December 11, 1955

Dr. A. Goureviteh
Bristol Laboratories
Syracuse 1, N.Y,

Dear Alex:

I am delighted to see how well the induction procedure has been sharpened
up. With a 10-- or higher-fold dncrease in yheld of plaques with 1 ug/al
of asaserine, you should certainly be fn a position to screen for comparable
agenta. There is no reason you should not proceed with the technique you have
worked out.

The reason I origihally suggested using 108 cells wm to allow the dilution
step that you (for other reasons) prefer to avoid. My reasoning was as Solloms.
Induction is a two-stage process 1) the "activation" of the prophage [whatever
this means} and 2) the growth of the phage and lysis of the treated cells. Step
1 can be conducted under a variety of coniitions: e.g., with UV even in buffer
suspension, but 2) requires that the cells be optimally situated for synthetic
processes. I was afraid that you might run into some brotha in which the anti-
biotic iteelf, or some othet constituent, would have so mich antibacterial acti-

vity that step 2) would be inhibited. I thought that one should separete the

activation step 1) by dilution #0 that step 2) would ocour in a noninhibitory
broth. This is only a premonition, and no antibiotic might actually work out
that way. You will have to decide yourself whether the risk of losing such
effects would be worth the added effort of a dilution step.

On the other hani, this problem suggests the possibility of looking for
still ancther kénd of antibiotic activity,namely for agents that will interfere
with step 2) of phage growth. The procedure would be to use cells that had been
induced previously with UV (or if you prefer asaserine) and test your broths
for the ability to inhibit the expected production of lambda. Streptomycin, 6.g.,
is known to have this effect (I don't know of any antibiotic, in fact, that
does not). I realize that you should not go off in all directions in a soreening
program, but if you have developed your procedure for this kind of test, you
might want to think about it. It would be of considerable interest, I think,
to identify 1) inhibitors of bacterla that do not prevent induction, and 2)
probably more exciting, non~bacterial—inhibitors that do prevent phage growth.
In addition, the inhibition test would be another check in looking for the more
ordinary kinds of antibacterial skbatednces. Let me know if you or Joe wants

to discuss this possibility any forsher and I won't elaborate any further now,

I ma swsume that your present screening with K-12 is done indepeniently of whether
you detect antibacterial activity of the broths.

It is too bad you have tke to gurse your cultures at home. Why not set up
a clock-controlimbdinrkis that runs cold water through a small thermostated—bath
until a given tims, then stops the water and turns on the heat? With an electric
interval timer, a solenoid valve, and a relay, it's easily done. Ajternatively
older cultures which are diluted 1:5 or 1:10 and regrown should be ready within a
couple of hours.

The lamda should be diluted either in a balanced salts solution, or preferably
ordinary Da&fco nutrient broth, rather than the buffer. Even distikled water would
probably give you reasonably clean results. I can't see why plate replications
should vary— are these inoculated simltaneously from the same pipette?

Weigle says a amall dose of U¥ (say 5 secs of a sterilamp [15Watt]} at 50 cm)
given to the sensitive bacteria before they are plated evens out the plaque morpho
logy considerably, if such a complication would be worth the trouble to you.

Sincerely,

Joshua Lederberg