July 31, 1950.

Dre Ge Be van Niel,
Hopkins “‘arine Station,
Pacific Grove, California.

eer Or. van Niel:

Forthwith, but under seprate cover, I am sending two cultures
from Escherichia coli, K-12, for recombination experiments:

58-161 B-M- (biotin, methionine)
W-1177 T-I-B,- Lac- Mal-Kyl-Gal-ara-Mtl- v," 37

(threonine, leucine, thiamin
lactose, maltose, d-xylose, l-arabinose, mannitol
phage Tl atreptomyoing

As we discussed, the easiest characters to classify anong prototropha
would be Lac and Mal, using EMB agar with 1% and 1% sugar respectively.
The other characters can be ighored. The easiest technique is as followa:

Inoculate separate cultures from slant into Pennaseay broth (or any other
nutrient broth without too much sugar), incubate overnight at 35-37 without
shaking or seration. Wash celle, mix equal aliquots of concentrated suspensions
and spread on thiamin supplemented plates of minimal ager, about 108 5 x 10
per plate. Use thick plates (25 m2 agar/10 om diameter plate). Incubate
at 3-37 for 48-72 hours. Pick prototrophsa, etc.

EMB plates should be incubated at 37 about 20 hours. "specially with
EMB Maltose, Malf tends to fade after a time.

If you wish to make up a more complicated experiment, you can also_scors
S¥ on the EB plates by streaking first a solution of streptomycin, 10° u/nl,
and cross-streaking the bacterial suspensions after the streptomycin has
dried into the agar. S 4e very closely linked to Mal.

I would anpreciate learning how this goes. Let me know if I can help
in any way.

Sincerely,

Joshua Lederberg