MEMORANDUM

BRISTOL LABORATORIES

 

UNIT OF BRISTOL-MYERS COMPANY

 

 

 

 

FROM__ Ae Gourevitch DATE December 5, 1955
To J. Lederberg SUBJECT cs
Consultantship Arrangement
C.c. J. Lein
Dear Joshuat

Joe suggested I send you the message at this time so that I can go into some
procedural details of our screening for phage induction.

Your suggestions were very helpful, and we modified our procedure to take them into
consideration. Let me first describe the procedure as we use it now and then discuss
some of the points of divergence with the one you suggested, and also the
difficulties we encounter.

We use a 12 to 16 hour old, nutrient broth culture of K-12, which we inoculate into
- M~9 medium at the rate of 10,000 cells per ml. We place 1 ml of this suspension in
NS 3/4" test tubes, and add 0.2 ml fermented broth. For control, 0.2 ml of non~

“ fermented broth is added. The broths are agitated for 2 hours on a Khan shaker. The
NY tubes are placed on the shaker with the tube axis slanted in the direction of
shaking, in order to increase aeration, After 2 hours agitation, 10 ml nutrient broth

(+ 0.5% NaCl) is added to each tube, and they are agitated 2 more hours. At that

\ ‘ time platings are done, without dilution, We use now plates containing nutrient agar
> + 0.5% NaCl + 50 we/ml streptomycin. For plating, we prepare soft (0.5% agar)

x } nutrient agar + 0.5% NaCl and dispense in Khan tubes. ‘The agar is melted and cooled
Ss C to 45°C, Prior to plating, 2 drops of a 24 hour culture of We3001 is added to each

tube, followed by 1 ml of treated broth. The contents of the tubes are mixed by
-, repeated inversion, and poured over a plate. Duplicate plates are used for each broth.
<* he plates are left right side up for about 45 minutes, after which they are incubated
“oy! at 37°C upside down.

. Under these conditions the non-fermented control will have anywhere between 10 and
100 plaques, and a large increase in plaque count over the antrol can be observed
visually. The presumptive positives can be then rerun with dilutions.

©
“One thing I should mention immediately, and that is the age of the K»l2 culture. We
, realize that 16 hours is too old. I am getting myself a little thermostatically
Ry; controlled bath to take home, so that I can inoculate cultures later at night and use
{ a younger culture, We noted that our controls come out much lower if a 10 hour culture
&) ig used instead of a 16 hour one.
~S
i)

= Before choosing this procedure we compared it with our old procedure (4 hour
.. incubation in stationary tubes) and with the procedure in which all incubation was
ne done on the shaker but omitting the addition of nutrient broth, The results with
~~ azaserine were as follows?
BRISTOL LABORATORIES INC. NO. 2

 

 

To: J. Lederberg December 5, 1955 New Antibiotics Screening
Program - Consultantship
Arrangement
Stationary 4 hour incubation 2 hour incubation on shaker in
incubation on shaker in M~9 M~9 followed by 2 hours in
nutrient broth
Control 200 2006 400
+1 ¥/ml azaserine 1800 4500 620,000

 

We decided to stick with M-9 because with the addition of fermentation broth the
difference between M-9 and your synthetic medium is negligible, The fermentation broth
contains yeast and cottonseed meal proteins, so that we really deal with supplemented
medium in the first place. Wewually do get higher plaque yields with the control
containing the non-fermented broth than all synthetic medium control,

One of the big differences between the procedure outlined and the one you sugeest lies
in the cell density during the induction. You suggest 108 cells per ml and we use 10.
My main consideration in cheosing the lower figure is due to the fact that under our
conditions no dilution is necessary. We usually can tell at a glance a 10 fold increase
in plaque counts, and consider these cultures as presumptive positives which are then
studied a little closer. This of course is only possible because we do not get too many
prssumptive positive broths coming throngh: this could not be assumed apriori. If you
know of any reason for our not using 1 cells, I would appreciate learning of it. I
actually can see some virtue in our method. The fact that we do not dilute not only
Saves time but also reduces the experimental error due to the dilution and the fluctuation
from tube to tube.

Now let me turn to some problems we encounter. Concerning the Haque size, we find that
the dryness of the plate surface is very critical. When we use old Plates, the plaques
come out very small, whereas fresh plates yield large and irregular plaques.

One of the things plaguing us is an Oceasional lack of replications In most cases,
replication is not too bad (we run 2 plates for each experiment), but sometimes it is
atrocious. Also there is quite often a discrepancy between different dilutions. We have
cases where non-diluted broth gives a count of 800 plaques per ml and the same broth
diluted 100 times -~ no plaques at all on both plates. As diluent we use Soerensen pH
7-0 buffer. Maybe we should use a different diluent?

Gourevidch

AG: jlg