J2 96 nate. APR 28 1955 ver. eC ab 325263 fiRe ? 0 D arn. Feat buy tare ayia. Dodite Fla Hi CR) x FAIO(H,*) + bserum —e —> Fiat H,> Pe A%—| FAG3 h* PAR qx HQ ke Suan. Gx=b\ Hi pel : (- Wt tt Gurtpee- i ~ Aulonste . z ? Novevain, 3 “ " . SON > 30 | : | | | . 257 WHIP? DBehumur }257 DATE: AO 5 St Davies allo orp: “obs x ty Hy bate us HOB Ybacpe | A Ma Mie adam [Hele Yarep> gin ren the sox ps 1, Currently enmeshed in bhe fate of £ granules. Can these really givr any importany information? By following a granule during the growth of a single cell, one might get a clue as to whether groth is interstitial or bipodar (jn a few cases). To distinguih, one might have to show increasing separation between two granules, before figgios in a single cell and thismay be difficult. It is already clear that 1) terminal granules usually remain terminal, and that this is the most common type, already suggesting a polerity in the cell. Occasionally, bi-antipolar cells are seen (more commonly that bi-synpolar), suggesting that the two poles share something distinct from the fissile center. However, the basic interest in the Z granule for the current problem is the possible correlation with E, and this, if anything is what should be pursued for now, Later it may be conveneint to try to bepeat experiments with a polar-flagellated organism. Another sideline is to use the shaihs in stiff medium to study other problems, chiefly lethalktym both spontaneous and UV. Also look for data en growth of branched cells. (Twort) 2. More pertinent: 1),look for divided E further. 2) diagnose E,S sells by viscous media, 3) transfer intermediate chains for electron microscopy 4) clean up serotypes of co-segregants-- collect more? 5) For 4 and others need to complete review of data and write up. Lab plags: what to doS? Things are a mess, 3. TODAY: Clean up what is accumulated to look at and photograph. Start new preps. of 93-——x w/wo TZ, Use for divided clones and for Z correlation. Motility selection: initials in methocel 1258D (Sat 5/7/55- Sun 5/8/55-——-) Use TZ staimed prepn. 5/6. 12n7 Chexked first with 1237A1+ for swarm motility. In this series, used 2% methocel 400, diluted c. 1/10 with penassay. a) use methocel for trap; b) isolate initials in broth trap, then trANSFER to mel. The latter was found ineffective (probably still too stiff); By 4 PM, had isolated 13 cells still sluggishly motile in mcl trap, and 7 addl. which were at a distance from reservoir but not now motile. swarm cells were sluggishly potile in this metho- cel conc., about 56-70% were directly inhibited. This oln. probably wets glass more effectively, at any rate it tends to spread, and a few of the mbbiles below may be contaminants from 1237Al+/ The motile residuals above were geet in individual drops of broth for class. as Sw. or E cells. wes found, in first group: second 6 swarms, 3 E, ang, 2 Ff. 4E lng 2% 4% which demonstrates strong selection against ~ cells Total. 68S TE 3ng Desailed counts: growth motiles 1. At 9 (104) 2 3% oS swarm, 50%? - 3 At 2 4 ong The occasion was also used to 5 At 12 (from Z cell, but Z nf) plant about 25 single motiles 6 nn $ 0,1 (removed before test below—per- zBp 7 mot cell haps should have been left in it) 3 20 as 16, sev. shakes, prob. aw for opngcoddance on immediate 9 aw ay and later motility of dividing Jo sw uw chain cell. About 12 usable cases 11 like & no discrepancies, some to one or » 12 500 eS two later divisions. As none gave 13 200 aw pw two mobiles, pres, none of these werexE , Of remminddr, most gave - 21 it h two app. nm at this division-- 22 ng it may be possible to reexamine © 23 he 18 these drops tomorrow. What is 2 it 2, significance of this crisis 25 ia 2 in termination? Is is growth in 26 da 15 fresh medium? (May still need 27 It 16 a good exhausted medium to keep cell size amall.) PS These were then used in tests for residual motility in mel. Unf., 1,5 were wasged in 54% mcl 15 (calc. vise 200) which proved also to inh. swarms. Further tests were then made with mcl 400, 1.3% and 1% (1:1 penassay), the latter being adopted as it permits almost full motility of motile swarms(from above). (This may be too. fluid for accurate discrimination against ¥, as will be saen). From E: 12,23,24 26, 27, adsegether cells reisolated which remained motyiere planted for further classification-> pr ute Ewes. Sven pro tere. a pubin pales ! DATE: —x SW 666 initials in methocel and Z chains ba serun. 9, 1955 REF: 1259 1 2 3 4 5 6 7 8 9 10 D: 20 40 50: Pres Note A. isol,. B. isol. tr in 6% d peepn. (alec anetior preph. unstz (1:1 & 38 isolations, 3 ng; E-clones were r able or riginally The jclone wa 7 hb out used. (st 2 9:45- ned = A. ye out of ) ined, plant out erate Z-stained initials. to c. single drops on isolation eg . Ditto for A-— p T. are inhibited and may ones. ed for sferred clones looked as if o initial cg. first, aps: pick ce. 4000 s did persist in DCG for results of platings(after but neither viable. had been separa swarms; onjy 4E, none interesting 233 43 5(sw). The therefore certainly not BW. celjs/ vered (owing to drying out LN; phage to 1:20, centr. thocel droplets. 3 | | droplets individually. : PM, some t out descendants initials | (somewha aorb i I i ve part once or twice at ntent. In B2, sib| to swarms motiles in E2 tes | | ce. 100+/10% but to veri and refr fresh isdl. C to 4:30 transfer latter as families bo t late now jor tes serum, though cells may co » planted out. 3 proved viablep swarms. picking to bro th) dn dimikashed therefore. 1% Methoce ‘Probably too thin.. norn Ae 22 motiles, | bed » all tse later proved , 1340) isolate initials ih 1% methocel 400 tsJ ntinue to owever, ot gave risé to to be ayrae fy originalY low assay. {2 Z, D PTT Ok / Otherwise ! | Fe 5/10 interm | but 2/4|\from B15 of se should DCG found Dl-3 all motile jbut with "all clusters"; & pure non-mébti numbers /. off inte. & serum: |at least) (28) from 5 clones were DO» However, two_ 8 were inhibited; spctfietiy Also saved 1259B1 (= b8). |Swarn- vast Det ediates were not sted swarm a by piating removed at ns = nonmot,a) | confusing clusters. Bl:. no le. Will have to | be recghecked on recorded, (not not cer definite eturn | record. A swarms H2b | E: 34 isolates planted w/o lineage afterward. 4B (9,11,15,16) | vF (1; 4y35151,657,35355y55251, 351545 --00) oe 7% 2 Dg. Only conclusion; medium not adequately selective. Try 14% methocel 400 (vies 1260) $f/2. Metd ow HbA eee O/$ O02 Tt ae BAAN | + laste, ‘Ds Bi, se Bab arabe © elnotine nbs, Cunt ! Chuskine © sevarine Saabs = D/ Ss! 2 pr GI , I D3 sf a” 4 6) 32 2] x B2b IC 17 ¥ s/t CU "anche “pg hd sVi3s¥ apeled mm MGA wy thi ‘gee had free 7" nln eh Mew aun) ame Utd agar | Qik ov gered Ieee, wd, 3/13 (OI, D2,/; D3,2) B/,2; B26;2.) Duele Shes ch 87% met Ba fn pt Arbt ethan f s/t. 5/16 ote COE Cost Senate , trlind anvanne, ¥ eh, EF VanlDhte* s € Oe = e4, deyhur ates » 7 D3 ~p) 2 oe OG Bi Lacks, chiolire, oppareh Borghi MO ALO Ae BRx fer nee Ag B26 Ge chuclne Qritmd JQ -: D(Y) 46 hule. DIQ) Chuckie, cette f merle D2 ual, tials | ant On gts D 30) Cuatro Anand tn D3) & % fm ans B11) f Claatie, tds o 1 catslidis | poritly anne oso | Bi Meo Audis. HBR unto ee po i P* pete WACDE by abe. abe. Zeoramn 5E ee Cpl ure Loadudl, sofas pathy | may 1 0 1955 No WA sane Heese Aura WA wr) Mh ome. [afaby heal wae t Dr Swen ‘Las te / | ml Jos, | Oot a . (e ° Gi] KI Yaya --x SW666 in metkocel 1260 Partitions . May 13. New prepn., unstained. (probably usual, about 90-120mins. ) Fuse drops 2:30 Collect to 3:30. Cf 1259D motile. No initial was nearly as active as 59D. Pick those hhat have mowed the furthest, not necessarily v. active now. Estimated yield, 10% of broth yield. Noye s to compensate for spreading of methocel solution, use cg. that has been greased (human), then flamed. This workesyd¢lwell, especially with labger drops, but smaller drops are too convex for best visualization. Intention was partly to look for early chains (E) in the methowel, but tie did not allow and most isolates were made to broth directly/(A, B resp.) Lineages were separated at No-3¢ As 1,2,3,6 ok, Partitions at nj: 14+ 31 635 ng snakes, Later transferred entifte clones to get fullest estimate of motiles. Al came out +(14):6 Sepn at n, = B1-14, 21-36, 4.ng. Mostly non E. Records show at first scanning: 23431 Uh 2:1 3 7320 0354;1 13338 3 2et 221 2:20 5 8w3 sw; sw3sw (1260B33 later DCG verified purity of each). 6:5 7:4 Underscores were rechecked (on ungreased slide!) and felowing definitobe Zalues for splits on theses 1:20 8:20 2:2 4:12 3:2 7: 26 Therefore no equal splits. General totals: E 5 ng 4 sw l E —_ Little if any selection for E in 14% metkocel.400. Need 33 2% which probably totally stops many motile cells. ee 21M sI\8 f x/24, (, 2 ; rly! J26] 3 4B Ligchatinf! ngtaler ia CoP hecunirelin oat ‘) HR paced pod sprilad shies ux (itt, 4 wn Bhr tte Ae en, TO lig oh Coe A | cee eee se bh , dec 30° | ) annals fo? 4. . 426,204, 4 2,50 3 20,10, 3,20,7,11, JUN 2 1955 © (2, 70, Seerdve | , CML withad ro Blas dybeor bcmrtn, OF | Wiiscore , Flat (12278)*) wer glee 6 bem (70% ?/) fod we vall, tif to toned “hare oe Nie — CMs part ened, bn An eotnuty furrle manly ptf ale nso tM Swan: maurkd ebonr f Lene on \mal, Ol wh 44 saouner AD fin Peed ‘el PP nb eos | (ove poke - phot hed prom hd ART orem ume 2'-heweo ? f > )26 2 EGO ey ie oe a ery pty. Yo ve fA lb afer oe Pe adaufr Ef, Hoag. Frere mky BE ehones, AZ B# a ow © ,| c Clow, \ ‘ Ae Sen AOS : | | 6 @ . ep lo ane Isa 3:34 awed 28:0 ( bere, By , 2,25 Bur aurum Aone C4 eluodapuat.. DCO ehuludl panty (Sint lege A 7 | & &® 5 Crt | eojuArel-S Whe =P nd Do Ly thule. poet Howe PEAS 2 anhid $( el ftp apni At By! Wukink 38 Of ) x swt bp manastod tad, oof |, ~> Yq. Wee ea EST ol Vd) BY 1/9 N41 Aa Vip. AS 3 ? Loy sg = 2 G G H] BZ). | w boob op. B33 O | » | 34 vp Al,A3 OF,01 ng. (ged mat) C2 4 | I 2 av. oie} X-32\ 2 4° "O fates ! 16 16 4 aye any 37> uke puns ron | | 4. Yo Yy nm ts (men) itel 4 C. | > uz >_ ‘t B Daal | a Ini Y [oA cama rsa. a0 OA wary DL : | 2 27 Ao be dukd og ofm'34 ! BY Y the lols tur Ss Ws 33 47. SB? ao LL 3 subset = prt A Mivd bunebe wi the peligeet . Th 23D @ $ WIAD. C3 73 6 yo 3B) 36 SkCaf DI Pe 7 lay tt I jo thal ug7 so + 3 448 o- /} i en Ss )) — 1S / Yq 17 SEP 9 1955 LUM b relbica> trans asarth ox ble 920° 22 f 7 pegat 295. Fer prbiremen Lopate Anrnerihe XE Hy, W302 #2, 137 Sg bth, ¢ remomtetc ae — pyri SEP 8 1955 Lesfow selon link ire poorwy HV A. D9 agers. f ages I i Moye] ryedn Ty bane 4C TT it la dat /