APPLIED MICROBIOLOGY, Dec. 1973, p. 850-854 Vol. 26, No.

6
Copyright 0 1973 American Society for Microbiology Printed in U.S.A.

Urease Activity of Enterobacteriaceae: Which

Medium to Choose
A. VUYE AND J. PIJCK
Laboratory for Pharmaceutical Microbiology, State University of Ghent, 9000 Ghent, Belgium

Detection and intensity of urease activity in enterobacteriaceae greatly varies

as a function of the media or techniques used, or both. A comparative
investigation on several solid and liquid media led us to the following conclu-
sions. (i) Detection of Proteus spp. can be adequately performed with the highly
selective solid medium described by Cook (1948), as well as with the different
liquid media described (Stuart standard and rapid media; Elek medium). (ii)
Detection of Klebsiella should be based upon urease production on solid media
with low buffer capacity (Christensen, 1946). (iii) For the identification of
Yersinia, either the solid Christensen urea agar or the rapid Elek technique give
optimal results.

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Urease activity of enterobacteriaceae is rou- TABLE 1. Urease activity of enterobacteriaceaea
tinely determined in the clinical bacteriological Enterobacteriaceae Urease activity
laboratory for differentiation and identification
purposes. Typical positive results are obtained Tribe I. Escherichieae
with Proteus and Klebsiella spp. as well as with Genus I. Escherichia ...........
non-enterobacteriaceae such as Yersinia, which Genus II. Shigella .............
are now more and more routinely searched for in Tribe II. Edwardsielleae
clinical specimens. Other species, however, Genus I. Edwardsiella ..........
show variable urease activities (Table 1). Fur- Tribe III. Salmonelleae
thermore, results vary as a function of media or Genus I. Salmonella ...........
techniques used, or both. In order to clearly Genus II. Arizona ..............
Genus III. Citrobacter .......... Diff (69.4% +;
standardize procedures and the interpretation 6.9% dc+)
of results, a comparative study was made with Tribe IV. Klebsiellae
different solid and liquid media as well as with Genus I. Klebsiella ............ + or -
media of varying buffer capacities. K. pneumoniae .............. 94.5% +
K.ozoenae .................. Diff (9.5% +;
MATERIALS AND METHODS 10.3% d+)
Cultures of enterobacteriaceae and Yersinia were K. rhinoscleromatis ..........
obtained from clinical specimens (feces, urine, ab- Genus II. Enterobacter ......... + or -
cesses, burns, etc.) and identified by using conven- E. cloacae ................. Diff (64.7% +)
tional methods (1-3, 7-10). A total of 89 cultures were E. aerogenes ................ - (2.7% +)
submitted to the comparative tests, which used differ- E. hafniae ..................
ent solid and liquid media composed and prepared as E. liquefaciens .............. Diff
in Table 2. Species having no known urease activity GenusIII. Pectobacterium. Diff
were intentionally neglected in this study (i.e., Esche- Genus IV. Serratia ............. Diff
richia, Shigella, Salmonella, etc.). Tribe V. Proteae
Solid media I, II, and III were inoculated by Genus I. Proteus ............... + (100% +)
streaking a loopful of pure culture onto the surface. Genus II. Providencia ..........
Liquid media IV and V were inoculated by adding a
Data were compiled from the literature (1, 8, 9).
seven drops of a suspension containing approximately
109 bacteria/ml. For the rapid Elek technique (me-
b Diff, differing activities.
c d, Delayed activity.
dium VI), 0.5 ml of medium was inoculated with a
loopful of a 24-h culture from a solid medium.
Incubation was performed throughout at 37 C, a was detected by color changes of the indicator (media
shaking incubator being used for the liquid media. I through V), or in absence of indicator (medium V
Incubation time varied as a function of the medium without indicator, and medium VI), by the addition of
and will be discussed later; urease activity (formation Nessler reagent.
of NH, from urea by the action of aminohydrolase) With the solid media, a quantitative interpretation
85(
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VOL. 26, 1973 UREASE ACTIVITY IN ENTEROBACTERIACEAE 851

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VOL. 26, 1973 UREASE ACTIVITY IN ENTEROBACTERIACEAE 853
of results (percentage of discoloration; Table 3) was TABLE 4. Results of urease activity studies with
obtained by evaluation of the length of medium liquid media
reddened after incubation (25, 50, 75, or 100%). To
obtain reproducible results, all tests were performed Medium Medium Medium
under strict standard conditions (10 ml of media, Organism Medium V Vb Vil
screw-capped tubes [20 by 160 mm] with deep butt IV (after (after (after
2 h) 2 h) 2 h)
and short slant).
Proteus sp. + (24 h) + + +
RESULTS Klebsiella - Diff _
The results obtained with solid media are Citrobacter - _ _
summarized in Table 3; numbers of each orga- Enterobacter - _ _
nism tested and percentage of positive reactions Serratia - -_ _
are indicated, as well as the time of onset and
Pectobacterium Diffa(48 h)' Diff
Diff _
Yersinia + (48 h)+ Diff Diff
the time needed for completion of positive
reaction. The same table summarizes the per- a
Diff, different reaction according to the strain.
centage of discoloration of the medium after Some strains were positive or delayed positive; others
final equilibrium had been reached. were negative.
Figure 1 illustrates the evolution of reaction b A positive reaction is shown by a dark-brown
intensity on medium I as a function of time for precipitate, and a negative reaction is shown by a
the most representative urease positive strains. yellow color or slight yellow precipitate. For the actual
Results obtained with liquid media are sum- test, a negative control (e.g., Escherichia coli as

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marized in Table 4. non-urease producer) and an uninoculated blank
should be included.
DISCUSSION
It appears from our results that several en- With the liquid or rapid media, or both,
terobacteriaceae other than Proteus and medium IV can be compared with the previous
Klebsiella possess a more or less intense urease solid media as to reaction speed: after 12 to 18 h
activity, detection of which varies with the of incubation, Proteus spp. reacted positively,
sensitivity and buffer capacity of the medium and longer incubation (24 to 48 h) gave rise to a
used. positive reaction for Yersinia; Klebisella re-
Comparative results obtained on solid media mained negative after 24 h of incubation.
(Table 3) show that media I and II had very Media V and VI allowed rapid urease identifi-
similar properties; only Citrobacter seemed to cation due to their low buffer capacity. When
react somewhat more quickly on medium II. reaction time was standardized to 2 h, clear-cut
Medium III, which had a higher buffer capac- positive results were obtained for Proteus spp.
ity, had a higher selectivity and should be When ammonia was detected with Nessler rea-
preferentially used for the identification of gent (medium V without indicator, and espe-
Proteus spp. after 24 h of incubation. Detection cially medium VI), Yersinia gave a strong
of urease activity in Klebsiella or Yersinia positive reaction, as did some Pectobacter spp.
should preferably be performed on medium I or Those species having an irregular urease ac-
II. tivity (i.e., Citrobacter, Enterobacter, etc.)
should be identified by other biochemical reac-
tions, since the urease criterion is irrevelant.
IIPOTEUS ELEUBSIELLA Summarizing the practical value of this com-
CL-ACAE-
l YERSINIA
parative study, we suggest the following proce-
dure: (i) for detection of Proteus, highly selec-
tive medium III or any of the rapid liquid
media; (ii) for detection of Klebsiella, solid
medium with low buffer capacity (medium I or
,CITROBACTER
II); and (iii) for detection of Yersinia, solid
medium I or liquid medium VI.
In practice, these investigations are of value,
Ca for instance, for the rapid differentiation of the
250.,,
,,-: . genus Proteus from the genus Salmonella (stool
examination): suspected gram-negative bacilli
OL tTIME. HRS.
which are lactose negative and H2S positive are
24 48 72 96 120 checked on urease activity in liquid medium VI;
FIG. 1. Intensity of urease activity in medium I as if the test is negative, there is possible evidence
a function of time. of Salmonella. This important differentiation
854 VUYE AND PIJCK
can be performed within 2 h. Furthermore, one
can characterize different lactose-positive (and,
in certain cases, lactose-negative) organisms by 4.
their delayed urease activity on Christensen
medium. By using Table 3, where the reaction
speeds are indicated, one can, for instance,
5.
recognize Klebsiella strains by their moderately
delayed urease reaction (onset of reaction after 6.
9 to 10 h as opposed to Proteus spp., which had
onset of reaction after 1 to 2 h). Table 3 gives a 7.
reliable estimation of reaction speeds of urease-
producing enterobacteriaceae other than 8.
Proteus spp. The urease test is thus a very
useful criterion for identification of these orga-
9.
nisms.
LITERATURE CITED 10.
1. Bailey, W. R., and E. G. Scott. 1970. Diagnostic micro-
biology. C. V. Mosby Co., St. Louis.
2. Blair, J. E., E. H. Lennette, and J. P. Truant (ed.) 1970.
Manual of clinical microbiology. American Society for 11.
Microbiology, Bethesda, Md.
3. Bodily, L. H., E. L. Updyke, and J. 0. Mason (ed.) 1970.
APPL. MICROBIOL.
Diagnostic procedures for bacterial, mycotic and para-
sitic infections. American Public Health Association,
New York.
Christensen, W. B. 1946. Urea decomposition as a means
of differentiating Proteus and paracolon cultures from
each other and from Salmonella and Shigella types. J.
Bacteriol. 52:461-466.
Cook, G. T. 1948. Urease and other biochemical reactions
of the Proteus group. J. Pathol. Bacteriol. 60:171-181.
Elek, S. D. 1948. Rapid identification of Proteus. J.
Pathol. Bacteriol. 60:183-192.
Ewing, W. H. 1962. Enterobacteriaceae. Biochemical
methods for group differentiation. In Public Health
Service Publ. no. 734, Washington, D.C.
Ewing, W. H. 1968. Differential reactions of Enterobac-
teriaceae. National Communicable Disease Center,
Atlanta, Ga.
Fife, M. A., W. H. Ewing, and R. R. Davis. 1965. The
biochemical reactions of the tribe Klebsiellae. National
Communicable Disease Center, Atlanta, Ga.
Pijck, J., M. Spoormans-Croux, R. Van den Bossche-Bilo,
and A. Vuye. 1972. Identification of Enterobacteriaceae
in the clinical microbiology laboratory, p. 390-396,
vol. 1. Medikon, European Press.
Stuart, C. A., E. Van Stratum, and R. Rustigian. 1945.
Further studies on urease production by Proteus and
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related organisms. J. Bacteriol. 49:437-444.