

Q23. Which unit is commonly used to express bacterial counts from plate counts?

  • CFU per milliliter (CFU/ml)
  • Weight percent
  • Optical density units only
  • International Units per ml for all microbes

Correct Answer: CFU per milliliter (CFU/ml)

Q24. During serial dilution for plate counts, which practice reduces counting errors?

  • Performing appropriate dilutions to obtain 30–300 colonies per plate
  • Plating undiluted high-concentration samples only
  • Mixing by shaking vigorously without sterile technique
  • Incubating at arbitrary temperatures

Correct Answer: Performing appropriate dilutions to obtain 30–300 colonies per plate

Q25. Which assay principle is used to determine potency of antibiotics that inhibit growth by measuring zones around wells containing different concentrations?

  • Agar well diffusion bioassay
  • Endotoxin gel-clot assay
  • Electron microscopy
  • Gram staining

Correct Answer: Agar well diffusion bioassay

Q26. In turbidimetric assays, Beer-Lambert law applies when:

  • Absorbance is directly proportional to cell concentration within a linear range
  • All particles are larger than 1 mm
  • There is no need for calibration
  • Only for colored chemical assays, not microbes

Correct Answer: Absorbance is directly proportional to cell concentration within a linear range

Q27. Which control ensures that reagents and media are sterile in a microbiological assay?

  • Sterility (negative) control with uninoculated medium
  • Positive growth control
  • Standard curve only
  • Endotoxin-positive control

Correct Answer: Sterility (negative) control with uninoculated medium

Q28. The term “limit of detection” (LOD) in microbiological assays refers to:

  • The lowest number of organisms that can be reliably detected but not necessarily quantified
  • The maximum number of organisms that can be present
  • A fixed value of 100 CFU/ml always
  • The concentration at which turbidity is highest

Correct Answer: The lowest number of organisms that can be reliably detected but not necessarily quantified

Q29. For endotoxin testing, a sample that gives a positive gel-clot result must be:

  • Considered contaminated with endotoxin above the test sensitivity limit
  • Ignored because gel-clot is unreliable
  • Replaced by a plate count
  • Automatically sterile

Correct Answer: Considered contaminated with endotoxin above the test sensitivity limit

Q30. In microbiological assay validation, “robustness” refers to:

  • The assay’s resilience to small deliberate variations in method parameters
  • The total number of steps in the assay
  • The assay’s sensitivity only
  • The physical strength of lab equipment

Correct Answer: The assay’s resilience to small deliberate variations in method parameters

Q31. A viable but non-culturable (VBNC) state in bacteria affects which assay most significantly?

  • Plate count methods, underestimating true viability
  • Endotoxin LAL assay
  • Disk diffusion showing larger zones
  • Turbidimetric assays falsely elevated

Correct Answer: Plate count methods, underestimating true viability

Q32. The main difference between MIC and MBC is that:

  • MIC prevents visible growth; MBC kills organisms leading to no regrowth
  • MIC measures endotoxin; MBC measures turbidity
  • MIC is always higher than MBC
  • MBC is used only for fungi

Correct Answer: MIC prevents visible growth; MBC kills organisms leading to no regrowth

Q33. When constructing a standard curve for a quantitative microbiological assay, which is essential?

  • Use of known concentrations of standard and plotting response vs log concentration over linear range
  • Only one concentration of standard
  • Use random values without replication
  • Plotting concentration vs time only

Correct Answer: Use of known concentrations of standard and plotting response vs log concentration over linear range

Q34. Which organism is commonly used as a performance check for aseptic technique because it is non-fastidious and easy to culture?

  • Bacillus subtilis or Staphylococcus aureus depending on context
  • Mycobacterium tuberculosis
  • Hepatitis virus
  • Prions

Correct Answer: Bacillus subtilis or Staphylococcus aureus depending on context

Q35. Which assay would be most appropriate to quantify bacterial endospores in a preparation?

  • Heat-shock followed by plate count for spore-forming units
  • Turbidimetric optical density only
  • LAL endotoxin assay
  • Disk diffusion

Correct Answer: Heat-shock followed by plate count for spore-forming units

Q36. In antimicrobial bioassays, why are replicates important?

  • They allow estimation of variability and increase statistical confidence in results
  • They always reduce incubation time
  • They are used only to waste reagents
  • They guarantee a higher potency value

Correct Answer: They allow estimation of variability and increase statistical confidence in results

Q37. The presence of detergents or preservatives in a sample can affect microbial assays by:

  • Inhibiting growth leading to false negatives unless neutralized
  • Enhancing colony formation always
  • Only changing pH without biological effect
  • Improving turbidity readings only

Correct Answer: Inhibiting growth leading to false negatives unless neutralized

Q38. Which measurement indicates that a microbiological assay response is linear across tested concentrations?

  • A correlation coefficient (R2) close to 1 in the calibration curve’s linear range
  • A single data point at mid-range
  • High turbidity at all concentrations
  • Large variance between replicates

Correct Answer: A correlation coefficient (R2) close to 1 in the calibration curve’s linear range

Q39. What is the role of neutralizers in microbiological testing of disinfectants?

  • They inactivate residual disinfectant to allow recovery of surviving organisms
  • They enhance disinfectant activity during testing
  • They act as growth promoters
  • They color the medium for visualization

Correct Answer: They inactivate residual disinfectant to allow recovery of surviving organisms

Q40. Which technique best quantifies viable but low-abundance pathogens in water samples?

  • Enrichment culture followed by selective plating or molecular methods
  • Direct turbidity measurement
  • Standard disk diffusion
  • Endotoxin gel-clot assay

Correct Answer: Enrichment culture followed by selective plating or molecular methods

Q41. Quality control strains (e.g., ATCC strains) are used in microbiological assays to:

  • Ensure assay performance and comparability between runs
  • Replace unknown clinical isolates
  • Act as preservatives
  • Measure endotoxin levels

Correct Answer: Ensure assay performance and comparability between runs

Q42. What does “quantal” response mean in biological assays?

  • The outcome is all-or-none (e.g., alive/dead or response/no response) for individual test units
  • The response is measured on a continuous scale only
  • Response increases smoothly without threshold
  • Only turbidity is measured

Correct Answer: The outcome is all-or-none (e.g., alive/dead or response/no response) for individual test units

Q43. Bioburden testing of raw materials primarily measures:

  • Total viable microbial load before further processing
  • Endotoxin levels exclusively
  • Potency of antibiotics
  • pH of the material

Correct Answer: Total viable microbial load before further processing

Q44. In a diffusion assay, poor diffusion of a test compound may lead to:

  • Underestimation of antimicrobial activity due to small zones
  • False positive endotoxin results
  • Excessive colony counts
  • Increased turbidity readings

Correct Answer: Underestimation of antimicrobial activity due to small zones

Q45. Which of the following best describes “accuracy” in a microbiological assay?

  • Closeness of measured values to the true or accepted reference value
  • Repeatability of results only
  • Speed of the assay
  • Ability to tolerate operator errors

Correct Answer: Closeness of measured values to the true or accepted reference value

Q46. For antibiotics with a time-dependent killing mechanism, which pharmacodynamic parameter is most relevant?

  • Time above MIC (T>MIC)
  • Peak concentration/MIC (Cmax/MIC)
  • Total endotoxin units
  • Area of inhibition zone only

Correct Answer: Time above MIC (T>MIC)

Q47. Which practice minimizes contamination during environmental monitoring in a sterile manufacturing area?

  • Use of settle plates, active air samplers, and proper personnel gowning with regular monitoring
  • Opening product containers randomly
  • Using non-sterile swabs only
  • Allowing unrestricted personnel movement

Correct Answer: Use of settle plates, active air samplers, and proper personnel gowning with regular monitoring

Q48. What is the main limitation of turbidimetric methods for microbial enumeration?

  • They cannot distinguish live from dead cells and have limited linear range
  • They provide exact CFU counts always
  • They detect only endotoxins
  • They are unaffected by cell clumping

Correct Answer: They cannot distinguish live from dead cells and have limited linear range

Q49. Which parameter must be included when reporting results of a validated microbiological assay?

  • Method name, sample ID, results with units, detection limits and acceptance criteria
  • Only the raw plate images without interpretation
  • Personal opinions of the operator
  • Unrelated chemical assay data

Correct Answer: Method name, sample ID, results with units, detection limits and acceptance criteria

Q50. When performing a zone of inhibition assay, why is it important to standardize inoculum density (e.g., 0.5 McFarland)?

  • To ensure reproducible lawn density so zone sizes reflect antimicrobial effect not inoculum variability
  • To increase endotoxin levels artificially
  • To avoid the need for incubation
  • To color the agar uniformly

Correct Answer: To ensure reproducible lawn density so zone sizes reflect antimicrobial effect not inoculum variability

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