Journal of Microbiological Methods 66 (2006) 572 – 575

www.elsevier.com/locate/jmicmeth

Note
A selective differential medium for Lactobacillus plantarum
Carmen Bujalance, Maria Jiménez-Valera, Encarnacion Moreno, Alfonso Ruiz-Bravo ⁎
Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada 18071, Spain
Received 3 October 2005; received in revised form 1 February 2006; accepted 9 February 2006
Available online 6 March 2006

Abstract

The quantification of exogenous lactobacilli in faecal samples is frequently required for the evaluation of the intestinal
colonization by probiotic bacteria. In this study, a selective and differential medium, designated LPSM, was developed for the
culture of exogenous Lactobacillus plantarum. In quantitative assays, LPSM showed a sensitivity similar to those of enriched and
Lactobacillus-adapted media. The presence of ciprofloxacin made LPSM inhibitory to most intestinal bacteria, including
endogenous acid lactic bacteria, whereas exogenous L. plantarum strains grew producing a yellow color caused by acid production
from sorbitol in the presence of bromocresol purple. The results showed that LPSM is suitable for detection and enumeration of L.
plantarum in faecal samples.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Ciprofloxacin; Intestinal microbiota; Lactobacillus plantarum; Probiotic bacteria; Selective medium

Probiotics are non-pathogenic microorganisms that,
when administered in adequate amounts, exert health
benefits on the host (Marteau et al., 2002; Reid et al.,
2003). Lactobacilli are among the first bacteria to be
described as probiotics. Strains of several Lactobacillus
species have proven to exert a range of health promoting
activities such as immunomodulation, enhancement of
resistance against pathogens, reduction of blood cho-
lesterol levels and others (Gill and Rutherfurd, 2001;
Rosenfeldt et al., 2002; Shu and Gill, 2002; Jones et al.,
2004).
The complexity of the intestinal microbiota, that
includes members from the genus Lactobacillus, makes
research on probiotic bacteria difficult (Savage, 1999;
Moreau and Gaboriau-Routhiau, 2000). There are some
selective media that support growth of lactobacilli and
some non-Lactobacillus species, including MRS Agar
⁎ Corresponding author. Fax: +34 958 246235.
E-mail address: aruizbr@ugr.es (A. Ruiz-Bravo).
0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2006.02.005
that is the most commonly used. However, these media
are not useful to distinguish between exogenous and
endogenous lactobacilli in faecal samples from humans
or animals fed probiotic lactobacilli. The interference of
endogenous lactobacilli is specially serious in the case
of assays in mouse models, since strains of Lactobacil-
lus species are predominant in the mouse gastrointes-
tinal tract (Moreau and Gaboriau-Routhiau, 2000).
Advances in molecular genetics of bacteria have
allowed more reliable methods for discrimination
between Lactobacillus species (Satokari et al., 2003;
Park and Itoh, 2003), but these methods require special
instrumentation and appropriate oligonucleotide probes
and do not discriminate between live and dead bacteria.
We hypothesized that, starting from the original
formulation of MRS medium, it is possible to design
modified media by adding selected antibiotics, an
appropriate sugar instead of dextrose and a pH indicator,
on which specific, exogenous lactobacilli can be
selectively cultured and recognized on the basis of
 C. Bujalance et al. / Journal of Microbiological Methods 66 (2006) 572–575 573

antibiotic resistance and sugar fermentation. In this the L. plantarum to ciprofloxacin and its ability to
report, we describe a selective and differential medium produce acid from sorbitol. The formulation per liter of
for accurate detection and enumeration of exogenous medium was Bacto proteose peptone (Difco) (10 g),
Lactobacillus plantarum from mouse faecal samples. Bacto beef extract (Difco) (10 g), Bacto yeast extract
Two catalase-negative, gram-positive strains were (Difco) (5 g), D-sorbitol (Sigma) (20 g), ciprofloxacin
isolated from commercial fermented milk samples on (Sigma) (4 mg), sodium acetate (5 g), ammonium citrate
Lactobacilli MRS agar (LMRS agar, Difco Laboratories, (2 g), potassium phosphate (2 g), magnesium sulfate
Francisco Soria Melguizo, Madrid, Spain) plates. Sugar (0.1 g), manganesum sulfate (0.05 g), bromocresol
fermentation pattern was determined using the API purple (0.02 g) and Bacto agar (Difco) (15 g). The
50 CH system (BioMérieux, Lyon, France), according to medium without ciprofloxacin was autoclaved for 15
the manufacturer's instructions. Results were recorded min at 121 °C, and cooled at 50 °C. Ciprofloxacin was
after 48 h at 37 °C and a biochemical profile was sterilized by filtration before being added to the cooled
obtained by the ApilabPlus software. Based on this medium. The pH of the medium was 6.0 ± 0.1. When
profile strain C1 (from yogourth) was identified as solidified, LPSM was an purple color.
Lactobacillus casei, and strain C4 (from kefir) as L. The sensitivity and specificity of LPSM were
plantarum. A gram-positive coccus isolated on Trypti- determined by comparison of four L. plantarum strains
case soy agar (TSA, Difco) from yogourth was with other selected bacteria grown on LPSM, LMRS
designated strain C5 and identified as Streptococcus agar and the enriched medium TSA. All strains were
thermophilus by the ApilabPlus software. Strain C16 grown on TSA tubes at 37 °C for 24 h. Bacteria were
was isolated from mouse faeces on LMRS agar and harvested, washed twice and resuspended in sterile
identified by API 50 CH as Lactobacillus fermentum. phosphate-buffered saline (PBS), and 10-fold serial
Strain C17 was a gram-negative bacillus isolated from dilutions (10− 1 to 10− 8) were prepared in PBS. 10-μl
mouse faeces on MacConkey agar (Difco) and identified volumes of undiluted samples and dilutions were plated
by API 20 E (BioMérieux) as Escherichia coli. Other onto LPSM, LMRS agar and TSA plates and incubated
bacterial strains used in this study are listed in Table 1 at 37 °C for colony enumeration. The results are
and were obtained from the Spanish Type Culture presented in Table 1. On LPSM, all four L. plantarum
Collection (CECT). strains produced large (diameter N 2 mm) yellow
The new selective differential medium designated colonies surrounded by a yellow halo after 48 h of
LPSM (L. plantarum selective medium) was developed incubation. The yellow color change in the medium was
for isolating and enumerating L. plantarum from faecal due to acid production from sorbitol. Recovery rates of
samples. LPSM design was based on the resistance of L. plantarum strains were similar in all three media. In
contrast, strains of L. casei and L. fermentum produced
similar colony counts on LMRS agar and TSA but they
Table 1 did not grow on LPSM above the detection level (102
Comparison of L. plantarum and other bacteria grown on several bacteria/ml). Strains of S. thermophilus, E. coli and
media
Salmonella enterica serovar typhimurium failed to form
Strain Average of CFU on the following colonies on LPSM and LMRS agar. Moreover, no
media
growth was observed when a loopful of suspensions of
LPSM LMRS agar TSA L. casei, L. fermentum, S. thermophilus, E. coli and S.
8 8
L. plantarum C4 1.0 × 10 1.9 × 10 2.2 × 108 enterica serovar typhimurium was streaked onto LPSM
L. plantarum ATCC8014 4.0 × 107 9.0 × 107 9.0 × 107 plates, supporting the selectivity of the medium (data
L. plantarum ATCC10241 6.0 × 107 5.6 × 107 6.0 × 107
not shown).
L. plantarum ATCC14431 5.9 × 107 5.1 × 107 6.0 × 107
L. casei C1 b102 4.5 × 107 5.0 × 107 To evaluate the sensitivity of LPSM to detect L.
L. casei ATCC393 b102 6.5 × 106 3.0 × 107 plantarum in heavily contaminated samples, faeces
L. fermentum C16 b102 7.0 × 105 2.0 × 106 from untreated mice were diluted in sterile PBS and
S. thermophilus C5 b102 b102 1.8 × 107 blended in a Vortex mixer, and 10 μl of appropriate
E. coli C17 b102 b102 2.8 × 108
dilutions of C4 suspensions in saline were added to the
E. coli ATCC25922 b102 b102 2.1 × 108
S. enterica serovar b102 b102 1.8 × 108 diluted faeces (1-ml samples). Samples were homoge-
typhimurium CECT4156 nized in a Vortex mixer and 10-fold serial dilutions
Each strain was suspended in saline and counted in triplicate on the
(10− 1 to 10− 8) of the homogenates were plated onto
corresponding media. The lowest detectable number of bacteria was LPSM plates and incubated at 37 °C for two days before
102 per ml of suspension. colony enumeration. The original dilutions of C4 in
574 C. Bujalance et al. / Journal of Microbiological Methods 66 (2006) 572–575
Table 2
Recovery of exogenous L. plantarum C4 from mouse faeces
Gavage Time (h) after Log 10 CFU per g of faeces
intragastric gavage on the indicated media
LMRS agar LPSM
C4 in milk 0 8.3 ± 0.44 b103
24 8.4 ± 0.36 6.38 ± 0.41
Milk 0 8.7 ± 0.31 b103
24 8.3 ± 0.45 b103
Results represent media ± S.D. from six mice. All C4 inoculated
animals gave positive coprocultures in LPSM at 24 h after gavage. The
lowest detectable number of bacteria was 103 per g of faeces.
saline were also plated onto LPMS, LMRS agar and
TSA plates. These experiments showed that LPSM did
not support the growth of faecal microbiota, whereas L.
plantarum produced CFU yields in accordance with the
viable bacteria counts of the original dilutions (data not
shown).
Finally, LPSM was evaluated for the recovery of L.
plantarum from faeces of L. plantarum-treated mice.
For the preparation of gavages, the L. plantarum
probiotic strain was grown in LMRS broth (Difco) at
37 °C for 20 h. Bacteria were harvested by centrifuga-
tion (2000 ×g, 15 min), washed twice in sterile PBS, and
resuspended in skimmed milk. For assessment of the
number of viable bacteria, suitable dilutions of the
bacterial suspensions were plated onto LPMS, LMRS
agar and TSA plates and CFU were recorded after 24
and 48 h at 37 °C. BALB/c female mice (Unit of Animal
Experimentation of the University of Granada, Spain)
received a dose of about 109 viable bacteria in 100 μl of
skimmed milk by intragastric route with a stainless steel
feeding needle (Harvard Apparatus, Edenbridge, UK)
and a 1-ml syringe. Control mice received skimmed
milk. Faecal samples were collected at the moment of
intragastric gavage (0 h) and 24 h later. Fresh faeces
were weighed, homogenized in sterile PBS, and suitable
dilutions of the homogenates were plated onto LPMS
and LMRS agar plates and incubated at 37 °C for two
days before colony enumeration. The experiments were
approved and supervised by the local ethic committee at
the University of Granada. Results in Table 2 confirm
the specificity of LPSM. Faecal excretion of L.
plantarum was detected in all the inoculated animals
at 24 h after the inoculation. Intestinal bacteria did not
grow on LPSM or eventually formed very small
colonies (diameterb 1 mm) without color changes that
were easily distinguishable from those of L. plantarum.
Our results showed that LPSM is suitable for
detection and enumeration of exogenous L. plantarum
in faecal samples. This medium provides a simple,
specific and sensitive tool to assess the intestinal
colonization by L. plantarum, whose probiotic potential
has been shown in several reports (Miettinen et al.,
1996; Herias et al., 1999; Coeuret et al., 2004). In
quantitative assays, LPSM has shown a sensitivity
similar to those of the enriched medium TSA and the
Lactobacillus-adapted medium MRS. A ciprofloxacin
concentration of 4 μg/ml made LPSM inhibitory to most
intestinal bacteria, including endogenous acid lactic
bacteria that grow on MRS agar, but had no adverse
effect on L. plantarum recovery. Some faecal bacteria
that eventually were able to grow on LPSM plates
formed pinpoint colorless colonies that were readily
distinguished from those of L. plantarum. We propose
that theoretical approach of LPSM design could be
applied to the design of selective and differential media
adapted to other Lactobacillus species with probiotic
potential.
Acknowledgements
This study was supported by Ministerio de Sanidad y
Consumo and the European Regional Development
Fund (Programa de Promoción de la Investigación
Biomédica y en Ciencias de la Salud, Proyecto
PI021774). C. Bujalance was supported by a predoctoral
grant from Junta de Andalucía.
References
Coeuret, V., Gueguen, M., Vernoux, J.P., 2004. In vitro screening of
potential probiotic activities of selected lactobacilli isolated from
unpasteurized milk products for incorporation into soft cheese.
J. Dairy Res. 71, 451–460.
Gill, H.S., Rutherfurd, K.J., 2001. Viability and dose–response on the
effects of the immunoenhancing lactic acid bacterium Lactobacil-
lus rhamnosus in mice. Br. J. Nutr. 86, 285–289.
Herias, M.V., Hessle, C., Telemo, E., Midvedt, T., Hanson, L.A., Wold,
A.E., 1999. Immunomodulatory effects of Lactobacillus plan-
tarum colonizing the intestine of gnotobiotic rats. Clin. Exp.
Immunol. 116, 283–290.
Jones, M.L., Chen, H., Ouyang, W., Metz, T., Prakash, S., 2004.
Microencapsulated genetically engineered Lactobacillus plan-
tarum 80 (pCBH1) for bile acid deconjugation and its implication
in lowering cholesterol. J. Biomed. Biotechnol. 2004, 61–69.
Marteau, P., Seksik, P., Jian, R., 2002. Probiotics and health: new facts
and ideas. Curr. Opin. Biotechnol. 13, 486–489.
Miettinen, M., Matikainen, S., Vuopio-Varkila, J., Varkila, K., 1996.
Production of tumor necrosis factor α, interleukin 6 and interleukin
10 is induced by lactic acid bacteria. Infect. Immun. 64, 5403–5405.
Moreau, M.C., Gaboriau-Routhiau, V., 2000. Influence of resident
intestinal microflora on the development and functions of the
intestinal-associated lymphoid tissue. In: Perdigon, G., Fuller,
R. (Eds.), Probiotics 3: Immunomodulation by the Gut Micro-
flora and Probiotics. Kluwer Academic Publishers, Dordrecht,
pp. 69–114.
 C. Bujalance et al. / Journal of Microbiological Methods 66 (2006) 572–575
Park, S.H., Itoh, K., 2003. Species-specific oligonucleotide probe for
the detection and identification of Lactobacillus isolated from
mouse faeces. J. Appl. Microbiol. 99, 51–57.
Reid, G., Jass, J., Sebulsky, M.T., McCormick, J.J., 2003. Potential use
of probiotics in clinical practice. Clin. Microbiol. Rev. 16,
658–672.
Rosenfeldt, V., Michaelsen, K.F., Jakobsen, M., Larsen, C.N., Moller,
P.L., Tvede, M., Weyrehter, H., Valerius, N.H., Paerregaard, A.,
2002. Effect of probiotic Lactobacillus strains on acute diarrhea in
a cohor nonhospitalized children attending day-care centers.
Pediatr. Infect. Dis. J. 21, 417–419.
575
Satokari, R.M., Vaughan, E.E., Smidt, H., Saarela, M., Matto, J., de
Vos, W.M., 2003. Molecular approaches for the detection and
identification of bifidobacteria and lactobacilli in the human
gastrointestinal tract. Syst. Appl. Microbiol. 26, 572–584.
Savage, D.C., 1999. Mucosal microbiota. In: Ogra, P.L., Mestecky, J.,
Lamm, M.E., Strober, W., Bienenstock, J. (Eds.), Mucosal
Immunology. Academic Press, San Diego, pp. 19–30.
Shu, Q., Gill, H.S., 2002. Immune protection mediated by the probiotic
Lactobacillus rhamnosus HN001 (DR20) against Escherichia coli
O157 : H7 infection in mice. FEMS Immunol. Med. Microbiol. 34,
59–64.