seHed 1958

F~disinfection experiments involving stocks other than 58-1461 p NS

K-12

Details on experiment #5320 are not very complete. K-12 was subcultured
massively from slant to gelatin motag. Swarms appeared as readily as with 58-141,
After 12 passages, highly motile K-12 was crossed with “1817 and W1177 via SRP.
Cultures were not mixed prior to plating on streptomycin, Prototroph recombinants
appeared in neither cross, although recombinants did appear in simultaneous cross of
K-12 (non-motile) x W1177, but not in K-12 (non-motile) by W1817. After 14 passages,
streaked out and single colony used to initiate W2285,

58-278

nN experiment #5334, considerable difficulty was had in obtaining motile
variants of 58-278 and S’ derivatives of same. This was in part due to inadvertant
use of high-% agar. However, one fairly motile culture was still FA (passage number ,
confused by the preceding difficulty). Even upon reinitiation of passage on known
0.4% agar, however, motility was obtained with difficulty and motile strains were
not obtained which seemed appropriate for testing F-status,

W1817

As observed elsewhere, W1217 and W1177 swarm less readily than 58-141 and do not
appear to reach as high a degree of motility. Further, growth aberrations of W1177
appeared upon repeated passage on motag. ‘hese factors may be related to the inability
to obtain F- W1817 by swarming, so far (tho, cf. W2202).

Exp.#5301: Subculture from broth to motag. At 24 hr. swarm radius (sr) 15 mm.
Subculture. At 46 hr. new swarm with sr of ll mm. At 90 hr. sr 21 mm. Subculture.

At 114 hr. subculture. At 117 hr. sr 1.5 mm. At 139 sr 21 mm. Subculture. At 158 hr

sr 20. At 140 hr sr 23. At 149 hr sr 30. Subculture. At 174 hr sr 2. At 177 hr sr 2.

At 191 hr sr 35. Subculture. At 203 hr sr 24, At about this time, crosses were attempted
among 41817, W1177, 58-141 and W1607 (motile and non-=motile of each). Although the

F- nature of motile 58-141 was first detected here no significant effect on the fertility
of W1817-motile could be seen. The prototroph yield was slightly lower in all crosses,
including that x58-141 F/,

Exp.#5331: After 23 passages of same line desc~ibed above, it was apparently as
motile as it would be, though still less than had been observed for 58-141. Crosses
with 58-141 and W1A07 gave 2000 and 1000 prototrophs regp., whereas simult. crosses
of non-motile W1817 gave only 1000 and 100 protos. Thus, if anything, fertility’ was
enhanced. This motile 1817 slanted as 38.

Exp.#5386: S8 reexamined, after almost 2 months. Still motile; now gave exactly
comparable crosses with 58-141, W1407, and 58141 F= as did 41817 non-motile. So if
enhanced fertility was real, no persistence,

It appears that this line was the only one that I followed exhaustively; perhaps
a repetition starting with another isolate would be successful.

W2202

Exp .#5337: considerable difficulty was had in getting this TLB, strain to swatm.
By the eigbh passage, however, it moved 8 mm in the first 7 hours and appeared to be
incapable of crossing with W1407 in a rather unsatisfactory test. After 10 passages
motility was somewhat greater and a satisfactory set of crosses revealed it to be
F- vis-a-vis W1407. However 2000 prototrophs resulted from crosses with 58-14]
(mostly mal-) as compared to only 200 prototrophs in a simult. cross of W1817 x 58-141
(mostly malf). Samples of eigth and tenth passages were saved, I believe, as W2235
and 2234 respect. “sther has further data on crosses of these. Thus, the phenomenon
applies to some TLB, strains at least.

W1896
F-removal. In expt .#5343, repeated inoculation of motag with separate colonies

~.Of W1895 verified a tendency to swarm readily, more so than 58-141. This could be

stgnificant. “apid appearance of what seem to be sequential mutants on one plate.
History of one extended passage: Inoc. on motag from single colony (1). By 24 hr

We
3 sequential swarms. Pick from fastest and subculture (M2). By 24 hr sr 32 mm. At

28 hr subcult. (M3). At 25 hr. sr 29 mm. Sube (M4). At 28 hr sr 42. Sube.(M5) At
4.5 hr sr 4. At 24 hr covers plate. Subec. (M4) and inoe for test cross. Still Hfr.
Subcultures M7-M12 confused by use of too conc, agar. Upon final return to 0.42%

agar, swarms readily. M14 moves 47 mm in 12 hr. Subc. (M18) At 14 hr covers plate,
Sube (M19) and inoe for cross. Cross x W117 yields 4000 protos; cross x W1177 gives
none. Str ak out and isolate 3 colonies. Cross each. two verify mas: cross, third
does not cross with W1817 (but moldy). Save one apparent F- as 42284. Subsequent
cross of W228) x W1817 gave only 500 Protos, but simult. 41895 x W1817 gave only 10008.

Reinfection. Expt .#5394: W228, (F-Lac¢) was grown with 41817 (Ff Lac~) and
W1941 (Hfr lac-) separately, for 44 hr. Loop inoculum from slants, grown until turbid.
Five lacf colonies from each mix were Pooled and crossed with W1817 and W1177. Both
pools gave results identical to 2284: 500 with W1817, none with W1177.. Thus no
evidence of reinfection. But no 58-141 x 41177 control; concurrent W1895 xW1177 gave
Hfr results, but only 1000 colonies with W1817. Repeated process again with same
essential results.

In expt .#5398, a repeat of preceding, three isolates from "1941 mix and four from
N1817 mix were tested vs. W1817 and W1177, Again all crossed with » not
giving mostly lac/ protos. Hfr crossed with W1177 but control 58-141 x #1177 cross
did nto go. Follow up by J.L. (#1055).

Crosses. In expt # 53107, W228, was crossed with 82057 (Hfr T-L-Mal- SS) and gave
only 7 prototrophs (sic). Three were mal-, four malf. ~rotos were purfied and :
crossed by SRP with W1177 and W817. None gave tkeombinants with 1177, but no adequate
control since 58-141 did not cross with W1177 at the time. 150-500 recomb. in crosses
with W1817. Most of the latter were lacf suggesting that the proto parent had been
F-. 10-30 lac~ recomb where proto parent had been malf; o-2 lac- recomh where proto
parent had been mal-. Repetition of cross using two mal~ protos showed no fertility
with W1177 in spite of simult. fertil. of latter with 58-1Al.

Het stcks.

In expt#5349, considerable difficulty was had in obtaining motility in N78,
W1325, and 41590. No g od swarms were ever obatined from 41590. The other two were
passed repeatedly (passage numbers meaningless since some were on harder agar).

After 13 passages, at least the last two on O.4% agar, W478 was tested adequately and
found to be F-. Saved as W2210. After 11 passages (mostly on 0.4%) W1325 was tested
and found to be F-. 800 protos vs. W1817, O vs. W1177.+ No 58-161 x W177 control
recorded however. Saved as W2301. -