APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1993, p. 607-609
Copyright X 1993, American Society for Microbiology
Development and Use of a Selective Medium for Isolation of
Leuconostoc spp. from Vegetables and Dairy Productst
NOREDDINE BENKERROUM,l MERIAM MISBAH,1 WILLIAM E. SANDINE,2* AND
ABDELRHAFOUR TANTAOUI ELARAKI'
Institut Agronomique et Veterinaire Hassan II, Departement de Microbiologie Alimentaire et de
Biotechnologie, Rabat-Instituts, Rabat, Morocco,.' and Department ofMicrobiology,
Oregon State University, Corvallis, Oregon 97331-38042
Received 3 August 1992/Accepted 11 November 1992
A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium
contained 1.0% glucose, 1.0o Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco),
0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25%
sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 jug ofvancomycin (Sigma) per ml, 0.20 jug of
tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium
was used successfully for isolation and enumeration ofLeuconostoc spp. in dairy products and vegetables. Of
116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as
members ofthe genus Leuconostoc. A total of89 ofthese isolates were identified to species; 13.5% ofthe isolates
were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leucono-
stoc mesenteroides subsp. dextranicum, 16.9%o were Leuconostoc mesenteroides subsp. paramesenteroides,
10.1% were Leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained
for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM
medium, we found no significant difference between the values obtained on the two media.
Leuconostocs are heterofermentative lactic acid bacteria
that occur naturally in milk, grass, herbage, grapes, and
many vegetables (23). Members of this group are used in
dairy fermentations to produce aroma compounds (3). At-
tempts have been made to develop media for the isolation
and enumeration of these organisms, and both selective and
differential media have been described; however, no medium
has proven to be satisfactory.
Comprehensive reviews of Leuconostoc differential and
selective media have been published by Garvie (9), Teuber
and Geis (23), and Cogan (3). Most media are based on the
ability of leuconostocs to utilize citrate, which is recognized
by the presence of halos around colonies growing on media
containing insoluble calcium citrate (3, 7, 20). Such differ-
entiation is not accurate since not all leuconostocs utilize
citrate; furthermore, other bacteria associated with green
plants, such as Lactobacillus species (12) and Lactococcus
lactis subsp. lactis biovar. diacetylactis, utilize citrate (19,
20). Vancomycin resistance in Leuconostoc species (6, 17,
21), as well as the sensitivity of Lactococcus species (6, 17)
and some lactobacilli (21) to this antibiotic, led us to consider
it for use in a Leuconostoc selective medium (LUSM medi-
um). In combination with other ingredients these agents
proved to be successful. In this paper we describe the use of
LUSM medium for isolating Leuconostoc species from
several natural sources and food products.
The microorganisms used in this study included bacteria
and yeasts (Table 1). These organisms were stored in litmus
milk at -20°C. Working cultures of lactococci were propa-
gated in M17G medium containing 0.5% glucose (22); other
organisms were propagated in MRS broth (4) by using 1%
t Technical paperno. 10033 of the Oregon Agricultural Experi-
ment Station and the Western Center for Dairy Foods Research.
inocula and overnight incubation at 30°C. The yeasts were
maintained on slants of nutrient agar (Biokar) at 4°C and
were propagated in nutrient broth (Biokar) incubated over-
night at 25°C.
The basal medium contained 1.0% glucose, 1.0% Bacto
Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat
extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate,
0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25%
sodium acetate, 0.1% (vol/vol) Tween 80, and 15% tomato
juice. Fresh tomatoes were blended in water (1:1, wt/vol) by
using a Moulinex blender. The juice was then centrifuged at
8,000 x g in a Sorvall centrifuge for 15 min and used directly
without filtration. The basal medium was sterilized by auto-
claving it at 121'C for 15 min. The final pH of the medium
varied from 5.3 to 5.8. If the medium pH was below 5.0, it
was adjusted with NaOH to pH 5.5. Stock solutions of
vancomycin (Sigma), tetracycline (Serva), and cysteine hy-
drochloride (BDH) were prepared as described below. Van-
comycin and cysteine hydrochloride were dissolved in water
to final concentrations of 10 mg/ml and 1 g/ml, respectively.
A tetracycline solution was prepared as described by Mani-
atis et al. (14) by dissolving tetracycline in ethanol to a final
concentration of S mg/ml. All of these solutions were filter
sterilized by using 0.22-,um-pore-size Millipore membrane
filters, and aliquots were stored at -20°C. The tetracycline
solution was protected from light by covering the container
with aluminum foil.
To prepare the complete medium, the basal medium was
melted and tempered to about 48°C. The vancomycin, tetra-
cycline, and cysteine hydrochloride solutions then were
added to final concentrations of 30 and 20 ,ug/ml and 0.5
Nine strains were tested for resistance to vancomycin
(Table 1). Vancomycin was added to MRS agar at concen-
trations of 30, 100, 300, and 500
preparations were poured into sterile petri dishes and al-
jug/ml), and the agar
Vol. 59, No. 2
APPL. ENVIRON. MICROBIOL.
TABLE 1. Bacteria and yeasts used in this study, their origins,
and their sensitivity to vancomycin
Lactococcus cremoris ACI
Lactococcus diacetylactis F7/22
Leuconostoc cremoris 225
Lactococcus lactis 7962
Lactococcus lactis LB11
Leuconostoc cremons 44-4
Leuconostoc cremoris CAF-7
Leuconostoc cremoris 104
Lactococcus diacetylactis BU2
Escherichia coli V517
Leuconostoc cremons M71
aFDRC, Federal Dairy Research Center, Kiel, Germany; OSU, Oregon
State University, Corvallis; IAV, Institute of Agronomy and Veterinary
Medicine Hassan II, Rabat, Morocco.
lowed to harden and dry. One drop (25 ,ul) of an overnight
culture of each test microorganism was spread onto the
surface of an MRS agar plate containing each concentration
of vancomycin. The plates were then incubated at 30°C and
checked for growth after 2 and 4 days.
All of the organisms used in this study (Table 1) were
propagated either in M17G medium (bacteria) or in nutrient
broth (yeasts). To determine the ability of each organism to
grow on LUSM medium, one 25-,lI drop from an overnight
culture (15 to 18 h) was spread onto the surface of the
medium, and the preparation was incubated at 30°C and
examined daily for growth for up to 4 days.
A total of 116 microorganisms were isolated from different
products, including vegetables, milk, and curdled milk.
Samples (11 g) of vegetables were blended with 99-ml
portions of sterile distilled water for 30 s, serial dilutions
were plated onto LUSM medium, and the preparations were
incubated at 30°C for 72 h. Serial dilutions of blended
vegetables and milk products were made in sterile 0.1%
peptone water. Colonies were selected from diverse loca-
tions on plates containing uncrowded colonies and were
purified on MRS agar. They were then stored in litmus milk
at -20°C until they were needed. Isolates were identified by
examining the following four Leuconostoc characteristics:
morphology, catalase production, gas production from glu-
cose, and arginine hydrolysis. Strains were identified to
species level as described by Garvie (9, 10). The ability of
the isolates to ferment the following sugars was also exam-
ined: lactose, sucrose, cellulose, arabinose, galactose, fruc-
tose, xylulose, trehalose, maltose, glucose, and esculin.
Comparisons of growth on MRS medium and LUSM
medium were performed with two strains, Leuconostoc
cremoris JLL8, and Leuconostoc dextranicum 181. These
strains were grown separately in MRS broth at 30°C for 16 to
18 h. The cultures were then serially diluted, and colonies
were counted by plating the organisms onto the two media.
Five replications were done with Leuconostoc cremoris
JLL8, and three replications were done with Leuconostoc
dextranicum 181, each in duplicate. The Student t test was
used to perform a statistical analysis of the data.
Plasmid analyses were carried out by using the procedure
of Anderson and McKay (1), as modified by Wyckoff et al.
Our results are shown in Table 1. All Lactococcus strains
were sensitive to vancomycin at concentrations less than 30
,ug/ml, while all Leuconostoc strains were sensitive to van-
comycin at concentrations less than 30 ,ug/ml, while all
Leuconostoc strains were resistant to this antibiotic at
concentrations greater than 500 ,ug/ml. The resistance of
leuconostocs to vancomycin is well established (6, 11, 17,
21). Orberg and Sandine (17) and Simpson et al. (21) found
that members of this genus are resistant to more than 2 mg of
vancomycin per ml. Of 17 strains of Leuconostoc spp.
studied by Orberg and Sandine (17), 3 without plasmids also
could withstand up to 2 mg of this drug per ml. Lactobacilli
(6, 21) and pediococci (21) also have been shown to be
resistant to this antibiotic, while Lactobacillus bulgaricus
tolerates concentrations only up to 100 ,ug/ml.
spp., Lactobacillus bulgaricus, and the
yeasts which we tested were all inhibited on LUSM. Al-
though Lactobacillus bulgaricus grew on MRS medium
containing vancomycin, it did not grew on LUSM medium,
suggesting that other ingredients of the medium were inhib-
itory for this organism.
With one exception, all of the isolates were gram-positive,
catalase-negative, gas-producing cocci which did not hydro-
lyze arginine and were considered leuconostocs. The excep-
tion did not produce gas and formed cocci assembled in
tetrads, characteristics that are typical of pediococci. A total
of 89 Leuconostoc strains were identified to the species
level. Our results showed that the most commonly isolated
Leuconostoc species was Leuconostoc oenos (40.4% of the
isolates), which may have been due to stimulation of this
organism by cysteine hydrochloride (9). Other Leuconostoc
species were isolated at the following frequencies: Leu-
11.2%; Leuconostoc mesenteroides subsp. mesenteroides,
7.9%; Leuconostoc mesenteroides subsp. paramesente-
roides, 16.9%; and Leuconostoc lactis, 10.1%. No lactoba-
cilli were found among the isolates despite the facts that
many lactobacilli are resistant to vancomycin and that lac-
tobacilli have the same natural habitat as leuconostocs (6, 8).
In isolating Leuconostoc from nature or food samples, the
combination of the presence of agents inhibitory to most
lactic acid bacteria other than Leuconostoc spp. and the
presence of agents stimulatory for Leuconostoc spp. in
LUSM medium selects for Leuconostoc spp. Sodium azide
has been reported to be inhibitory for most lactic acid
bacteria, including lactobacilli (13), but not for leuconostocs
(15) or Lactococcus diacetylactis (20a). Therefore,
compound was used by Mayeux et al. (15) as a basis for the
formulation of a Leuconostoc selective medium. Tetracy-
cline has also been shown to be inhibitory for lactococci at
concentrations less than 10 ,ug/ml (16). To our knowledge, no
data are available on the effect of this antibiotic on lactoba-
cilli or pediococci; nonetheless, tetracycline has been used
in media that are selective for leuconostocs (16, 18).
The major contribution ofvancomycin in our medium is to
inhibit lactococci and homofermentative lactobacilli (21).
The latter group also is inhibited by sorbic acid, and the
sensitivity of these organisms to sorbate increases at pH
values below 6.3 (21). In contrast, sorbic acid was shown to
stimulate the growth of leuconostocs (5); it is also inhibitory
for yeasts (2).
No significant difference (P < 0.05) was found between the
Leuconostoc counts on MRS medium and LUSM medium.
For strain JLL8 the values obtained on MRS medium and on
LUSM medium were 2.0 x 108 + 1.7 x 108 and 1.8 x 108 +
1.3 x 108 CFU/ml, respectively (means + standard devia-
tions of five determinations performed in duplicate). The
values obtained for strain 181 on MRS medium and on
LUSM medium were 1.9 x 108 + 2.7 x 107 and 1.6 x 108
3.4 x 107 CFU/ml, respectively (means + standard devia-
tions of three determinations performed in duplicate). For
Leuconostoc dextranicum 181, a suitable colony size on
LUSM medium was obtained after 3 to 5 days of incubation,
while on MRS medium only 24 h of incubation was required.
In view of these results, LUSM medium is recommended for
the isolation and the enumeration of leuconostocs in mixed
starter cultures or in fermented products containing a het-
This studywas supported in part by U.S. Agency for International
Development project 608-0160 and by a grant from the Western
Center for Dairy Foods Research.
We are grateful to H. Neve and M. Teuber (Federal Dairy
Research Center, Kiel, Germany) for supplying some strains used in
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