RECONSTRUCTION EXPERIMENT OF THE TRANSDUCTION BETWEEN SINGLE

H~PHASE It SALMONELLA, II
Report by Tetsuo Iino
April 10, 1956

Tn the previous reconstruction experiment, the possibilities that the
masked transduction types are not recovered because of inhibition dy the
antiserum added to selective media, and that the appearance of exceptional types
is caused by the phase variation in recipient cultures during experiment, have
not been tested. The present experiment was designed to test these posibilities.

Materials aud experimental procedures were listed in fable 1.

The average no. of colonies on each series of plates are shown in Table 2,
and the no. of swarms recovered from brushes are shown in Table 3, with their
antigen types and galactese character,

From the data of Table 2, numbers of each type of cells in original culture,
in mixed suspension and in brushes are calculated as follows:

Volume of loop = 2,8 x 10 - = 5.2.x 107? (m1)

W7
Com, of original cultpres (per ml)

b:(1,.2)Gal* = 65.2 x52 10? = 3.3 x 10°
(b):1,.2Gal” = 37.6 x5 x10? = 1.9 x 10°
4: 1.2Gal* = 47.4 x 5 x 107 = 2.4 x 10”

Conc, in mixed suspencton (per ml)

65.2 x G25 =B8.2x 102

6
37.6x Gsfe = 4.7 x 10°

i:1.2 Gal? = 2.4 x 10% x 0.5 = 1.2 x 107

b:(1.2)Gal*

(b):1.2Gal™

No. of cells in 15 brushes
pi(1.2)@al” = 8,2 x 10? x 5.2 x 10x 15 x 32 = 61

i
8 # "1 i "! =
8 xe 3

le 4

b:(1.2)Gal = 4.7 107 x 5.22 10x 15 x x 2

(b):1.2 Gal®

- 19
b):1.2 Gal7 = ' " " " — =
(b)31.2 Ga f ' x 50 35
The final results are summarized in Table 4. ‘the results clearly show the
masking of Gal (b):1.2 cella by anti i,1.2 mot agar plate, and the growth

of swarms of exceptional type (Gal” swarms in this experimeat) due to phase
variation happened during the coumse of experiment. The ratio of Gal*b: (1,2)
to Gal™ b:(1.2) recovered by auti i1,1.2 mot agar selection in this experiment
agree quite well with the correspoadiug ratio in transduction experimeut

(see Table 4. X2 = 0.07%, 0.7 <p ¢ 0.8).
Table 1.

Procedures of the reconstruction experiment.

Sel. typhimuriun

 

 

 

b(1.2)Gal* (b)1.2 Gal™ i:1.2 Galt
a ae atreak on FIMB-gal. oe from stabs |
ae eee antigen test by slide agglutination ----
worsen nese overnight culture in penassay broth media wane enna

0.01 ml to 10 ml saline 0.01 ml to 10 ml saline

0.01 ml to 10 ml saline 0.01 ml to 10 nl saline
|

 

spread on EMB—gal,. spread on EMB~gal.
plates, 0.02 ml per Plates, 0.02 ml per
vlate (plate-I) plate (plate-II)
0.25 ml 0.25 wl 0.5 ml

SN
>

0.01 ml to 10 ml saline 1 locp to 10 ml saline brush by loop on anti-
, 1,1.2 mot agar pjates
0.01 ml to 10 ml saline 0.01 ml to 10 ml saline

spread on EMB-gal. plates, spread on’ EMB-gal. plates,
0.02 ml per plata 0.02 ml per plate
(plate-III) (plate-IV)

* 5 plates were used for the epkeay count.
Table 2.

Average mmber of colonies per plate (average of 5 plates)

Plate no, Culture

I b3(1.2)Gait
II (B):1.2Gal~
III 4:1.2 Galt

(0.01 ma pipette)

IV 4:1,2 Gal
(loop)

Average no. Antigen type of colonies

65.2 b:19/20 1.2:1/20
37.6 b: 1/20 1.2:19/20
47 4 i: 6/20 1.2:14/20

24.8

 

Table 3.

Number of swarms and their galactose character.

 

 

Brush no, 1234 5 6 7? 8 910111213 W115 Total

Gal"b:(1.2) 5 2231797643573 5 6

Gal b:(1.2) © 01009090043212000008000 2

Total 523317986435 5735 "A
Table 4.

Summary of the results

Number of cells of each types

Calculated from colony
count of EMB-plateé

7 (per 35 brushes)

Gal b(1.2) 61
Gal*(d)1.2 3
Gal*$(1.2) 2

Gal “(b)1.2 35

Recovered from 15 brushes Corresponding ue. from

by anti 1,1.2 mot agar transduction experiment
selection (no. of brush differ)
69 133
/ /
2 3
/ /