Phone :

Li .
SLOane 4.277 ister Institute,

Chelsea Bridge Road,
London, S.W.1.

43th July, 1954.

Dear Dr. Lederberg,

Thank you for your letter of the 6th July. I am writing in
a hurry, so that this letter may reach you before you leave for
your holiday on July 20th.

(1) The parcel containing the collection of special strains
of the typhoid-paratyphoid group of organisms will be dispatched
within the next 8 or 10 days. Since the package will not contain
any phages or sera, it will be sent by ordinary surface mail,
together with the List giving full details of the strains. The
parcel will probably arrive about mid-August and may safely be
left unopened.at room-temperature for several weeks, until your
Department starts full work again.

(2) You may remember that in the joint paper with Craigie
on typhoid phage-typing (Lancet, 1947, on page 12 of the reprint)
we advised against drying of the type cultures for the time being.
For reasons similar to those mentioned in my letter of the 29th June,
and also for experimental purposes, I decided to have the complete
sets of type strains of the internationally recognized phage types
of Salm. typhi and Salm.paratyphi B also dried and kept in safe
custody in several Institutes in different parts of the world.
These dried cultures probably need not be tested earlier than
perhaps after some 10 or 20 years. I shall send you a complete
set of these cultures (in dried ampoules only) at the same time
the cultures mentioned under (1) are sent.

(3) For Gram-negative organisms like Salmonella, Shigella,
Ee coli, Proteus etc. I advise keeping the stock collection at
room-temperature in preference to storage at 29 CG. to 40°C, This
procedure also is followed at the N.C.T.C. at Colindale.

Since you mentioned in your letter that your collection is
being maintained partly tin nutrient agar stabs' I would like to
refer you to my old paper in the J.Hyg.Camb., 1938, 38, 750. You
will find on page 759 that strong emphasis is laid on the necessity
of using different nutrient agar medium for the stabs in which the
cultures are preserved, as contrasted with the medium employed for
surface growth. It is essential to maxe sure that the agar-stab
medium be completely free from any fermentable substances (see page
759, line 6), otherwise the cultures will not survive long enough,

 
or most of the surviving organisms will be found to be ‘frought
variants, In this country a commercial meat extract 'Lab Lemco!
has been found suitable for this special purpose. The same
product would be a very poor ingredient of agar medium employed
for surface growth. I enclose a copy of a brief note on Lemco
agar I used to send to my correspondents years ago.

If you look up my letter dated 10th February, 1953,
page 2, lines 8 to 12, you will see that the cultures that were
prepared for you in January 1953 were derived from Lemco-agar
stab cultures dated 1935 or 1936, that is to say, they had survived
for 18 years at room-temperature.

(4) The nineteen special strains shown in the enclosed List,
that are widely employed in agglutination tests, virulence tests
and in the preparation of T.A.B, vaccine, have been maintained
exclusively on or in agar medium throughout their whole period of
laboratory existence. The ampouled dried cultures, of course,
were also prepared from agar-grown cultures.

It is only since the introduction of routine phage typing
of Salm.typhi and Salm.paratyphi B (about 1940) that I have been
using Dorset egg medium. This has been and is being employed
exclusively for the preservation of those strains that are used
in routine phage typing or in experimental wor with phage. The
reason being that trought variation occurs much more readily on,
or in, agar medium thah it does on Dorset egg medium, It is thus
possible to employ in bacteriophage tests stock cultures from
Dorset egg slants without plating and colony selection at very
short intervals, whereas this would be unavoidable with agar-
grown stock cultures.

(5) The history of strain 0901, which goes under the name
Felix and Olitzki, is as follows: Broth cultures of H901 were
stored in the dark at room-temperature and the ageing cultures
were examined at regular intervals over periods of weeks and months
(during 1925 in Jerusalem), This procedure had been employed
successfully in earlier work on antigenic variation in Proteus X
atrains (see Ztschr. Immun Forsch., 1922, 35, 57, particularly
page 61 and Tables I and VI). ‘

You may be interested in that paper on Proteus X strains
also for two other reasons (see Summary, pp. 95-96) :

(a) Variants of tintermediate' antigenic constitution
were described, indicating that the phenomenon was due to gradual

 
-3-

destruction (or suppression) of the original antigenic components
and gradual development of different antigenic components.

(b) Qualitative changes in the H antigen were not observed,

(6) The reference to the earlier attempts at isolating a
stable O-form of Salm.typhi is J.Immunology, 1924, 9,115. The
essential difference between 'real' O-forms and 'apparent' O-forms
is dicussed on pages 156-157 of that paper.

(7) The re-examination at regular intervals of an ageing
broth culture is, of course, an extremely simple experimental
manipulation. I would certainly recommend it as the most promising
general method of isolating stable O-forms in Gram-negative organisms.

When broth cultures stoppered with cotton-wool plugs are kept,
in tubes or in larger vessels, for weeks or months at room-temperature,
the volume gradually shrinks and the concentration of the various
components of the medium gradually increases. This probably is the
factor stimulating variation.

The examination of the ageing cultures may be continued
until the volume of the liquid has been reduced to 10% or 5% of
its original size. I used to continue with the examination until
the contents of the tubes had reached a semi-solid state.

I do not think it advisable to accelerate the process by
keeping the cultures at 37° C. instead of at room-temperature. On
the other hand, in experiments with the different Gram-negative
organisms it may be useful to employ a number of different liquid
media in addition to the customary broth. The eve of the summer
vacation may be just the right time to set up such an experiment
and thus gain a month or two.

(8) With regard to the E. coli strains N.C.T.C. Nos 122 and 123
I am afraid the only person who could give details of their antecedents
is Dr. Cowan at the N.C.7T.C. When the N.C.T.C. was transferred from
the Lister Institute to the P.H.L.S. at Colindale none of the records
were left at the Lister Institute. JI am writing to Dr. Cowan asking
him to do his best and to try to trace for you the history of these
cultures.

Please do not bother to answer this letter while you are busy
preparing for your holiday. ‘

With best wishes and kind regards,

Professor Joshua Lederberg, Yours very sincerely,
Department of Genetics,

University of Wisconsin,
Madison 6, Wisconsin, Yelegs