RECONSTRUCTION EXPERIMENT OF THE TRANSDUCTION BETWEEA SINGLE H-PHASE Iii SALMONELLA. Report by fFetsuo Tino Feb. 6, 1956 It has been known that H-antisera inhibite in some extent the movement of the cells which have no corresponding antigen. If such inhibition happens when antisera are used for the selection of H-transduction, they may inhibite the development of the transduction swarms, which have ro specific antigen corresponding them, and may cause the discrepancies of the result from what is expected. In order to test such possibilities, reconstruction experiments of the transduction between single phase cultures were performed. MATERIALS AND METHODS. The experimental procedures were tabulated in Table 1. e803 b(zenx) Gal” and SW 803 (b)senx Gal* are the same strains which have been used or obtained on the tuansduction between single phase donor and the mixed recipient culture of Gal-labeled single phases. In the 2nd experiment, TM2 i(1,2) Galt, TM2 (i) 1,2 Gal~, TM2 b(1,2) Gal* and ™M2 (i)enx Gal~ were used instked of the four strains used in the lst experiment, and anti-i, -1,2 MGA plates were used for the selection instead of anti-b, -enx MGA plates. RESULTS AND DISCUSSION. The results are summarized in Table 2. In most cases the yield of the swarm are lower than the expected, except experiment 1 where there is no significant differences between the obsebrved and the expected. ‘The degree of decrease may be controlled by several factors as follows: 1). Experimental error during the dilution of the cultures may cause some degree of diversity of he results. The fructuation of the loop volume fron experiment to experiment suggest this possibilities. However, the strong bias to the underestimate cannot be explained simply by the experimental error. ( When the average loop volume of the four experiments is used for the calculation, expected numbers of swarms change as in Table 3, which suggest the possibility that the experimental error is remarkable as a cause of the higher estimate in the experiment 1 rather than the lower estimate in others.) 2). The inhibitory effect of the antisera is clear on the experiment 3 and 4: the higher the concentration of antisera in a plate, the less proportions of swarms are recovered. The quantitative relationship between them is not clear from thése experiments. 3). The condition of the media, especially the humidity of the MGA- surface, allow the teakjual spread in various degrees. In general, the more the plate surface im humid the wider the brush spread ard the recovery of swarms decrease, even if the concentration of the antisera is the same. 4), As far as the present results concern, Gal* cells decreased more than Gal™ celle regardless the phase of H-antigens. However, the review of the foregoing transduction experiment does not show such regularity, so the generality of this phenomeron is still in question. In conclusion, the method emploied for the single phase transduction experiment may results the overlook of a part of transduction in various degrees by the interference of several factors mentioned above. This loss mast be considered carefully when the frequencies of the transductions are discussed quantitatively. Especially, when the transduction efficiency of the H, and H, is discussed from the ratie of H)-transduced type and H,- transducedotype in "phase 2 --x phase 1" transduction, the third variable “the efficiency that the transducion clone can develope as swarmy" must be introduced. Table 1. Procedures of the reconstruction experiment. Sal. abony Sw803 4 (enx)Gal~ (b)1 ate 4----------streak on EMBigal. plates from stab eee eee ~m---antigen test by slide agglutination - 0.01 mi 0.01 ml 10 ml saline 10 ml saline 0.01 ml ——~ 0.01 ml 10 ml saline 10 ml saline 676? spread on EMB-gal Plates, 0.02 ml per plate (plate-I) 2 Gal” overnight culture in penassay broth] media spread on FMB-gal plates, 8.02 ml per plate Te (bd) enx Gal” 6703=n% Pieter) mix 0.25 ml each — 0.61 mi 1_loop brush on anti- 10 ml saline 10 nl saline b,-enx MGA plates 0.01 ml 0.05 ml | (or 0.01 ml) 10 ml saline 10 ml saline @r0t=nt spread on FMB=gal plates, 0.02 ml per plate (plate-III) spread on EMB-gal plates, 0.02 ml per plate (plate-I¥) * 3 to 5 plates were used for the spread of each dilutions. Formla to caluculate leop volume and expected ~umber of swarms: Loop volume (L) = (number of colonies per plate-IV)x10~2 (number of colonies per plate-III)x5 Expected mumber of swarms of "i(enx)Gal” ’= —):25__spunber of coionies per plate-~I) xL Expected number of swarms of "(b)1,2Ga1TM& 9.23 x (mumber of colonies per plate-II) x L Table 2. Results of the reconstruction experiment of Heantigen transduction between single phase cultures. a). Experiment with T™2. Number of coloni lat No. of swarms No.of antisera 1 2 Spin Sete ; a CLOOp) ~ er brush Experiment (amount per b(1,2)(i)enx 1(1,2)(4)1,2 41(1,2)(41)1,2 att 32) \iJenk loop volume plate) Gal” Gal” Gal Gal Gal Gal __ Gal? Gal eo 1 anti-i O.4ml 52.0 36.6 21.0 16.6 46.0 41.5 2.8 2.3 anti-1,2 0.4ml expected---- 3.1 2.2 4. 7x107 & observed to expected90_ 108 2 anti-i 0.4 ml 68.6 54.8 25.6 26.0 20.6 14.8 4.0 5.2 anti-1,2 0.4m1 expected——-- 7.3 5.8 8.5xl0~ 90 _% observed to expecteds5 * 6.01 ml of suspension was transfered instead ef 0.05 mloon 2nd dilution bd). Experiment with SW803. Ne. of antisera panes ef colonies per plat No. of swarns Experiment (amount per 1 3(pipette) 4(loop) per brush plate) Loge) (0) 24 2 b(enx)(b) enx nage? (pen i (enx) fo) h.2 106p volume Gal Gal? Gal” Gal Ga ‘eal Gal” 3 anti-b 0.75ml 86.0 103.7 25.3 29.3 87.5 -91.0 Ze 3 4 6. 5x1079 2 anti-enx 0.4m1 expected----- 7.0 8 % ovserved to expected" 30 27 3 5 4 anti-b 0.3 ml 36.0 62.6 20.0 25.0 73.4 81.8 2.0 3 3 % observed to expected---- 65 61 Table 3. Index of recovered % betweer two cultures. Experimental number 1 2 3 4 Index Of phase-i to phase-2 0.86 0.61 1.1 1.6 + — Index of Gal to Gal 0.86 O.ol N.91 0. Ft