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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1978, p. 199-201 Vol. 35, No. 1
Copyright © 1978 American Society for Microbiology Printed in U.S.A.

NOTES

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Dulcitol-Malonate-Phenylalanine Agar for the Identification of
Salmonella and Other Enterobacteriaceae
SAMUEL ESKENAZI AND ALLAN M. LITTELL*
Food and Drug Administration, Department of Health, Education, and Welfare, Brooklyn, New York 11232

Received for publication 23 September 1977

An agar medium combining dulcitol fermentation, malonate utilization, and

phenylalanine deamination was evaluated with 229 isolates representing 19 gen-
era. All reactions agreed with those obtained on conventional media.

Numerous composite, tubed media have been complex in the presence of ferric ions. Shaw and
developed and are currently used in clinical, Clark (2) found that testing for this product was
quality control, and other laboratories involved compatible with the malonate test and devel-
in microbial identification. The purpose of these oped a medium incorporating both substrates.
media is to combine several biochemical reac- Therefore, phenylalanine was included in our
tions into one test system, with the resulting composite medium.
savings in time and equipment, without loss of The dulcitol-malonate-phenylalanine agar
sensitivity. (DMP agar) we propose is patterned after the
For identification of Enterobacteriaceae, medium described by Bonev. Salicin was deleted
three composite, tubed media have been de- from the formulation and phenylalanine was
scribed (1-3) that combine the principle of mal- added. All other ingredients are the same, except
onate utilization with other biochemical reac- the agar concentration is reduced from 8 to 5
tions. The most recent one, reported in 1976 by g/liter. The formulation of DMP agar is shown
Bonev (1), combines dulcitol and salicin fermen- in Table 1. The medium is dispensed in tubes
and after autoclaving is allowed to solidify in a
tation with malonate utilization for use in differ-
entiation within the genus Salmonella. The in- vertical position (i.e., not as slants). The medium
itial purpose of our studies was to develop and is inoculated by stabbing once with a needle
evaluate a medium combining dulcitol and mal- through to the bottom of the tube.
onate into a single test that would be useful for In preliminary studies, it was found that by
differentiating Salmonella arizonae from other reducing the agar concentration a faster and
salmonellae. S. arizonae uses malonate but not more distinct acid reaction was obtained with
dulcitol. With most other salmonellae the re- dulcitol fermentation. This is probably due to
verse is true. With these two carbon sources more rapid growth and an enhanced diffusion
together in an agar medium, dulcitol fermenta- of acid end products. Reactions in the unslanted
tion results in an acid reaction, whereas malo- tube were easier to read and more distinct than
nate utilization produces an alkaline reaction. those observed on agar slants.
The two reactions can easily be distinguished The organisms used in evaluation of the me-
with bromothymol blue incorporated in the me- dium were obtained from the culture collections
dium as an indicator. of the Food and Drug Administration, Brooklyn,
A third biochemical test was included in the N.Y., and from the National Center for Disease
medium so that it could be used for differentiat- Control, Atlanta, Ga., or they were routine iso-
ing Proteus from Salmonella. Typically, urease lates from food samples analyzed at the Brook-
and phenylalanine deaminase production are lyn Food and Drug Administration Laboratories.
used to differentiate these two genera. Testing The cultures were grown on brain heart infusion
for the former enzyme activity is incompatible agar slants for 24 h before testing.
with the malonate-dulcitol system described The appearances of typical reactions on DMP
above because the alkaline reaction would be agar are described in Table 2. An organism that
confused with malonate utilization. One of the does not use malonate or dulcitol will leave the
products of the phenylalanine deaminase reac- medium unchanged in color. If malonate but
tion, phenylpyruvic acid, gives a green-colored not dulcitol is utilized, an alkaline reaction oc-
199
200 NOTES APPL. ENVIRON. MICROBIOL.
TABLE 1. Formulation of DMP agar-
Ingredient Amt (g)
Ammonium sulfate. 2
Dipotassium phosphate" 0.6
Monopotassium phosphateb 0.4

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Sodium chloride" 2
Sodium malonateb 3
Bromothymol blue" 0.025
Yeast extract. 1
Dulcitol ........................... 1
DL-Phenylalanine .2
Agar ............................... 5
Distilled water 1,000 ml
aAdd ingredients, heat to boiling, adjust pH to 6.7
(green color), dispense 10 ml into tubes, and autoclave
for 15 min at 121°C. Allow to solidify in an unslanted
position.
bThese components represent malonate broth
(Difco) that can be purchased commercially and used
as a base for DMP agar.

TABLE 2. Description of typical reactions on DMP
agar at 24 h
Appearance
Reaction
Top Butt
Malonate -
Dulcitol- Green Green
Malonate + Blue Green
Dulcitol -
Malonate - Yellow Yellow
Dulcitol +
Malonate + Blue Yelow
Dulcitol +
curs in the top portion of the medium, turning
the indicator blue and leaving the butt relatively
unchanged. If only dulcitol is utilized, a charac-
teristic of most Salmonella, the entire medium
will turn yellow, as a result of acid formation.
Those organisms capable of utilizing both mal-
onate and dulcitol produce a blue alkaline reac-
tion in the top portion of the medium and a
yellow acid reaction in the butt of the medium.
After reading the malonate and dulcitol reac-
tions, the phenylalanine test is initiated by add-
ing 0.2 ml of 0.1 N HCl and 0.2 ml of a 10%
ferric chloride solution to the tube. After 5 min,
the phenylalanine deaminase reaction develops
as shown in Fig. 1. The tube on the left was
inoculated with a Proteus strain that produces
phenylpyruvic acid from phenylalanine. The
positive reaction is seen as a dark ring at the
interface of the reagents and medium due to
complexing of phenylpyruvic acid with the ferric
ion. The tube on the right is a negative Salmo-
FIG. 1. Phenylalanine deaminase reaction. The
tube on the left shows a positive phenylalanine de-
aminase reaction. Note the dark ring at the interface
of the medium and the reagents.
nella strain that does not produce the ring. We
found that refrigerating the medium before re-
agent addition gave a sharper reaction in a pos-
itive phenylalanine deaminase test. This is prob-
ably caused by limited diffusion of the colored
complex through firming of the agar medium.
Occasionally, a slow dulcitol-fermenting or-
ganism will be encountered. Therefore, it may
be necessary to incubate tubes with questionable
reactions an additional 24 h before testing for
phenylalanine deaminase.
The ability of the medium to produce char-
acteristic and reproducible reactions was tested
by inoculating tubes of the medium with known
cultures of Enterobacteriaceae and other
groups. A total of 229 isolates representing 19
genera of bacteria were used. In each case, tubes
of DMP agar were inoculated concurrently with
the conventionally used dulcitol and malonate
broths and phenylalanine agar (Table 3). In all
cases, the reactions on DMP agar agreed with
those of dulcitol and malonate broths and phen-
ylalanine agar.
DMP agar has been included as an additional
medium along with the malonate and dulcitol
broths normally used for Salmonella analysis
in the Brooklyn Food and Drug Administration
Laboratory. A total of 47 isolates from 15 food
samples have been tested. Other analysts were
involved in inoculating and reading the reactions
on DMP agar and the three conventional media.
In all cases, the reactions on DMP agar agreed
with those of the dulcitol and malonate broths
and phenylalanine agar.
VOL. 35, 1978 NOTES 201
TABLE 3. Results obtained by conventional media in comparison with DMP agar
Results of conventional media DMP agar reactions
Organism Total no. of
strain Phenylala- Phenylala-
Dulcitol Malonate nine deami- Top Butt nine deami-
nase nase

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Salnonella 70 + - - Yellow Yellow -
Escherichia coli 4 + - - Yellow Yellow -
Citrobacter 6 + - - Yellow Yellow -

Arizona 16 - + - Blue Green -

Citrobacter 1 - + - Blue Green -

Enterobacter 20 - + - Blue Green -
Klebsiella 3 - + - Blue Green -
Citrobacter 1 + + - Blue Yellow -
Klebsiella 1 + + - Blue Yellow -
Enterobacter 17 + + - Blue Yellow -
Citrobacter 4 - - - Green Green -
Shigella 14 - - - Green Green -

Klebsiella 2 - - - Green Green -

Edwardsiella 5 - - - Green Green -
Enterobacter 20 - - - Green Green -
Serratia 17 - - - Green Green -

Proteus 8 - - + Green Green +

Providencia 2 - - + Green Green +
Pseudomonas 1 - + - Blue Green -
Aeromonas 1 - + - Blue Green -

Streptococcus 1 - - - Green Green -

Staphylococcus 2 - - - Green Green -
Micrococcus 3 - - - Green Green -
Sarcina 3 - - - Green Green -

Gaffkya 1 - - - Green Green -

Brevibacteriun 2 - - - Green Green -

Bacillus 4 - - - Green Green -

In summary, we have developed an agar tube
medium that combines in a single test dulcitol
fermentation, malonate utilization, and a test
for phenylalanine deaminase. On the basis of
the work reported here, DMP agar proved
equally as sensitive as the medium used for the
individual test.
(This paper was presented at the 77th Annual
Meeting of the American Society for Microbiol-
ogy, 8-13 May 1977, New Orleans, La.)
We wish to thank James D. Macmillan of the Food and
Drug Administration and Rutgers University for his invalua-
ble assistance in the preparation of this manuscript and Philip
McLaughlin, Thomas Mood, and Daniel Quagliaro, MicSec
NYK-DO, for their photographic assistance.
LITERATURE CITED
1. Bonev, S. I. 1976. DMS agar, a new composite tube
medium for the differentiation within the genus Sal-
monella. Int. J. Syst. Bacteriol. 26:79-81.
2. Shaw, C., and P. H. Clarke. 1955. Biochemical classifi-
cation of Proteus and Providence cultures. J. Gen.
Microbiol. 13:155-161.
3. Stroup, J. R. 1972. Malonate Dulcito Lysine Iron Agar-a
new differential medium for the identification of Sal-
monella subgenera 1-111. J. Assoc. Off. Anal. Chem.
55:214-218.