August 25, 1956

Dear Sol:

Thank you very much for the lipase and various instructions. I had nbt been
able to get underway as fast as I had hoped, but these experiments are on the
agendad for this week. We are between assistants in the lab. now and this has
slowed things up. Also, I've been experimenting with the projiferation of proto-
plaste -viz., the L~form problem, and begin to see my way into it. Proteus
works much more easily than E. coli, but the pattern is pretty much tha same.

In liquid medium, the protoplasts never divide, but can get very large of courses.
In certain (still not too consistent) conditions, in agar, most of the protop]iats
can form "L-colonies". My observations so far point to budding as the medhanisn
of proliferation, and the granules as lytic degeneration products, but the systen
needs a lot more work to be quantitated properly. The hypertonic medium is, I am
sure, the basic key to the problem; I find 1t tard to understand how Dienes & Co.
got as far as thay did without it.

I am enclosing another batch of cultures which should be more satisfactory for
you.

-3133, W-3134 and W-3090 represent the Lac, cds tron (cf. FUL Genetics 1952).
I would recommend these in preference to any of your Lac, 8s now in hand,
3186 represents Lac (ef. CSH 1951). I have sent a culture that carries a

maltose-suppressor, eo the phenotype is lactose— glucose— maltose+; it is essentially
the same as W-327 (Doudoroff et al 1949) but prototrophic. Keep 1t on maltose to
minimize selection for reversions. :

We4ll-20 are a batch of Lac- mutants Esther got from the M- Constiththve, W-1301,
which was selected with naolactose. They are all different from our basic testers
but may be miwteankex iso-cistronic with some. We don't know much more about
them offhand. W-2417 is carrying Lac 4b’ The tubes are rather old; if more than a

couple don't grow out let us know hut they should be ok.

Yours,