September 27, 1966 .

Dear Dr. Dianes:

Firet let me thank you for directing not only the various cultures,
but also Dr. Sharp, to the lab. here. I had a most enjoyable and infor
mative discussion with hig.

We are finally learning how to grow£ the L colonies of FE. coli K-12.
The experience with Proteus was invaluable for this purpose. Then we
managed to handle ancther ooli strain, and finally K-12 iteelf. There
were a number of ridiculous, empirical variables: the exact recipe for
the nukrient broth base, the freshness of the Mgt+peniciliin solutions,
and worst of all the immxtak batch of Difco agar. Finally, we found a lot
of New Zealand agar which is the best of all.

Since writing that note for PNAS, which should be in print at any moaent
I have been finding that the use of penioillin + a protective (hypertonic)
medium has been anticipated several times for the production of "protoplaste".
The only distinction I oan claim for the data of that note are 1) the use
of BE. coli, 2) the semantios, which was not entirely clear to m then, ani 3)
the quantitetive and enaymological features. [There is, for example, a report
by Bonifas, Schw. Ztechr.Path. Bakt.,17,525 "Influw nce de la pression cemotique
sarele maintien en milieu liquide penicillins, d'une souche de Proteus sous
sa Form 1", which we must both have missed until recently — ay lead to it
was the aiteer good account of Liebermeister & Kellenberger, quoted in the
enclomire j.

I have also had many discussions, including with Dr. Sharp, conwebning |
the implication for mechaniam of penicillin action. His data on the carbo-
hydrate content of stabilised L-forms are very impressive, but I gather you
and he do not necessarily agree with ay prhposal. The mechanism of stabilisas
tion of L-forms is a provocative problem, and a feature which has no direct
explanation from this proposal, but I feel 1t is a separate probleg. If we
can get such steble L{s from the ooli mutants, it deuld be possible to define
their genetic basis. I had hoped that the {{aplications from the one sentence
in the discussion in the PNAS note would be sufficiently clear, but evidently
they are sot to a number of people I have talked with— and there is still
so much nonsense about penicillin floating around, all based on studies of

disrupted cells! At any rage, I am planning to submit the enclosed as a note
for J. Bact.

With best regards,
Yrs. sincerely,

Joshua Lederberg