Noveriber 1?, 1953

Dr, R, G, E. “urray
Department of Bacteriology
University of Western Ontario
Victoria Hospital

Lomion, Ontario, Canzda

My dear Murray:

Thank you very much for your courtesy in senting the reprints and draft just
received,

I have not yet had time to study your reuarks more carefully, but am excited
to see how well they have anticipated the conclusions that I am just now beginning
to crystalize from studies with /, coli K 12, I was impressed to notice in our
material that after Giemsa staining every so often unhydrolysec cells would show
a red mclear staining acuinst a very dark blue-purple cytoplasm. The melei,
unfortunately, not too sharp, appeared here penerally sac-!1kc and not like the
discrete granule after hydrolysis. Perhaps most of the nuclei are vascular
consisting of a hypo~chromatic "matrix" in which the Feulgen-positive acid-
resistant granules arc embedded, I am, however, inclined to regard ticse
granules as possibly hyper-chromatic blocks in the chromosome spilin;, I
could not readily reconcile most of the cramile firures with a mitovic se-
quence, but if the figures arc coup rable to the chromocenters of interphase
miclet of hisher Soras the contracicitions may still be resolvable, .On the other
hand, occas sionnl figures cre compelling rominisce::t of mitotic seperation, How=
ever, I do not see spindles, If there is something co.warable co mitosis (and
of course, there has to be 4f only to comprehend haploid and diploid K 12) per-
haps the spindle or its mechanical analerc is confined within the meclear menbrane
as it is in so many Protista. (Ore of the clef reasons I had been suspicious of -
attempts to force all of the «remle firures into a mitotic mold is thet I could
often see connections between the separate "chrouosoncs.") Ina few fortun te
cases these scem to have been strung out as in the enclosed prints, (3y the way,
I had taoucht that the Giemsa technique ealled for diluting the strain in rether
cLlute buffer at pH 7. I had a let of trouble on tiis count when I swrht to use
strong enough buffer to actually control the pH of the stain solution, This should
be about 5 not 7. I now use the commercial siz:in Ciluted in 1/10 pil 5 phosphate
buffer 1:10 for 10 to 15 mimutes, Abopon (‘luted 2:1 in buffer and filtered at %)
degrees has proved to be an indispensable mountin: agent (Sollowine Delaporte),
with occasional trouble from erystallivation,

We still ere primarily concerned with the sul:iy problem rether than these
questions of muclear structure though £ “ind it diffic 1t to see how we will
be able to avoid the litter, Thouvh w have little new direct cytolorical
evidence on mating, we now have Sairly divect demonstrations that the synkeryons
have the full veretie complenent o° ~eth parents althourh there ere leter climin: tions
th: t influence segregation ratios,

Yours very sinecercly,

JL/ew Joshua Lederberr