CARNEGIE INSTITUTION OF WASHINGTON

DEPARTMENT OF*GENETICS

COLD SPRING HARBOR, LONG ISLAND, N. Y.

March 13, 1950

Dr. Joshua Lederberg
Department of Genetics
University of Wisconsin
Nadison, Wisconsin

Dear Joshua:

Thanks so much for your letter -- I am genuinely excited
by your results, and wanted to lose no time in telling you so.
I think you are putting bacterial genetics on a sound basis,
and my hat is off to you.

I would like to let you know something about how I have
handled the agglutionation problem. In my earlier work with
acriflavine, I of course ran into it, and found some wavs of
getting around it. More recently, I have managed to overcome
it completely, I think. In using heavy cell suspensions and
fast-killing concentrations of acriflavine, agglutination is
likely to produce just the kind of complications you mention,
With lower coficentrations, while agglutination occurs, there
is a gradual unclumping, and 24-hour exposures give fairly
homogeneous suspensions. Tests with mixtures showed no complications
under these conditions. I made some measurements of the rate
of infection by phage of acriflavine survivors, however, and
found a significant slow-down, which naturally worried me. All
this made me spend considerable time working out a method of
verifying the indications of mutagenicity of acriflavine.

I am planning to publish results of this work soon.

Essentially, the method is as follows: About 107 bacteria
are inoculated into 5 ml. of 0.01% acriflavine in broth, in each
of 50-100 tubes. There is no detectable agglutination with
suspenstions of this density, and if there is some that escapes
detection, subsequent operations nullify it. After 4 hours of
incubation, when survival is about 0.1%, sodium nucleate is
added to each of the tubes to give a concentration of about 0.2%.
The nucleate instantly knocks out the acriflavine, and the
survivors begin normal division after a Slightly prolonged lag.
The tubes are then incubated 24 hours, and assayed to determine
the no. of Tl-resistant mutants per 108 bacteria. What ya have
is the growth of 10% survivors of acriflavine treatment in broth
containing nucleate-inactivated acriflavine. The final growth
is entirely homogeneous, and infection-rate by phage entirely
normal. Controls consist of 50-100 tubes containing both
acriflevine and nucleate, inoculated with 104 untreated bacteria,
incubated 24 hours and assayed in the same way. The controls
grow exactly as they would in broth alone, and give a frequency
distribution of mutants euyasWy comparable to that given by
& qomeuteib series of broth cultures. The distribution in the

ParnLar
&

experimental series, however, is strikingly different --~ most of
the cultures have enormous numbers of mutents, and I have some
evidence that both zero pointg and delayed mutations contribute

to the final yield. Advantages of this method are a) agglutination
is completely eliminated, and 2) the final test with phage is

on bacteria that have gone through an essentially normal culture
cycle after treatment, and normal infection rate is assured.

Thus, I have little doubt at present that acriflavine is truly
mutegenic.

Incidentally, Braun has found that agglutination with
acriflavine is a good indication of rough-smooth differences,
and B/r, which is relatively smooth, gives much less of it
than rougher strains like MMB. If K-12 is rough, you will

get. much more agglutination than we get with B/r.

I am no longer working with chemical mutagens, having
been caught up in the problem of delayed mutetions and segregation,
but if you have any compounds that you would like tested, I
would be glad to give them a whirl. I think, though, that this
would have t@ be slow and careful work, as each compound presents
its own special hazards in mutagenicity tests, and to my mind,
requires a great deal of work to arrive at any certein answer.
I have a good deal of confidence in acriflavine now, but it
took a great deal of work to get it.

As far as urethan is concerned, Bryson spent a long time
on it, and other carbamates. He found enormgous selective
survival of phage-resistent mutants, and was therefore unable to
conclude anything about its mutegenicity in this system.
Latarjet published results with it ( Comptes Rendus - CXLIII,
June 1949, p. 776). He apparently got large mutagenic effects,
and mentioned specifically that he did not observe the selective
effects that Bryson got -- which is curiogs indeed, since they
used exactly the same material and methods, as far as I could tell.

Hope you'll send something on your work in for the next
MGB -- the April issue will be quite good, I think, and very
international.

With best regards,

Sincerely
Evelyn M. Witkig
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