Replica plat­ing

Pic­tures Con­sid­ered #41

by Elio

Fig­ure 1. Replica plat­ing for the iso­la­tion of aux­otrophic colonies. A, Ini­tial plate; B, Replica; both on com­plete agar. C. Sec­ond replica on min­i­mal agar. The ar­row des­ig­nate the aux­otrophic colonies which fail to grow on min­i­mal medium. The res­o­lu­tion of these repli­cas is of fair to av­er­age qual­ity.

In the early stages of mod­ern mol­e­c­u­lar ge­net­ics, say 1942‒1952, the time came to set­tle ex­per­i­men­tally the old ques­tion about whether mu­ta­tions in bac­te­ria arise spon­ta­neously or are in­duced by the en­vi­ron­ment. Call this Mendelian vs. Lamar­ck­ian evo­lu­tion. By then, the spon­ta­neous oc­cur­rence was gen­er­ally fa­vored, be­ing con­firmed by sev­eral pieces of work such as the clas­sic fluc­tu­a­tion test of Luria and Del­brück. Other clever ex­per­i­ments added cre­dence to this con­clu­sion. How­ever, they were all open to the ob­jec­tion that the mu­tants arose af­ter con­tact with the se­lec­tive con­di­tion, such as ex­po­sure to an­tibi­otics or phages. How can one test whether the mu­tants were present with­out ex­pos­ing the bac­te­ria to the se­lec­tive con­di­tion? Not easy to fig­ure out, but that's pre­cisely what ge­neti­cists Joshua and Es­ther Leder­berg did. For a bit of his­tory of the two, see here.

Here's the idea: Grow your bac­te­ria so that they pro­duce iso­lated colonies on an agar plate. Call this the "mas­ter plate." Now make a replica of it by touch­ing the sur­face of the agar with a suit­able ma­te­r­ial. The Leder­bergs chose vel­veteen, which be­cause its fuzz acts like hav­ing a myr­iad of in­oc­u­lat­ing nee­dles. Now touch the vel­veteen on two new plates, one con­tain­ing a se­lec­tive agent (e.g., an an­tibi­otic), the other not. In­cu­bate. The plate with the an­tibi­otic will have colonies of re­sis­tant mu­tants. Yes, they will have been ex­posed to the agent, but those on the sis­ter plate will not. With a lit­tle tri­an­gu­la­tion, look at the sis­ter plate and pick the colonies that cor­re­spond to the ones grow­ing on the "se­lec­tive" plate. These will be re­sis­tant with­out hav­ing pre­vi­ously been ex­posed to the agent (there's a re­ally nice tu­to­r­ial on YouTube).

You want to pick nits? Ar­gue that the mu­tants will even­tu­ally have to be tested in the pres­ence of the se­lec­tive agent. True, but prac­ti­cally none of the other colonies on the sec­ond plate will be re­sis­tant. Want to be ex­tra-mod­ern? In­stead of mea­sur­ing re­sis­tance to the an­tibi­otic, grow the re­sis­tant colonies in its ab­sence and mea­sure their level of an en­zyme known to be in­volve in re­sis­tance (e. g., a beta-lac­ta­mase if you had used a beta-lac­tam an­tibi­otic). One can also em­ploy a num­ber of other se­lec­tive con­di­tions, e. g, re­sis­tance to a phage or loss of abil­ity to grow on a min­i­mal medium (aux­otrophic mu­tants).

This el­e­gant brain­child went a long way to­wards quelling the heat of the dis­cus­sion. The choice seemed clear: mu­tants arise spon­ta­neously and not due to ex­po­sure to the se­lec­tive agent. And so the de­bate rested for a cou­ple of decades, only to be re­vived by newer and rather con­tro­ver­sial no­tions, such as that epi­ge­netic phe­nom­ena lead to Lamar­ck­ian evo­lu­tion. For a dis­cus­sion of cur­rent views of this ques­tion, see here. So, there you have it, some ar­gu­ments seem never to cease. But the el­e­gance of the Leder­bergs' con­tri­bu­tion re­mains for all to ad­mire.

 

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