Doermann was another researcher working in microbial genetics. He and Luria were well acquainted through the Cold Spring Harbor
Symposia, and throughout the 1950s, they occasionally taught a training course in phage biology there. Here he replied to
a query from Luria about technical problems in experiments with mutant phages in recombination studies.
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1956-02-21 (February 21, 1956)
Doermann, A. H.
University of Rochester
Luria, Salvador E.
Original Repository: American Philosophical Society. Library. Salvador Luria Papers
I have your letter mailed Sunday, and will answer it to the best of my ability (which is pretty small in this case, at least)
immediately before the letter has a chance to get buried. Unfortunately you did not tell me very much about your experimental
procedure, so I am guessing about it.
We have not had any difficulty which seemed serious in connection with reduced burst size using T4rII's. I guess that
your experiments are usually single infection experiments. Ours, of course are multiple infection. Also, we never do the
experiment where no r+ is expected to appear in the cells. We usually expect recombinants in our rxr crosses (an exception
noted in the data given below). To give you some idea about our experiments, I can list a few data:
CROSS; Burst Size; Remarks; r+ in yield
r145 x r168; 279; These are Benzer's rII's; 0.001
r205r271 x r320; 249; These are Benzer's rII's; 0.00027
r67 x r47tu41; 340; These are T4D mutants; 0.1
r67 x tu42b; 404; These are T4D mutants; 0.5
r67 x tu45; 302; These are T4D mutants; 0.5
If there is a significant difference between the first two and the last three experiments above, it could easily be that it
is due to the differences between Benzer's T4 and ours. On the other hand, it doesn't seem enough to be alarming.
Both of the Benzer rII crosses are with mutants exclusively in the A segment, so the parental particles should not have a
complete functional unit as far as the r gene is concerned. This is the closest thing we have to your experiments, I suppose.
If you are interested in the manner in which we do our experiments, the protocol follows: (everything is done at 30 C, except
plates incubated at 37)
Bacteria: We make 1:1000 dilution of overnight aerated B in broth (0.8% NB + 0.5% NaCl) and aerate at 30 C for 140 minutes.
Concentrate 20x by cfgn. Should be 2 x 10^8. Add CN to make 0.001M.
[Handwritten note: Which B?]
Phage: Multiplicities are aimed at 6-7 of each parent. Loss of infectious bacteria is negligible under these conditions.
Adsorption permitted for 4 minutes. Then serum added to adsorption tube. The serum goes for 5 minutes, and then dilute
to growth tubes. The rest is standard.
One thing that occurs to me which might be causing you some trouble is that you might be using rII phages which have been
propagated on K=12s. In our experience, such phage stocks cause a great deal of loss of bacteria, so that assay of bacteria
by colony count before infection is considerably lower than infected bacteria titer. This is true even at multiplicities
as low as a total of 5 phage per cell, and possibly considerably lower. B-grown or S-grown stocks do not do this. It seems
possible that one might encounter lowered burst sizes from such a stock too.
If the difficulty is not due to one or the other of these differences, I don't know what to suggest. If you continue
to have trouble after checking these possibilities (broth composition, bacterial host culture, phage stocks) I would be glad
to run a single infection experiment here to see whether we have your difficulties too, or whether we have just been luckier
and can then investigate other differences.
Bert regards to you and the others in Urbana. Also to Zella and Danny.