Seventy Years on from the Luria and Delbrûck Fluctuation Analysis: A Comparison of three Methods for Estimating Mutation Rate

Abstract

Seventy years ago, Luria and Delbrûck discovered fluctuation assay for estimating mutation rates. While this method is slightly dated, it is one of the few methods for estimating mutation rates in batch culture. Mutation rates when determined expose information on cellular processes and fundamental mutagenic mechanisms. Formerly, inferences drawn from fluctuation assay were sufficient to answer a specific question inbacterial genetics. However, contemporary interpretation of results goes far beyond the motive originally intended. As the fluctuation assay has gained popularity in various scientific disciplines, analyses of results obtained are not same. This study aims to compare the estimation of mutation rates using the Poison distribution (Po) method with, the Ma-Sarka Sandri maximum likelihood estimator and the Lea-Coulson median estimator. Mycobacterium smegmatismc 2 155was used as a model organism for Mycobacterium tuberculosis, and spontaneous mutations that arose in stationary phase cells exposed to antibiotic stress were investigated. Ten to twenty-four parallel cultures were tested with various anti-tuberculosis drugs; isoniazid, kanamycin, rifampicin and streptomycin. Minimum Inhibitory Concentration (MIC) of the drugs were also determinedto be; 8 ìg/mL, 0.24 ìg/mL, 16 ìg/mL and 0.5 ìg/mL for isoniazid, kanamycin, rifampicin and streptomycin respectively. The mutation rates obtained with the methods were very similar. To improve the power of deductions drawn from fluctuation assay, efforts should be made to experimentally determine the relative fitness of wild-type to mutant bacteria.This comparison is only a guide providing evidence regarding the authenticity of some of the methods currently available to researchers interested in estimating bacterial mutation rates.
Keywords: antibiotic resistance, mutation rate, fluctuation assay, fluctuation analysis calculator.