APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1987, p. 211-213
1
0099-2240/87/010211-03$02.00/0
Copyright © 1987, American Society for Microbiology
Specific and Sensitive Plate Assay for Bacterial Lipases
GISELA KOUKER AND KARL-ERICH JAEGER*
Lehrstuhlffur Biologie der Mikroorganismen, Ruhr-Universitat, D4630 Bochum, Federal Republic of Germany
Received 30 July 1986/Accepted 7 October 1986
A plate assay to detect bacterial lipase (EC 3.1.1.3) in a medium containing trioleoylglycerol and the
fluorescent dye rhodamine B is presented. Substrate hydrolysis causes the formation of orange fluorescent halos
around bacterial colonies visible upon UV irradiation. The logarithm of lipase activity from cell-free culture
supernatants is linearly correlated with the diameter of halos, thereby allowing quantitation of lipase activities
ranging from 1 to 30 nkat.
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are
produced by various microorganisms either alone or to-
gether with esterases (carboxylic-ester hydrolases; EC
3.1.1.1) (2). Screening of lipase producers on agar plates is
frequently done by using tributyrin (9) or Tween 80 (12) as a
substrate. Modifications of these assays use Tween 80 in
combination with Nile-blue (7) or neat's-foot oil, with Cu2+
salts (13). These substrates are not suitable to detect true
lipases because they are hydrolyzed by esterases, too. The
existence of lipases has to be verified by applying time-
consuming methods, e.g., a titrimetric test (11) with
trioleoylglycerol, which is an ideal lipase substrate (6).
Fungal lipase can be visualized after ultrathin-layer
isoelectric focusing when the focusing gel is overlaid by an
agar gel containing trioleoylglycerol and the fluorescent dye
rhodamine B (5). We adapted this technique to develop a
specific and sensitive plate assay suitable either to identify
lipase-producing bacteria or to quantify lipase activity in
culture supernatants.
Olive oil as an inexpensive substitute for trioleoylglycerol
(6) was purified by passage through a column of neutral
alumina in a solvent mixture of ether and petroleum ether
(1:10 [vol/vol]) (2). Rhodamine B (1 mg ml-'; Sigma Chem-
ical Co., Munich, Federal Republic of Germany) was dis-
solved in distilled water and sterilized by filtration. Growth
medium contained (per liter): nutrient broth, 8 g; sodium
chloride, 4 g; and agar, 10 g. The medium was adjusted to pH
7.0, autoclaved, and cooled to about 60°C. Then 31.25 ml of
olive oil (2.5% [wt/vol])-10 ml of rhodamine B solution
(0.001% [wt/vol]) was added with vigorous stirring and
emulsified by mixing for 1 min with an Ultra-Turrax homog-
enizer (Janke & Kunkel KG, Staufen, Federal Republic of
Germany). After the medium was allowed to stand for 10 min
at 60°C to reduce foaming, 20 ml of medium was poured into
each plastic petri dish. Pseudomonas aeruginosa PAO was
from B. W. Holloway, Clayton, Australia, P. aeruginosa
PAC 1R was from P. Meadow, London, England, and
mucoid P. aeruginosa strains were from J. Wingender,
Bochum, Federal Republic of Germany. All other bacterial
strains were from our own culture collection. Lipase-
producing strains were identified on spread plates after
incubation for 48 h at 37°C. To quantify lipase activity,
3-mm-diameter holes were punched into the agar and filled
with 10 ,ul of cell-free culture supernatant. The plates were
incubated for 16 h at 37°C. Lipase activity was assayed
* Corresponding author.
211
Vol. 53, No.
titrimetrically with purified olive oil as substrate (4), with 1
nkat representing 60 nmol of oleic acid released per min.
Agar plates containing trioleoylglycerol and rhodamine B
appear opaque and are pink colored. Lipase production is
monitored by irradiating plates with UV light at 350 nm.
After 16 h of incubation bacterial colonies began to show an
orange fluorescence; with continuing incubation time orange
fluorescent halos were formed around the colonies of lipase-
producing strains, e.g., P. aeruginosa PAC 1R and Staphy-
lococcus aureus (Fig. 1A, sectors 1 and 2). Strains not
producing lipase, such as Escherichia coli, accumulated
rhodamine B, i.e., formed pink colored colonies, but did not
show orange fluorescence upon UV irradiation (Fig. 1A,
sector 3). Examination of P. aeruginosa ATCC 9027, PAO,
and PAC 1R, Serratia marcescens, S. aureus, and Bacillus
subtilis on rhodamine B plates identified these strains as
lipase producers, in accordance with the results of a
titrimetric assay of culture supernatants. Production of exo-
polysaccharide did not interfere with a positive reaction on
rhodamine B plates, as was shown when mucoid, i.e.,
alginate-producing, P. aeruginosa strains were screened.
Plate methods to identify lipase-producing microorganisms
with trioleoylglycerol as substrate used pH indicators such
as Nile-blue sulfate (3) or Victoria blue B (1) which respond
to any changes in pH and inhibit growth of certain bacterial
species; another method requires an additional developing
step with OS04 (8). The rhodamine B plate method is
insensitive to pH changes and allows reisolation of organ-
isms which show no inhibition of growth or change of
physiological properties.
A direct quantitation of lipase activity is difficult, presum-
ably because of the low amounts of lipase molecules released
by a single colony. Quantification can be done, however, by
filling culture supernatants into holes punched into the agar,
as was shown by testing a culture supernatant from P.
aeruginosa PAC 1R (Fig. 1B). The logarithm of lipase
activity is linearly related to the zone diameter (Fig. 2),
thereby fullfilling the requirement of a valid agar diffusion
assay (9). A linear regression analysis revealed a correlation
coefficient of 0.991. The specificity of the assay was shown
when esterase (carboxylic-ester hydrolase) was tested and
no fluorescent halos were formed (Fig. 1B). The sensitivity
of the assay is demonstrated by the fact that the lowest
detection limit was about 1 nkat of lipase activity, whereas
about 20 nkat was needed for the titrimetric assay.
The molecular mechanism underlying the formation of
fluorescent products generated from trioleoylglycerol hydro-
lysis by lipase is unknown. A method for determination of
free fatty acids in solution has been described in which
212
FIG. 1. Identification and quantitation of lipase activity on rho-
damine B agar plates. After incubation for 48 (A) or 16 (B) h at 37°C,
plates were subjected to UV irradiation (350 nm) and photographed.
(A) P. aeruginosa PAC 1R (sector 1) and S. aureus (sector 2) formed
orange fluorescent halos around the colonies, indicating production
of lipase, whereas E. coli (sector 3) did not. The faint widespread
halos in sector 1 resulted from greenish blue fluorescence of
pyocyanin produced by P. aeruginosa PAC 1R and could easily be
distinguished from orange fluorescent halos close around the colo-
nies. (B) Lipase (L) from culture supernatant of P. aeruginosa PAC
1R and esterase (E) (Sigma) were filled into holes no. 1 to 4, with
each hole containing 10 Ll of the following enzyme activities
(nanokatals): 1, 1.6; 2, 8; 3, 16; and 4, 32.
rhodamine B is used in the presence of uranyl ions, yielding
orange fluorescent complexes with an excitation wavelength
of 350 nm (10). This corresponds to our observations with
immobilized rhodamine B, as does the detectable concentra-
tion range, which is given as 50 to 400 nmol of fatty acid. The
mechanism suggested by the authors is a complex formation
between cationic rhodamine B and the uranyl-fatty acid ion
(10). We tested possible reaction products of trioleoyl-
glycerol hydrolysis by lipase, i.e., mono- and dioleoyl-
glycerol, oleic acid, and sodium oleat (data not shown). All
products when solubilized in petroleum ether and filled into
holes in rhodamine B plates gave a positive reaction, yield-
ing orange fluorescent complexes. A conceivable mechanism
may be the generation of excited dimers of rhodamine B
APPL. ENVIRON. MICROBIOL.
I I I
0
3
00
E 21
E .0
1
F
. Y A
0
2.0 2.5 3.0
log lipase activity
FIG. 2. Relation between the logarithm of lipase activity and the
diameter of halos formed on rhodamine B agar plates. Culture
supernatant of P. aeruginosa PAC 1R (10 RIl) representing lipase
activities in the range of 100 to 1,000 nmol of oleate min' (1.7 to
16.7 nkat) was pipetted into holes, and the plates were incubated for
16 h at 37°C. AX represents the diameter of the orange fluorescent
halo minus the diameter of the hole. All values are mean values from
triplicate determinations.
which fluoresce at longer wavelengths than the excited
monomer (excimer fluorescence).
The plate assay described here is suitable to detect lipase-
producing bacterial species and to quantify lipase activity in
culture supernatants. In our laboratory this method allowed
us to identify lipase-negative mutants that still produce
esterase as well as to isolate lipase-hyperproducing strains
from a mutagen-treated P. aeruginosa culture.
We thank Fabian Linden, Bochum, Federal Republic of Ger-
many,for preparing the photographs.
LITERATURE CITED
1. Alford, J. A., and E. E. Steinle. 1967. A double layered plate
method for the detection of microbial lipolysis. J. Appl. Bacte-
riol. 30:488-494.
2. Brockerhoff, H., and R. G. Jensen. 1974. Lipolytic enzymes, p.
25-34. Academic Press, Inc., New York.
3. Collins, M. A., and B. W. Hammer. 1934. The action of certain
bacteria on some simple tri-glycerides and natural fats, as
shown by Nile-blue sulphate. J. Bacteriol. 27:473-485.
4. Decker, L. A. 1977. Worthington enzyme manual, p. 125-126.
Worthington Biochemical Corp., Freehold, N.J.
5. Hofelmann, M., R. Kittsteiner-Eberle, and P. Schreier. 1983.
Ultra thin-layer agar gels: a novel print technique for ultra
thin-layer isoelectric focusing of enzymes. Anal. Biochem.
128:217-222.
6. Jensen, R. G. 1983. Detection and determination of lipase
(acylglycerol hydrolase) activity from various sources. Lipids
18:650-657.
7. Karnetova, J., J. Mateju, T. Rezanka, P. Prochazka, M.
Nohynek, and J. Rokos. 1984. Estimation of lipase activity by
the diffusion plate method. Folia Microbiol. 29:346-347.
8. Kuimova, T. F., E. A. Shabanova, and G. A. Kazakov. 1975.
Rapid selection of microorganisms forming extracellular lipases.
Mikrobiologiya 44:365-366.
VOL. 53, 1987
9. Lawrence, R. C., T. F. Fryer, and B. Reiter. 1967. Rapid method
for the quantitative estimation of microbial lipases. Nature
(London) 213:1264-1265.
10. MacKenzie, R. D., T. R. Blohm, E. M. Auxier, and A. C. Luther.
1967. Rapid colorimetric micromethod for free fatty acids. J.
Lipid Res. 8:589-597.
11. Peled, N., and M. C. Krenz. 1981. A new assay of microbial
lipases with emulsified trioleoyl glycerol. Anal. Biochem.
NOTES 213
112:219-222.
12. Sierra, G. 1957. A simple method for the detection of lipolytic
activity of micro-organisms and some observations on the
influence of the contact between cells and fatty substances.
Antonie van Leeuwenhoek J. Microbiol. Serol. 23:15-22.
13. Wand, H., A. Gabert, and H. Uhlig. 1980. Einfache Methode
zum Nachweis mikrobieller Lipase. Z. Allg. Mikrobiol. 20:
291-293.