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Microbiology JoVE (Open Access) Citable · DOI

Measuring Microbial Mutation Rates with the Fluctuation Assay

DOI: 10.3791/60406-v
What you'll learn
  • ✓ Design and execute a fluctuation assay to measure microbial mutation rates
  • ✓ Analyze mutant colony counts across parallel cultures using statistical methods
  • ✓ Interpret how genotype and environment influence spontaneous mutation frequencies
Protocol

Biopharma Insights Here, a protocol is presented to perform a fluctuation assay and estimate microbial mutation rate using phenotypic markers. This protocol will enable researchers to assay mutations in diverse microbes and environments, determining how genotype and ecological context affect spontaneous mutation rates.

Difficulty
intermediate
Total time
~5 days (Day 1 inoculation through Day 5 analysis)
Biosafety
BSL-1

Steps

1
Inoculate and acclimate microbial cultures

Prepare overnight cultures of microorganisms in selective growth medium and allow them to acclimate to experimental conditions. This establishes a consistent genetic and physiological baseline before mutation assay begins.

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2
Generate mutant phenotypes in parallel cultures

Distribute acclimated cultures across multiple independent culture flasks to allow spontaneous mutations to accumulate. Parallel cultures enable statistical estimation of mutation rates from variation in mutant frequencies.

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3
Plate cultures on selective and non-selective media

Plate aliquots from each culture on both selective agar (to identify mutants) and non-selective agar (to determine total viable cell count). Colony counts from both media types are required to calculate mutation frequency.

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4
Incubate and enumerate bacterial colonies

Allow agar plates to incubate for sufficient time to permit visible colony growth. Count mutant and total colonies across all plates to generate the dataset for mutation rate estimation.

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5
Calculate mutation rate from fluctuation data

Apply statistical analysis (e.g., median method or maximum likelihood estimation) to mutant colony counts across parallel cultures to derive the per-cell, per-generation mutation rate for the phenotypic marker.

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