AN EXPERIMENT OW THE DIFFUSION OF SALMONELLA CELL SUSPENSIONS
THROUGH FILTER FAPER STRIPS.

Report by Tetsuo Lino ( January 12, 1958)

The peport deals with a preliminary experiment to separate the
mixtures of two different serotypes or of motile and non-motile cells by

differential diffusion on filter paper strips.

Laterials and Methods.

In the experiment 1, the following two stable monophasic strains
were used: a Gal7 Flat b-type strain obtained from 5. abony SW803 -x
5+ para B SW6oo, and a Gal* Fle’ i-type strain, SW1180. Each of them
was grown overnight in penassay broth and their Gal and antigen characters
were confirmed on the samples which were streaked or sto EB-gal phates
before the cultures were mixed.

A 8mm wide filter paper roll (for paper chromatography) was cut 60
160mm long pieces, and each of them was hung in a test tube (ismm assis)
with metal cap, as shown in figure 1. Five such test tubes were prepared.
In the test tubes 2 and 3, 0.2 ml of 1/100 dilution of anti-b and anti-i
strums were dropped correspondingly at the bottow of the tubes( the serum
does not touch the filter papers}. In the tubes 4 and 5, 0.02 ml of 1/10
dilution of anti-b and anti-i serun sotugmtions were correspondingly
dropped on the filter paper strips at 5 om from the lower end. ‘he serua
solution diffused 2 cm up and down from the point spotted. the filter
papers were stayea until they were dried.

0.5 ml of the each test cultures were mixed in 10 ml of water, and
2 ul of them was dropped at the bottom of each tubes ( ca 0.5 ma of the
lower ends of the filter papers were immersed in the cell suspensions).
they were kept at room temperature, and let the cell suspensions to diffuse
through the filter papers. After one end a half hours ( the water has
reached at the upver end of the filter papers), filter papers were tekken

out from the tubes, cut in two and printed on EMB-sal plates. Each print
was devided into five sections and was strecked as shown in figure 2.
ithe proportion of two types of celis along the filter paper strips was
scorec from the ratio of Galt? and Gel” colonies grown from each sections
of the EMB-—gal plates.

vhe same experiment wes repeated with the mixture of a Galt Fla”
strain, SW1157, and e Gal~Flet i-type strain obtained from S. typhimurium
TM2 =x S. paratyphi B SWoo6. Anti-b test tubes were excluded from this

experiment.

Results and Conclusions.

‘The results were summarized in table 1. In the first experiment,
the proportion of i-tyse cells decreases at the upper part of the papers
in every tubes. that is, the b-type cells diffuse faster than the i~type
cells. ‘the effect of antiserum could not be aemonstrated. Whether the
diitferenee of the diffusion between the two different serotypes relates
to the negative charge of the cells or their motility is not clear,

The experiment 2 siiows that Flat i-type cells diffuse faster than Fla
cells. Anti-i serums did not afrect the result. The method is, therefore,
not useful immediately for the screening of non-motile cells . It is
suggested to use the higher concentration of antiserums for the farther

experiments.
Table 1

the distribution of two cell types along the filter papers.

 

 

 

 

 

 

 

SECTION ON 7 Number of Galt and Gal™ cells on each sections
A FILTER i (%o)
PAPER + =f + =] + -] 4+ ~{ + - + w-l + =| + o
upper end
5 1 3} 0 QO 0 0 0 0 QO 0 0 1 0 0 0 0
(25)
4 4 13 0 1 0 6 0 0 2 16 0 6 0 0 0 2
(24) (11)
3 54 58] 18 46] 20 23/17 19) 40 73 4 88 8 46 9 42
(48) |(28) (47) 447) (35) (4.5) |(15) (18)
2 5D 24; 59 42) 26 45] 45 53117 22 14 ef} 11 821 3 50
(70) |(58) = |(37)~——s*|(46) (44) (34) = {(12) (38)
1 T2 42) 42 21) 77 321 47 %60| 73 62 30 68, 17 74) 28 57
lower end (63) |(60) (72) ~—*4|(57) (54) (31) {(19) 33:
OQ 48 45 835 134
(51) (38)
Tube no. 1 2 5 4 5 6 7 8
Antiserum anti-b| anti-i anti-b| anti-i / | anti-i | amti-i
in susp. in susp.on filt.on filt. in susp.on filt.

Mixture of

Flat b Gal” & Flat i Galt

 

 

 

Fla-(i)Galt & Plat i Gal~

 
Fig. 1

metal cap
Wire holder

test tube

 

: filter paper strip
i (fold longitudinaly)

 

 

cell mixture

 

 

 

Fig. 2

 
        
   
 

EMB-gal plate

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print of filter
paper strip