Japan. J. Microbiol.

Vol. 14 (4), 279-284, 1970

Determination

of Pseudomonas

by Biochemical
II.

Acylamidase
the

Test,

Identification

Test

aeruginosa

Methods

a Modified

Biochemical

of Pseudomonas

Test

for

aeruginosa

Taketoshi ARAI, Masako OTAKE, Seiji ENOMOTO, Sachiko GOTO,

and Shogo KUWAHARA
Departmentof Microbiology,ShowaPharmaceutical College,Tokyo, and Department
of Microbiology,Toho UniversitySchoolof Medicine,Tokyo
(Receivedfor publication,January 9, 1970)
ABSTRACT
A rapid and simple test method for the detection of acylamidase activity of Pseudomonas aeruginosa was devised. One loopful of a nutrient agar overnight culture of a
test organism was inoculated into 1 ml of a test medium consisting of 0.2% KH2PO4,
0.01% MgSO4.7H2O, 0.5% NaCl and 0.1% acetamide (final pH 6.8). After aerobic incubation at 37C for 6 hr, one drop of Nessler's reagent was dropped into the test medium. A
reddish-brown sediment appeared immediately if results were positive. Of 40 test strains
of P. aeruginosa 39 gave strongly positive results. A strain showed a weakly positive result
after 6 hr incubation, but the reaction became stronger after 18 hr culture. Other species
of Pseudomonas, and various species of bacteria such as genera Vibrio and Aeromonas,
and family Enterobacteriaceae were negative in this test. From these experimental results,
the acylamidase test was considered to be highly specific for strains of P. aeruginosa, and
therefore useful as a reliable method for the identification of this species.

In a previous paper [1] an improved

technique was reported for microbial gluconate oxidation. Although this reaction
was considered to be one of the most reliable tests for the speciation of P. aeruginosa, there exist strains of this species
which, though rare, are negative for gluconate, oxidation. Further some other species
of Pseudomonas and Enterobacteriaceae

activity
was fairly specific for P. aeruginosa.
Consequently,
attempts
were made
to de-

gave positive results in this reaction.

In 1960, Kelly and Clarke [4] reported
the presence of an aliphatic amidase which
was capable of hydrolysing either acetamide or propionamide. It appeared likely,
from our experiences, that this enzymatic

ence with

vise a simple
the detection
this

and reproducible
of acylamidase

was accomplished

medium

method
activity,

by using

containing

acetamide

for
and

a synthetic
as the

sub-

strate
for enzymatic
action,
and Nessler's
reagent
was used as the test reagent.
The
our

present

paper

Basal

279

our

experi-

its specificity,

and

results.
MATERIALS

tion

reports

this procedure,

medium.

of the following

AND METHODS
An

inorganic
composition

salts

solu-

was used

280

T.

ARAI,

M.

OTAKE,

S.

ENOMOTO,

S. GOTO

as the basal media.

KH2PO4
NaC1
MgSO4.7H20
Redistilledwater

AND S. KUWAHARA

Table

2.0g
5.0 g
0.1 g
1000ml

1.

List

of

the

test

strain

pH 6.8

To this basal solution acetamide was added

in various concentrations, the reaction was
again adjusted to 6.8, and each solution
dispensed into 15 x 170 mm test tubes in
1 ml amounts, and then autoclaved at
121 C for 15 min.
Strains used. A total of 78 strains of 18
species as listed in Table 1 were used.
Thirteen strains of P. aeruginosa were isolated from patients at the Central Laboratory of Toho University Hospital. Aeromonas and V. parahaemolyticus
strains
were kindly supplied by the Yokohama
Quarantine Station, and some strains of
enterobacteria by Tokyo-to Laboratories
for Medical Sciences. All strains were subcultured on nutrient agar slants wiht successive transfers at one month intervals.
Strains of V. parahaemolyticus were cultured in a nutrient broth or agar medium
containing 3% NaCl.
Method for detection of acylamidase activity. One loopful of a nutrient agar overnight culture of the test organism was inoculated into 1 ml of the acetamide medium,and after incubation at 37 C, aerobically for 6-18 hr, one drop of Nessler's reagent was added to the culture. A reddishbrown sediment was immediately produced
if a positive result appeared.
The intensity of the Nessler reaction
was expressed as +, ++, and +
Nessler's reagent was prepared as follows: One gram of HgC12 was added to 6
ml of distilled water and heated to dissolve
completely. Separately, 2.5 g of KI was dissolved in 6 ml of distilled water, and this
was mixed with the HgC12 solution. Then
6 g of KOH was dissolved in 6 ml of distilled water, and added to the above solu-

tion.
tilled
tion

After
water
filtered

stirring

well,

was added,
before

13 ml more
and

the

total

of dissolu-

use.
RESULTS

Determination of the Composition of the

Basal Inorganic Salts Solution
a) Examination for the presence of acylamidase activity with aqueous acetamide
solution. A 1% aqueous acetamide solution without any additional salt component was autoclaved, and then 1 ml of this
solution was inoculated with one loopful
of an overnight culture of P. aeruginosa,
incubated at 37 C for various incubation
times and the presence of ammonia produced by acylamidase activity was checked
with Nessler's reagent.
As shown in Table 2, Nessler reaction
was weakly positive at 6 hr, and strongly
positive at 24 hr, although no bacterial
growth was observed.
b) Effect of the addition of MgSO4 to the
test medium. To the basal composition
mentioned above and to the one lacking
MgSO4 0.1% acetamide was added and,
after sterilization, they were inoculated in

MODIFIED

BIOCHEMICAL

TEST

FOR

IDENTIFICATION

the same way as above and incubated to

investigate the effect of magnesium ion on
growth and acylamidase activity. As seen
in Table 3, the addition of MgSO4 to
0.01% caused only slight differences in
Nessler's reaction, although a definite increase in bacterial growth was noted with
the addition of MgSO4.
c) Effect of the addition of sodium acetate
to the test medium. To the basal composition sodium acetate was incorporated to
0.1 and 0.5% respectively besides 0.1%
acetamide, and the Nessler's reaction was
compared with that lacking acetate. As
shown in Table 4, the addition of sodium
acetate did not exert any affect on the
acylamidase activity.

OF

P.

AERUGINOSA

281

Relation
between the Amount of Acetamide and the Acylamidase Activity
Acetamide

was added

to the basal com-

position to various concentrations,

and the
effect of the concentration
of acetamide on
intensity of Nessler reaction was examined.
The results are given in Table 5.
Nessler reaction
tended to be weaker
when the concentration
of acetamide was
below 0.02%. When it was 0.05%, the results varied depending
on the strain used.
When it was above 0.1%, all the test strains
of P. aeruginosa gave strongly positive reaction. Consequently,
0.1 % was considered
the optimal concentration
of acetamide for
the test medium.
Relation between
and Intensity

Table 2. Acylamidase test using 0.1%

aqueous acetamide solution

the Size of the Inoculum

of Nessler Reaction

Cells of P. aeruginosa

culture

on nutri-

ent agar slant were suspended

in sterile
saline so as to contain 109 viable cells per
ml, and serial ten-fold dilution were prepared from this original suspension. Each
dilution, in an amount of 0.05 ml, was inoculated into 1 ml of acetamide medium,
Table

3.

Effect
sulfate

of
on

the
the

addition

of

acylamidase

Table

5.

magnesium

Table

4.

test

Relation
and

between
the

Effect

acetate

intensity

the
of

concentration
Nessler

reaction

on

of

of

the

the

acetamide

addition

acylamidase

of
test

sodium

282

T.

ARAI,

M.

Table

OTAKE,

6.

S.

Relation

ENOMOTO,

between

intensity

Table

7.

of

per ml of test medium gave a negative

reaction, 107 cells per ml of test medium
gave varying results depending
on strains,
and above 108 viable cells per ml of test
medium was required to produce the positive results. According to our experimental
results, one loopful from a nutrient
agar
slant with a standard platinum loop contained approximately
109 viable cells, and
therefore one loopful inoculum to 1 ml of
acetamide medium was quite sufficient to
result

the

size

Nessler

GOTO

of

AND S.

inoculum

after

only

6 hr

Relation between Time of Incubation

Intensity of Nessler Reaction

and

One loopful of an overnight

nutrient agar of the test strain

culture on
of P. aeru-

ginosa was inoculated

into 1 ml of acetamide medium, and incubated
at 37C for
various times of incubation to examine the
effect of incubation
time on the acylami-

KUWAHARA

and

the

reaction

Relation
between
the time of incubation
intensity
of Nessler reaction

and Nessler reaction was examined.

The
results are shown in Table 6. As seen in
this table, the effect of the inoculum size
was quite negligible after 24 hr culture, but
at 6 hr, an inoculum size of below 106 cells

give a positive
incubation.

S.

and

dase activity. The results are shown in

Table 7. After incubation for more than
5 hr, the reaction was always strongly positive regardless of the incubation time.
Results of Acylamidase Test of 40 Clinical
Isolates of P. aeruginosa and Related
Species
Fourty fresh clinical isolates of P. aeruginosa were investigated for their acylamidase activity by the test method mentioned above. After 6 hr incubation all the
test strains showed strongly positive results
except for one strain, which gave weakly
positive reaction at 6 hr, but turned more
strongly positive after 18 hr incubation.
Seven strains of P. flu orescens, and each
one strain of P. fragi, P. chlororaphis and
P. putrefaciens were cultured at 18C for
various times of incubation, and tested for
their acylamidase activity. As shown in
Table 8, the reaction was negative in all
the test strains after 48 hr incubation, and
even after 72 hr of incubation only one
strain gave a weakly positive reaction, and
3 strains showed doubtful results.
Results of acylamidase test with related

MODIFIED

BIOCHEMICAL

TEST

FOR

Table 8. Acylamidase
activities
of various
species of Pseudomonas
other than
Pseudomonas

IDENTIFICATION

Table

OF

9.

P.

AERUGINOSA

Acylamidase

activities

gram-negative

bacteria

283

of

related

aeruginosa

bacterial species, such as Aeromonas

spp.
V. parahaemolyticus,
Achromobacter,
E.
coli, Klebsiella, Enterobacter,
Citrobacter
and Proteus group are given in Table 9. All
of the test strains gave negative results not
only at 6 hr but also even after 18 hr
incubation.
Thus, acylamidase activity appeared
to
be highly specific to P. aeruginosa, and the
test highly discriminatory.
DISCUSSION

Biihlmann and his coworkers [2] described a test method for the deamination
of acetamide by P. aeruginosa using a
modification of Christensen's urea broth
[3]. However, this method required at least
12 hr incubation to give clear-cut results.
The use of an inorganic salts solution containing acetamide as a sole source of carbon and nitrogen in our present investigation resulted in the more rapid detection
of acylamidase activity. We used Nessler's
reagent as the test reagent to detect the
presence of ammonia resulting from enzymatic action.
Kelly and Kornberg [6] detected amidase
activity from cells grown in a synthetic
medium containing acetate as a sole carbon
source. In our present experiments, how-

ever, we could not confirm any affect of the

addition of acetate to the acetamide medium. Although
acylamidase
activity could
be detected by inoculating
a large amount
of resting cells into a 1% aqueous solution
of acetamide,
more distinct
and reproducible results were obtained when a test
strain was inoculated
into the above described medium and incubated at 37C for
more than 6 hr.
Since it is well known that alkaline earth
metals cause sediment production by Nessl-

284

T. ARAI,

M. OTAKE,

S. ENOMOTO,

er's reagent,

it was of considerable

im-

portance to determine if the addition of

Mg ion exerted any effect on the test.
Actually, a slight pale yellowish turbidity
appeared in the control medium several
minutes after the addition of Nessler's
reagent.
When the reaction was negative, however, the medium was colorless and transparent immediately after the addition of
the reagent, and, if positive, red-brownish
sediment was produced rapidly so that the
determination could be made quite easily.
In the Biihlmann's method, acetamide
was added to a final concentration of 1%
to the test medium. However, in our
present test method, 0.1% was sufficient
for successful result, presumably because a
synthetic medium containing acetamide as
a sole nutrient source was used as test
medium and method to detect ammonia
directly was employed.
Kelly and Kornberg [6] recommended
the use of Seitz filtration of the acetamide
solution as a method of sterilization. But,
in our present experiments, the compositions containing acetamide was sterilized by
autoclaving at 121C for 15 min, enabling
the preparation of the test medium to be
done easily. Since the Nessler reaction was
confirmed to be negative in the control
medium, the effect of sterilization by heating seemed negligible for the present
object.
Buhlmann and his coworkers reported
that out of 49 strains of P. aeruginosa 45
were strongly positive and 4 strains were
weakly positive after incubation at 37C
for 12 hr. In our experiments, all except
one of the 40 test strains gave strongly
positive results after 6 hr incubation at 37C
when a large size of inoculum was used.
If the Nessler reaction was performed after
24 hr incubation, the results were invariably positive with the strains of P. aeru-

S. GOTO

AND S. KUWAHARA

ginosa regardless of the size of inoculum.

According to Bahlmann's
paper 12
strains of other species of Pseudomonas
were all negative in acylamidase test. In
our experiments, all the test strains of
P. fluorescens, P. fragi and P. chlororaphis
were negative in this reaction even after
48 hr incubation, and related genera such
as Aeromonas, Achromobacter, and V.
parahaemolyticus were also all negative
after incubation at 37C for 24 hr. Thus the
acylamidase test devised by the authors
was found to be highly specific to P. aeruginosa, and is therefore considered to be
useful as a means of the identification of
this species.
ACKNOWLEDGEMENT
We want to express our deep thanks for the
supply of test strains to Dr. K. Shimizu of the
Central Laboratory of Tokyo UniversityHospital,
Dr. H. Abe of YokohamaQuarantine Station and
Mr. Kudoh of Tokyo-to Laboratoriesfor Medical
Sciences.
REFERENCES
[ 1] Arai, T., Enomoto,S., and Kuwahara,S. 1970.
Determinationof Pseudoinonasaeruginosa
by biochemicaltest methodsI. An improved
methodfor gluconateoxidationtest.Japan.
J. Microbiol.14: 49-56.
[ 2 ] Bilhlmann,X., Vischer,W. A., and Bruhin,
H. 1961. Identification of apyocyagenic
strains of Pseudoinonasaeruginosa.J. Bacteriol. 82: 787-788.
[ 3 ] Christensen,W. B. 1946.Urea decomposition
as a means of differentiatingProteus and
Paracolon cultures from each other and
from Salmonella and Shigella types. J.
Bacteriol.52: 461-466.
[ 4 ] Kelly, M., and Clarke, P. H. 1960.Amidase
production by Pseudoinonas aeruginosa.
Biochem.J. 74: 21.
[ 5] Kelly,M., and Clarke,P. H. 1962.An inducible amidaseproduced by a strain of Pseudomonasaeruginosa.J. Gen. Microbiol.27:
305-316.
[ 6 ] Kelly,M., and Kornberg,H. L. 1962. Discontinuity of amidase formation by Pseudomonas aeruginosa.Biochim.Biophys.Acta
59: 517-519.