You are working in a microbiology laboratory that studies amino acid auxotrophs,

Question

You are working in a microbiology laboratory that studies amino acid auxotrophs, and you have recently isolated a mutant strain of your favorite organism, Iwana confusia, that cannot make its own histidine. It cannot make the histidine that it needs to grow because of a mutation in one of the genes that encodes enzymes involved in histidine synthesis. The organism grows fine when histidine is added to the media. Your absent-minded labmate accidentally combined your only culture of the mutant with a culture of the wild-type I. confusia which can make its own histidine. In fact, the organisms are identical, except for their different abilities to make this amino acid.

Knowing what you do about bacterial selection (growing organisms under conditions that preclude the growth of others), and the action of penicillin (blocks peptidoglycan cross-linking), and knowing that penicillin is only effective on cells that are active in the process of making peptidoglycan, describe a way that you might use penicillin to separate re-isolate your mutant from the mixture.

Outline the steps you would take (including growth of the strain(s) on any liquid or solid media), starting with the mixture of bacteria in the test tube, and ending with a pure culture of your mutant. You don’t need to mention every ingredient in the media, but you should indicate components that are pertinent to this problem.

Explanation / Answer

A mutant will grow only when you provide enrichment to the growth condition. As mentioned in the question, the histidine auxotroph will grow when you provide histidine to the minimal media, but the wild type 1 confusia will grow if histidine is not provided also.

Here with the use of penicillin we can easily separate the wild type cells from the mutant one. Penicillin can kill only the growing cells by inhibiting the cell wall biosynthesis, but at the same time penicillin cannot kill the nongrowing cells.

Imagine, you have kept the both wild type strain and mutant strain on minimal media (without histidine), in this scenario only wild type strain confusia I will try to grow and penicillin present in that media will try to kill all the wild type strain, but it will not kill mutant strain as it is not growing on the minimal medium without histidine.

At the end, you will be left only with the viable mutant strain. Like this penicillin will kill about 99% of wild type of cells during each round.

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