PRACTICAL LABORATORY TEST FOR THE IDENTIFICATION OF

PSEUDOMONAS AERUGINOSA
W. L. GABY AND C. HADLEY
Division of Microbiology, Hahnemann Medical College and Hospital, Philadelphia, Pennsylvania
Received for publication March 15, 1957

Despite the efforts made to identify Pseudo- Smith and Stotz, 1954). These techniques have
monas aeruginosa by saccharolytic methods, this been modified and applied toward a practical
gram-negative microorganism remains predomi- laboratory test for the identification of P.
nately a proteolytic bacillus with weak saccharo- aeruginosa.
lytic activity. Pigmentation, growth temperature, METHODS AND MATERIALS
pellicle formation and other minor characteristics
are unsatisfactory laboratory diagnostic criteria Cultures of the microorganisms used in the pre-
(Gaby, 1946, 1955; Gaby and Free, 1953). Need- liminary screening tests were obtained from the
less to say it is highly probable that numerous P. departmental stock culture collection and the di-
aeruginosa and P. aeruginosa-like cultures have agnostic laboratory. All cultures were grown on
been erroneously identified. This is undesirable nutrient or blood agar plates, in nutrient broth
not only from a diagnostic point of view but one (4.5 ml) for 18 to 24 hr at 37 C or room tempera-
has only to examine the classification of this genus ture (24 to 28 C) depending upon the optimal
to understand the taxonomical problems in- growth requirements of the organism. All cultures
volved. were checked biochemically and stains for flagella
The need for a diagnostic procedure which can were made on all doubtful strains.
be carried out routinely in the laboratory for the The reagents used in the test were 1 per cent
identification of nonpigmented strains of P. aeru- a-naphthol in 95 per cent ethanol and a 1 per
ginosa is obvious. Preliminary experiments car- cent aqueous solution of p-aminodimethylaniline
ried out in this laboratory indicated that while oxalate (Difco). The solutions were prepared
the cells of P. aeruginosa contain a peroxidase fresh weekly because of the autooxidation of p-
and catalase, other gram-negative bacilli similar aminodimethylaniline oxalate, but can be used
to P. aeruginosa contain similar enzymes which for at least 2 weeks.
makes their utilization in a practical laboratory To detect cytochrome oxidase in broth cultures
test extremely difficult. However, it appeared of the microorganisms, 0.2 ml of the a-naphthol
that P. aeruginosa contained an abundance of solution and 0.3 ml of p-aminodimethylaniline
cytochrome oxidase. This enzyme, also referred oxalate were added to each tube. The tubes were
to as Atmungsferment, indophenol oxidase, nadi immediately shaken vigorously to insure mixing
oxidase, cytochrome c oxidase, cytochrome a3 ox- and the thorough oxygenation of the culture. The
idase, and cytochrome a oxidase, will oxidize appearance of a blue color is indicative of the
dimethyl-p-phenylenediamine in the presence of presence of cytochrome oxidase in the bacterial
molecular oxygen and cytochrome c, and upon cells. To detect the presence of the enzyme in
the addition of a-naphthol, indophenol blue is microorganisms cultured on agar plates, equal
formed. Keilin (1929) and Frei et al. (1934) have amounts of the above reagents are mixed and
shown that several fungi and bacteria contain a several drops allowed to flow over isolated
cytochrome-cytochrome oxidase system. (See colonies.
also the reviews by Smith, 1954; Clark et al.,
1955.) The vast majority of the studies on these RESULTS
compounds, however, have been carried out with The cytochrome oxidase activity of 57 stock
extracts of animal tissue in an attempt to charac- cultures is summarized in table 1. Morphologi-
terize the various components of the enzymes cally and biochemically the stock cultures were
(Wainio and Cooperstein, 1956; Estabrook, 1956; correctly labeled and not contaminated. Broth
Henderson and Rawlenson, 1956; Holton, 1955; cultures of P. aeruginosa gave a positive reaction
356
1957] IDENTIFICATION OF P. AERUGINOSA 357
TABLE 1 American Type Culture Collection tested by this
Cytochrome oxidase activity of 67 stock cultures method were found in most instances to contain
Number Cyto- comparable amounts of cytochrome oxidase.
Microorganisms of Species chrome
or Strains Oxidase These species however are being studied in more
Tested Test detail by manometric techniques.
The test worked equally as well on cultures ob-
Klebsiella ............ 6 * tained from clinical specimens. The results sum-
Proteus ......................... 6 -
marized in table 2 show a typical group of
Aerobacter ..................... 4 -
nonpigmented, gram-negative, bacilli isolated in
Salmonella ..................... 12 -
Shigella ........................ 5 -
the clinical laboratory. All of these cultures pro-
Escherichia coli . ................ 2 - duced a pellicle when grown in a liquid culture
Paracolon ...................... 2 - media and all had a similar biochemical pattern
Vibrio . ........................ 2 - when examined routinely. Of particular interest
Serratia marcescens .. 1 - were cultures no. 1034, 1306, and 1335. Colonies
......3
Staphylococcus ........... - of these cultures on blood agar after 18 to 24 hr
Bacillus ........................ 3 - incubation at 37 C were small and nondescript.
Bacillus anthracis .. 1 -
Culture no. 1306 (urine specimen) was tentatively
Alcaligenes ..................... 4 4t identified as P. aeruginosa. Cultures no. 1335
Pseudomonas aeruginosa......... 6 +1 (stool specimen) and no. 1034 (blood at autopsy)
Pseudomonas aeruginosa, 3 drops
1% KCN . .................... 1 were tentatively identified as Alcaligenes. How-
ever, cultures 1034 and 1335 gave positive cyto-
* Colorless. chrome oxidase tests while 1306 was negative.
t Blue color in 2-5 min. An extensive morphological, and biochemical ex-
t Blue color in 10-30 sec. amination of the cultures indicated that 1306 was
a paracolon bacillus similar to type 29911, while
(blue color) in 15 to 30 sec, indicating a readily
available high cellular concentration of cyto- TABLE 2
chrome oxidase. Broth cultures of Alcaligenes Cytochrome oxidase activity of several representative
faecalis gave a positive reaction in from 2 to 5 cultures isolated from clinical specimens
min. The blue color developed slowly and was Gran-Negative Cyto-
easily differentiated from that produced by P. Bacilli* (Speci- Glucose
men Numbers)
Flagellar Stain chrome
Oxidase
aeruginosa. All of the other stock cultures were
negative when tested for the presence of cyto- 796 - Lophotrichous, few
chrome oxidase by this method. As partial evi- monotrichous
dence that this reaction is due to an oxidative 1034 - Monotrichous +
mechanism present in P. aeruginosa the results 1105 - Lophotrichous and
show that KCN inhibited the formation of indo- amphitrichous
phenol blue by this microorganism. Additional 1306t + Peritrichous
evidence that this reaction is due to cytochrome 1335 + Monotrichous +
oxidase has been shown in this laboratory by the 7 days
1530 - Nonmotile +
use of manometric techniques carried out on a
1704 - Nonmotile +
mammalian cytochrome c-hydroquinone system 13347 - Lophotrichous, few -
and intact P. aeruginosa cells (Gaby, unpublished amphitrichous
data). 13575 - Lophotrichous, few -
Colonies of P. aeruginosa on blood or nutrient amphitrichous
agar turn blue in 15 to 30 sec when exposed to a 13650 - Monotrichous, few +
solution containing equal parts of a-naphthol and lophotrichous
p-aminodimethylaniline oxalate, whereas the col- * All cultures gave negative lactose and xylose
onies of other gram-negative bacilli are unaf- fermentations, and gelatin liquefaction, reduced
fected. By this procedure it has been possible to nitrates to nitrites, and were motile (except
identify and isolate colonies of P. aeruginosa from no. 1704 and 1530).
a plate containing a mixed population. Eight t This culture was urease -, methyl red +,
other species of Pseudomonas obtained from the H2S + (weak), indole +, and sucrose + (late).
358 GABY AND HADLEY
1034 and 1335 were nonpigmented P. aeruginosa
strains. Cultures 796, 1105, 13347, and 13575
were identified as Alcaligenes. Numerous other
known clinical cultures, including P. aeruginosa,
were tested by this procedure and in every in-
stance the reaction has been specific. No doubtful
reactions have been encountered.
DISCUSSION
Due to the high concentration of cytochrome
oxidase within the cells of P. aeruginosa it has
been possible to develop a practical laboratory
test based on the formation of indophenol blue
from the oxidation of p-aminodimethylaniline
oxalate in the presence of a-naphthol. The test is
recommended particularly for nutrient broth cul-
tures of nonpigmented gram-negative bacilli re-
sembling P. aeruginosa. On blood or nutrient agar
plates, colonies of P. aeruginosa also give a posi-
tive test when exposed to an equal mixture of the
reagents. In the clinical laboratory all gram-nega-
tive nonlactose fermenters are of interest and
require inoculation into at least the basic bio-
chemical media. The time and expense required
for the inclusion of a nutrient broth culture tube
in this routine is negligible. Since P. aeruginosa is
the only species of Pseudomonas known to be
pathogenic for man, the test is specific when car-
ried out on cultures obtained from human speci-
mens. Although other species of Pseudomonas will
give a positive reaction it is not certain at this
time if the presence of cytochrome oxidase is
characteristic of all members of the genus. This
problem is being investigated at the present time.
SUMMARY
A practical and specific laboratory test has
been described for the identification of Pseudo-
monas aeruginosa isolated from clinical specimens.
The test is based on the high concentration of
cytochrome oxidase present in the cells of P.
[VOL. 74
aeruginosa. Other bacilli with similar biochemical
reactions were negative for cytochrome oxidase
when tested by the method described.
REFERENCES
CLARK, W. M., KAPLAN, N. O., AND KAMER, M.D.
1955 Symposium on electron transport in the
metabolism of microorganisms. Bacteriol.
Revs., 19, 234-262.
ESTABROOK, R. W. 1956 The low temperature
spectra of hemoproteins. I. Apparatus and
its application to a study of cytochrome c.
J. Biol. Chem., 223, 781-794.
FREI, W., REIDMULLER, L., AND ALMASY, F. 1934
tYber Cytochrom und das Atmungssystem der
Bakterien. Biochem. Z., 274, 253-267.
GABY, W. L. 1946 A study of the dissociative
behavior of Pseudomonas aeruginosa. J.
Bacteriol., 51, 217-234.
GABY, W. L. AND FREE, E. 1953 Occurrence and
identification of non-pigmented strains of
Pseudomonas aeruginosa in the clinical labora-
tory. J. Bacteriol. 65, 746.
GABY, W. L. 1955 Taxonomic problems relating
to the identification of species within the
genus Pseudomonas. Intern. Bull. Bacteriol.
Nomen. Taxon., 5, 153-160.
HENDERSON, R. W. AND RAWLINSON, W. A. 1956
Potentiometric and other studies on prepara-
tions of cytochrome c from ox and horse heart
muscle. Biochem. J. (London), 62, 21-29.
HOLTON, F. A. 1955 Spectrophotometric studies
of the cytochrome system of heart muscle.
Biochem. J. (London), 61, 46-61.
KEILIN, D. 1929 Cytochrome and respiratory
enzymes. Proc. Roy. Soc. (London) Series
B., 104, 206-252.
SMITH, L. 1954 Bacterial cytochromes. Bac-
teriol. Revs., 18, 106-130.
SMITH, L. AND STOTZ, E. 1954 Purification of
cytochrome c oxidase. J. Biol. Chem., 209,
819-828.
WAINIO, W. W. AND COOPERSTEIN, S. J. 1956
Some controversial aspects of the mammalian
cytochrome. Advances in Enzymol., 17,
329-392.