I had delayed answering your letter of July 24 with a view to waiting until I could arrive at a completely satisfactory and
reproducible method of shocking E. coli protoplasts. Actually, I am not completely satisfied yet, but we have a procedure
which, at least in our hands, gives us excellent results of four out of five times. The procedure is briefly described as
Cells are grown up overnight in the Difco Pen-assay medium. In the morning they are diluted by a factor of three with fresh
medium containing sufficient sucrose and penicillin to yield a final concentration of 18% for the former and 1000 units/ml
for the latter. The culture is then reincubated with shaking, and within a period of 2 1/2 hours, complete conversion to
protoplasts is achieved. For synthetic activity we find that it is not advisable to extend this period much longer. Thus,
serious losses in enzyme-forming ability occur with an additional 3/4 of an hour incubation.
The protoplasts obtained are spun down and resuspended in our shocking medium, which is as follows: 18% sucrose, 0.6% HDP
(Mg salt), 1% AA, 10 to the -3 M MgCl2, 10 to the -4 M MnCl2, and 1000 units/ml penicillin. To this is added 10 volumes of
distilled water, suddenly with shaking. Within 1/2 minute, sufficient sucrose is added (in the form of a solution) to give
a final concentration of 10%. The resulting material is then spun and the pellets recovered and washed with 10% sucrose.
It is important to avoid the presence of inorganic phosphate at any stage subsequent to the production of the shockate. One
may use Tris at a low level (.01M) as a suitable buffering agent.
I am sending under separate cover a copy of my Johns Hopkins paper which I finally had mimeographed. I would appreciate hearing
from you your opinion, particularly with respect to the first portion of the manuscript in which I say openly some obvious
things that perhaps need be explicitly stated. I recognize, however, that it is symptomatic of an infliction unique to pedagogues,
characterized by the compulsion to raise the obvious to heights of originality.