Novenber 15, 1948.

Dr. i Re Zelle,
Dept. Bx terlolosy,
Cornell University,
ithaca ’ 4 od e

Dear Yax,

“Lease excuse m tandiness in answering, but when I returned last Monday
from New Haven, I found that I had to recover the helerozyzotes again; the
stock siants had lost much of their viability, and what was left was mostly
segregated. However, this is coming along vary well now, and it nicht be profi-
table to send then out.

The media now used are a) F..Belactose (or xylose) :

Da r . I usualip keep the cyes and phosphate as a
ee N2Case) dry mix. (£8 «ix)
NaCl 5
K HPO, 2
Ecsin Y oh
Uethylene Blue 0065
Agar 16
Lactose 19
b) Synthetle ~' (FS)~ lactose
Sodiun succinate 56 {Asparagine can be used instead if desired)
Ammon. sulfate 5
NaCl i
Agar 15
Lactose 10
EiBoix 205

For "complete" slants: nutrient agur plus .3% yeast extract.
For minimal, (i.e. 2@x T(0) ) see my naper in Genetics.

When you give me the word that you can start handling cultures, I will send
you H-72 which as a protebroph heterozygous for Lac and for Xyi, and for nutritional
factors I haven't checked on yet. It was obtained from a cross between Bele x
ToL@B, ~Lac, Mal) -GaleXyl-arab-V,". H designates the unknown factor which leads to
the apnearance of heterozygotic prototrophs in crosses and nay be a chromosomal
wberration. He72 is not heterozygotic for the other sugars, but is # for then.
-2-

You may find handling and preserving these cultures sonewhat ticklish at
first/ I'm still working on means of ‘alleviating these difficulties. I don't
know how lyophilization would wors.

You will receive H-72 on a T(0) slant. Immediately upon receipt, the culture
should be emulsified in water and streaked out concurrently on "MB and EUS Lac.
If the bulk of the cells are still heterozygous, the colonies on FuB will be
mosaic f and =; if not they will be intact = or 4, On FuS you should get, after
40-50 hours a number of Laes colénies. These should be emlsified ani streaked
in the saie way, until you establish a line of transfers which gives 4 colonies
on =MS and mosaics on "2B. The most reliable way of keeping the hetervuzygutes
is by such transfers once or twice weekly. Single colonies serially transferred
on FMS, several colonies being chosen from¢ each plate and taken both to TAB
and "ho and carrying through the lines which give moséics on TB. The line is
thus carried on =MS, and the isolates tested cn each transfer on Tf. I've carried
thea this way for 2 or more transfers julte satisfactorgly, while nass cultures
on slants segregate out very quickly. ‘hen the line is established, you will
find that only two or three colonies have to be transferred and cheeed on
each transfer to be sure of carrying along a heterozygote.

Mhen:heterozygotic colonies are inoculated into complete broth, they rapidly
segregate out, and at the end of growth only a very few will still produce nosaic
colonies on !){B. In mininal liquid medium, you ray get variable luck; soxnetines
finding mostly heterozygotes after growth. The most reliable source ur is single
colonies from FS.

I would suggest that before we get together personally, you try jaunt hand
at carrying and testing these cultures. Perhaps, it would be well to try to Isolate
a few single heterozygote cells, to and in minimal uedhum. That would be a good
time for a conference to plam further work. I hone to have a preliminary account
of this work written up before many more weaks.

As I don't have ary classes, I would be glad to take the time to see you at
Ithaca if that were acre convenient. But we don't have any funds for that kind of
thing, sv I could travel in that direction only by invitation. You mav be better
fixed,in that reppect, to come here.

Your estinate of two weeks to have your lab ready :nakes me a little envious.
Ifve been here over a year; and have still to move into my new lab, which needs
omly plusbing and painting now-~ another two months probably!

Yours elincerely,

Joshua Lederberg