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A NEW CULTURE MEDIUM FOR THE ISOLATION OF 
BACILLUS TYPHOSUS FROM STOOLS* 

PLATE 20 

J. E. H L T-H ARRIS AND OSCAR TeAGUE 

From the Quarantine Laboratory, Health Officer's Department, Port of New York 

Of the numerous methods which have been recommended for the 
isolation of typhoid bacilli from feces the one at present most widely 
used is that devised by Endo in 1903. It allows the development of 
a high percentage of the typhoid organisms inoculated on it, but does 
not inhibit to any practical extent the growth of those fecal bacteria 
which develop on ordinary nutrient agar. The great value of the 
method lies in its sharp differentiation between the lactose-ferment- 
ing organisms and those organisms which do not ferment lactose. 
The colonies of the lactose-fermenting organisms are colored red after 
24 hours' incubation, while the other colonies remain colorless. We 
have found that better results are obtained if both lactose and saccha- 
rose are added to the Endo medium, since certain members of the 
colon-bacillus group ferment saccharose more rapidly than lactose 
and hence their colonies take on the red color sooner and can no longer 
be regarded as slow colonies; the typhoid colony is without color in 
24 hours, as on the Endo medium containing lactose alone. 

The chief disadvantage of the Endo medium lies in the fact that 
the red color is not confined to colonies of B. coli themselves, but 
spreads out through the medium adjacent to them. If the colonies of 
B. coli are close together the whole plate soon becomes red and then 
colorless colonies on it can no longer be distinguished from the red 
ones. We have tested a great number of stains separately and in 
combinations of varying strengths with the view of overcoming this 
difficulty while at the same time preserving the effectiveness of the 
medium for the sure growth of typhoid. In every instance where 
the acid or the basic fuchsin was tried, the color diffused into the 
medium around the red colonies, so that it was difficult, if not impos- 
sible, to recognize the typhoid colonies which may have lain in these 
areas. We have finally devised a medium that gives even better dif- 

* Received for publication November 24, 1915. 



Medium for Isolation of Typhoid Bacilli from Stools 597 

ferentiation between the lactose-fermenting colonies and those that do 
not ferment lactose than the Endo plate and at the same time remains 
unchanged in the areas between the colonies. It consists of a com- 
bination of methylene blue and eosin in nutrient agar containing 
lactose and saccharose. On plates of this medium after 18 hours' 
incubation the colonies of typhoid are colorless and transparent, 
while those of B. coli are a deep black and do not transmit light. The 
medium immediately around the colonies of B. coli remains practically 
unchanged. Hence the plate is workable even when the colonies are 
close together. More of the feces, therefore, can be safely inoculated 
on this medium than on the Endo plate and it is to that extent more 
delicate and to be preferred. 

The medium is prepared as follows : Nutrient agar is made in the 
usual way, containing 1.5% agar, 1% Witte's peptone, 0.5% sodium 
chlorid, and 0.5% Liebig's meat extract, to the liter of distilled water. 
It is cleared with egg-white, placed in flasks, and sterilized in the 
Arnold sterilizer on 3 successive days. The reaction is brought to 
+0.8. The agar is melted and saccharose (.5%) and lactose (.5%) 
are added. The medium is then heated for 10 minutes in the Arnold. 
To every 50 c.c. of the medium are added 1 c.c. of 2% yellowish eosin 
and 1 c.c. of 0.5% methylene blue. We always add the eosin first and 
then the methylene blue. The mixture is shaken and plates are poured. 
The surface of the medium is dried in the usual way before the plates 
are inoculated. We have also obtained excellent results by substitut- 
ing for Liebig's extract, meat infusion rendered free from sugar by 
incubation with B. coli. 

Stock solutions of 2% eosin and 0.5% methylene blue in distilled 
water are kept in the dark. We have not sterilized these solutions, 
as we found that they could be kept in the ice-box for weeks without 
causing contaminations of the medium. Ordinarily we do not heat 
the agar after the dyes are added, but we have demonstrated that 
the stained agar can be heated a half hour in the Arnold sterilizer 
without injury. 

Eosin and methylene blue in distilled water in the proportion used, 
give precipitation ; in the medium the agar acts as a "Schutzkolloid," 
preventing the formation of a precipitate. 

The following experiment shows that methylene blue in even twice 
the amount contained in the medium as described, whether alone or in 
combination with eosin, does not inhibit the growth of the typhoid 



598 J. E. Holt-Harris and Oscar Teague 

bacillus. A strain of B. typhosus freshly isolated from the blood of a 
patient by culturing in bile was used. Two suspensions of the bacilli 
were prepared in salt solution and 1 loop of each suspension was 
inoculated on half of a plate, as shown in Table 1. The colonies 
were counted after 24 hours' incubation. They were of practically 
the same size on all the plates. The same nutrient agar with a reaction 
of + 0.7 was used throughout. 

TABLE 1 
Experiment Showing that Typhoid Bacilli are not Inhibited by Methylene Blue 



Medium Used 



Plain nutrient agar 

Agar + methylene blue (0.02%) 

Agar -f eosin (0.04%) + methylene blue (0.02%). 
Agar + eosin (0.04%) 




The following experiment was carried out to determine whether 
there is any inhibition of the typhoid bacillus when a typhoid stool 
is inoculated on the plate. A portion of the typhoid stool was rubbed 
up in salt solution and filtered first through a thin layer of absorbent 
cotton and then through filter paper. This filtrate was shaken and 
dilutions of 1 : 10 and 1 : 100 were prepared in salt solution. After 
this treatment it may be assumed that the typhoid bacilli present are 
distributed uniformly in the suspensions of the feces. One loop of 
each of the dilutions was inoculated on an Endo plate, on our methy- 
lene-blue eosin plate, and on & plain agar plate. The series of plates 
containing a convenient number of colonies was worked up in each 
instance with the results shown in Table 2. 

Table 2 shows conclusively that typhoid bacilli in stools develop as 
readily on the methylene-blue eosin plate as on Endo plates or plain 
agar. 

The chief advantage of this medium over the Endo plate, as 
already mentioned, consists in the fact that the colonies of B. coli 
become sharply differentiated from the typhoid bacilli without affect- 
ing the medium lying between the colonies, so that a typhoid colony 
can be readily recognized tho lying in close proximity to numbers 
of colonies of B. coli. There is of course a sharp limit to the amount 
of feces that can be safely inoculated on this medium, as is true of 



Medium for Isolation of Typhoid Bacilli from Stools 599 



every other medium. If the plate contains tens of thousands of colo- 
nies, each individual colony of B. coli remains very small and is 
poorly, if at all, differentiated from the typhoid colonies. It is cer- 
tain, however, that this plate will stand a heavier inoculation than 
the Endo or the Conradi Drigalski plates. 

The differentiation between the colonies of B. coli and those of 
the organisms that fail to ferment lactose is evident by reflected, as 
well as by transmitted, light; in selecting the suspicious colonies for 
fishing we use both, but rely chiefly on the picture afforded by the 

transmitted light. 

table 2 

Experiment Showing that Typhoid Bacilli Inoculated from Stools are not Inhibited 

by Methylene Blue 



Patient 



Medium 



Total 

Number of 

Colonies 



Number of 
Typhoid 
Colonies 



J. L 

M. M. ... 

M. O. ... 
M. B 



Endo . 

Methylene-blue, eosin 

Plain agar 

Endo 

Methylene-blue, eosin 

Plain agar 

Endo 

Methylene-blue, eosin 

Plain agar 

Endo 

Methylene-blue, eosin 



31 
42 
38 
29 
28 
37 
11 
12 
4 
44 
35 



1 

3 

3 

15 

16 

22 

4 

4 

2 

21 

20 



In addition to this main advantage the methylene-blue eosin plate 
possesses the following minor advantages: 

1. The colonies of B. coli are differentiated earlier on this plate 
than on the Endo plate; that is, if the two plates are inoculated at 
the same time, the colonies of B. coli on the methylene-blue eosin 
plate in some instances will have taken on black centers while those 
on the Endo plate are still colorless or have merely a pink tinge. 

2. A greater percentage of the colorless colonies turn out to be 
typhoid on the methylene-blue eosin plate than on the Endo plate. 
This is probably due in part to the fact that the former medium con- 
tains both lactose and saccharose, while the latter contains lactose 
alone, and in part to the fact that some of the organisms producing 
colorless colonies on the Endo plate fail to grow on the methylene- 
blue eosin plate. 

3. There is complete inhibition of certain organisms which form 
small colonies on the Endo plates. 



600 J. E. Holt-Harris and Oscar Teague 

4. Certain bacteria which give colorless colonies on the Endo 
medium yield colonies with blue centers and transparent peripheries 
on our plate. 

5. The Endo plate gradually turns pink on exposure to light; our 
plate remains unchanged. We have left it exposed to diffuse daylight 
for a period of 3 hours before inoculation without causing any notice- 
able deterioration. 

6. The Endo agar is adjusted to + 0.2, a reaction too alkaline to 
permit of the optimal growth of the typhoid bacillus. Our medium 
yields good results in agar, the reaction of which is best suited for 
typhoid. Slight variations in the reaction of the nutrient agar from 
which it is made, do not affect the efficacy of the methylene-blue eosin 
medium. 

7. Since the medium around the colonies remains unchanged any- 
way, there is no reason for making the agar stiffer than it is in ordi- 
nary use. 

Experiments were made also with the eosin methylene-blue com- 
bination of stains in nutrient agar with the addition of lactose, 
saccharose, and dextrose, and with the addition of dextrose alone, 
with a view to differentiating between typhoid and other non-lactose- 
fermenting colonies. There is no marked difference between the result 
given by the 3 sugars from that given by dextrose alone. On a plate 
containing the stains with 0.25% dextrose in agar, typhoid colonies 
grow well and are unmistakably differentiated from other colorless 
colonies when examined under the low power of the microscope, for 
they have peculiar blue centers which look like fine matted blue 
hairs. However, as this picture was not constant when typhoid stools 
were used, we decided to abandon the use of dextrose and depend on 
the colorless transparent colony on the eosin methylene-blue plate 
containing saccharose and lactose for diagnosis. If this plate be incu- 
bated for 48 hours, the typhoid colonies, even here, will often assume 
the centers described. 

We have used this medium for the examination of scores of 
typhoid stools and mixtures of normal stools and typhoid cultures 
with very satisfactory results. 



Plate 20 




Reproduction of a methylene-blue eosin plate inoculated with a typhoid stool 
and incubated for 18 hours. The colonies of B. coli are black. The typhoid 
colonies are transparent. The largest transparent colonies are not typhoid.