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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.
