Welcome to the ScienceDuo blog by Chris Wallis and Rhiannon Morris. Screeds on science and sanity from two people who understand neither.

Classic genetics experiments, Luria-Delbrück’s fluctuation assay and the nature of random mutations in evolution

In this post I wanted to cover a few things related to the classic genetics paper by Luria and Delbrück which won them the 1969 Nobel Prize in Physiology or Medicine and underlies one of the important parts of evolutionary theory, the random nature of mutations.

Firstly, before the Luria-Delbrück experiment in 1943 it was argued by many biologists that unlike eukaryotes, bacteria could somehow develop specific adaptive mutations depending on their environment, a kind of directed mutation. Others argued that mutations in bacteria were largely random with respect to adaptation or phenotypic utility. This was the puzzle that Luria and Delbrück set out to solve; are mutations random or directed? To do so they designed an ingeniously simple experiment called the fluctuation test.

The experiment runs as follows. A number of single independent colonies of E.coli are inoculated and grown for some period time and then plated onto agar containing T1 phage, a virus that destroys E.coli. These plates are then incubated over-night. The next day the number of colonies that have become resistant to phage are counted and analysed. Luria and Delbrück recognized that there are two ways resistant colonies could form, either mutations occurred during the initial non-selective growth phase (no phage) or they occurred in response to phage during growth on phage laced plates (Figure 1A.). They also recognized that the way to tell which explanation was correct was to look at the distributions of resistant colonies (Figure 1B &C). If mutations arose in response to selection then they reasoned that they should be roughly equal numbers of survivors on each plate distributed according to a Poisson distribution with the mean equal to the variance. If on the other hand there was a fairly constant background rate of random mutations then there should be a huge variation between the numbers of resistant colonies on each plate, with the variance being larger than the mean. This is because early mutations would spread quickly and many cells and their offspring would become resistant, or late mutations would confer resistant on only a small number of cells that didn’t have time to reproduce before being plated. This is what Luria and Delbrück found, most of the mutations arose during the non-selective growth phase instead of during the selection phase.

Figure 1. A. Cells are plated and grown for N generations and then plated onto selective phage containing media. Blue spots represent cells with resistant mutations. B. Resistant colonies are counted and analysed for variance. C. The difference between spontaneous and induced mutations can be seen from the various distributions (Kulesa et al., 2012.).


So this is experiment and many others confirmed one of the central tenets of evolution, that mutations occur in the absence of selection and evolution itself doesn’t act towards any pre-defined end. Later work has challenged this, and the idea of directed mutations and self-genetic engineering has also recently become popular among quacks and new aged loons. Some of the scientific criticism of the idea of random mutations is based on the work of John Cairns, Julie Overbaugh, and Stephan Miller. They showed that lac+ reversion mutations in E.coli actually does occur in response to selection for lactose metabolism. This really is a strange result when you think about it; how on earth does a bacteria “know” precisely how to fix a mutation in a Lac gene?  The answer is still not completely understood, but later work did show that in fact these mutations are not directed and only occur when the Lac gene is present on a plasmid and not on the chromosome. Some explanations are that under stressful conditions bacteria induce hypermutation via the SOS response, here error prone enzymes introduce extra mutations all over the genome (or plasmids) which increases the probability of a useful adaptive mutation occurring. This is not the entire story but for anyone interested in the details I strongly recommend a paper entitled Adaptive Mutation in Escherichia coli by Patricia L. Foster who worked with Cairns.

Foster, P. L. (2004). Adaptive mutation in Escherichia coli. Journal of Bacteriology, 186(15), 4846-4852.

Kulesa, A., Krzywinski, M., Blainey, P., & Altman, N. (2015). Points of significance: sampling distributions and the bootstrap. Nature methods, 12(6), 477-478.

Luria, S. E., & Delbrück, M. (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics, 28(6), 491.


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This entry was posted on May 17, 2016 by in Uncategorized.
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