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THE LILLY RESEARCH LABORATORIES

ELI LILLY AND COMPANY
INDIANAPOLIS 6, IND., U.S.A. April 27, 1954

Dr. Joshua Lederberg
Dept. of Genetics
College of Agriculture
University of Wisconsin
Madison 6, Wisconsin

Dear Dr. Lederberg:

On behalf of our staff I want to tell you that we enjoyed your
visit here and are hoping you will want to drop in again when
you have time to meet more of our research people,

On the attached table you will find a list of media which we
have found useful in cultivation of streptomycetes, Three are
chemically defined or contain only starch as an ingredient of
questionable purity. The other three are complex, The numbers
in parentheses have the following significance: Streptomycetes,
in our experience, rather frequently sporulate well on only one
of the two general classes of media included here. Strains pre-
ferring inorganic nitrogen may often do best on numbers 1 and 25
less well on 3, and sporulate inversely as the medium number
rises. Conversely, some (relatively few) prefer a very rich
medium such as Emerson's (6) and sporulate progressively less on
the lower-numbered substrates of the series, Of course, all
variations may occur. It might be mentioned that starch is often
the best carbon source for sporulation. Medium 1 with starch
(weight for weight) in place of sucrose is one of the best "syn-
thetic" media we have used.

The above considerations might have application in the handling
of mutants. I have isolated spontaneous variants (in cultural
characteristics) which differed greatly from the parent and from
each other on such a series of media, You may face the same pro-
blem.

Max Stark agrees that we have no information concerning sporulation
"run-down" in cultures used here. In a few experiments with differ-
ent streptomycetes, production "run-down" has uniformly occurred

in a dozen transfers or less, but sporulation was not significantly
affected. The work of Williams and McCoy is the best in this area
so far as I know. One or two tricks may help: Sporulation of cer-
tain strains is depressed by water on the agar surface, They will
do much better if a dry transfer is made (avoid suspensions) and
well dried slants are inoculated. If sporulation "run-down" should

occur in spite of precautions, you might plate out on several media
-2-

and select a more vigorous colony. As an alternative or supple-
mentary measure, we frequently pass the strain through a culture
generation in sterile soil (witchcraft!). Simply cover the bottom
of an Erlenmeyer flask with soil, sterilize and inoculate with a
suspension of the organism, Enough water should be added to moisten
the soil thoroughly, and I usually spread the "mud" with a sterile
glass rod so as to form a fairly thin layer on the inner wall of
the lower part of the flask, Incubate at 25-30°C for one to four
weeks and look for a fine white film covering the soil surface.
When this is observed, recover the culture by direct transfer or
by plating.

Incidentally, you will get best plating results by the "surface-
inoculum" method, for submerged actinomycete colonies don't tell

you much, If many plates must be inoculated with a single dilution,
we usually make the dilution in melted and cooled 0.25 per cent agar
held at 42-45°C,. One or two ml of such a dilution can quickly be
spread over a plate by vigorous tipping or swirling, thus avoiding
the use of glass spreaders, This works best if two people cooperate.
The 0.25 per cent agar dries out quickly during incubation,

My apologies for such a long-winded letter, Where I have included
trivial details already known to you, please ignore them, I hope
at least a little of this will be valuable. If we can help in any
other way, please let us know.

Sincerely

CC CZ

R. CG, Pittenger
Department of
Microbiological Research

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