LAMVAB—A new selective medium for the isolation of lactobacilli from faeces

doi:10.1016/S0167-7012(97)00025-0

Journal of Microbiological Methods

Volume 29, Issue 2, 1 May 1997, Pages 77-84

https://doi.org/10.1016/S0167-7012(97)00025-0 Get rights and content

Abstract

The development of a new selective medium, LAMVAB, for the isolation of Lactobacillus species from faeces is described. The medium is highly selective due to a low pH and the presence of vancomycin (20 mg l⁻¹). In addition, the medium contains cysteine–HCl to enhance anaerobic conditions, and bromocresol green as a pH indicator. Competing faecal flora is inhibited by the low pH (enterobacteria, bacteroides and other Gram-negative anaerobes, clostridia) and the vancomycin (enterococci, bifidobacteria, clostridia). Characterization of the strains isolated on LAMVAB from human faeces included all common faecal lactobacilli and no other lactic acid bacteria. In one sample, vancomycin-resistant cocci were present and in another sample, yeasts were isolated. It can be concluded that the new medium is highly selective and permits the growth of all common faecal lactobacilli. The new medium can also be applied as a selective medium for lactobacilli in probiotics, which contain mixed populations of lactobacilli, bifidobacteria and enterococci.

Introduction

Lactobacilli are Gram-positive anaerobic or facultative aerobic rods, which can be isolated from a large number of sources. Lactobacilli can be isolated from the human and animal body (oral cavity, stomach, intestines, vagina), plants and material of plant origin, sewage and fermented products. Lactobacilli are strictly fermentative and acid-resistant. Growth is enhanced by acidic conditions. The final pH in broth cultures is generally below 4.5 [1].

Lactobacilli are considered to have beneficial effects on human and animal health. Therefore, several species of lactobacilli are used as probiotics. Worldwide, many products with probiotic lactobacilli are marketed 2, 3. To have a beneficial effect on the health of the host, it is necessary that the probiotic strain is able to survive in the gastro-intestinal tract [2]. It is therefore necessary to determine the number of lactobacilli in faeces and, if possible, to identify the different species directly. This can be done using specific selective media, DNA probes or immunological techniques.

Lactobacilli are generally isolated on rich, slightly acidic media, such as MRS, LBS and Rogosa agars 4, 5, 6. For most (fermented) food products, these media allow selective isolation of lactobacilli, as they are generally the most abundant group of bacteria present.

In faeces, however, lactobacilli are present in relatively low numbers 7, 8. In faecal samples, several lactobacilli have been isolated (Table 1), in humans, the main lactobacilli are Lact. acidophilus, Lact. casei and Lact. paracasei [1]. The counts of lactobacilli in faeces are highly variable between species and persons, even within a single person 4, 7, 8. The media mentioned above are not selective, as growth of other acid-resistant bacteria is not restricted and, in particular, bifidobacteria, streptococci and enterococci are able to grow on these media 9, 10. Growth of other intestinal bacteria is restricted, but not negligible. It is therefore necessary to have a highly selective medium, which allows the isolation of low numbers of lactobacilli in the presence of high numbers of bifidobacteria, streptococci and/or enterococci.

In this report, we describe the development of a new selective medium for the isolation of faecal lactobacilli.

Lactobacilli are highly acid-resistant, with growth being possible at an initial pH of 5.0 [1]. Most other intestinal bacteria are not able to grow at this acidic pH. Bifidobacteria, enterococci and streptococci are not inhibited 11, 12, 13. Contrary to other Gram-positive bacteria, most lactobacilli are resistant to the antibiotic vancomycin, while Gram-negative bacteria are generally resistant 14, 15, 16, 17. Vancomycin has already been applied previously for the selective isolation of lactobacilli from brewery products [18]. It is not useful as a selective agent in dairy products containing Leuconostoc or Pediococcus species, as these are vancomycin resistant 14, 19, 20. Enterococci, streptococci and bifidobacteria are susceptible to vancomycin 21, 22, 23. Furthermore, resistance to vancomycin is not inducible and only a few vancomycin-resistant enterococci have been isolated [24]. Vancomycin-resistant bifidobacteria have not been reported.

Although lactobacilli are aerotolerant, anaerobic conditions favour their isolation and growth. It was decided to add cysteine as a reducing agent to improve the isolation of lactobacilli.

The final medium consisted of MRS agar, with vancomycin, low pH (5.0) and cysteine as selective and elective agents, and was named Lactobacillus Anaerobic MRS with Vancomycin and Bromocresol green, or LAMVAB.

Section snippets

LAMVAB

The medium consists of three different components:

Solution A: MRS broth (Merck 1.10661), 104.4 g/l, with cysteine–HCl (0.5 g/l) and bromocresol green (0.05 g/l). The pH of this solution was adjusted to 5.0±0.1 using 4 M HCl before autoclaving. Solution B: Agar, 40 g/l. Solution C: Vancomycin hydrochloride (local pharmacy, >95% purity), 2 mg/ml in water. Solution C should be kept at 0–4°C and was found to be stable for at least three months.

Solutions A and B are sterilized at 121°C for 15 min.

Growth of different intestinal bacteria on LAMVAB

None of the intestinal strains, except for the lactobacilli and one strain of Streptococcus gordonii were able to grow on LAMVAB. All lactobacilli from all origins were able to grow on LAMVAB. Growth was determined as colonies over 1 mm in diameter and a yellow discoloration of the medium, due to acid production. Most colonies were green or blue, due to the uptake of bromocresol green into the cell. As not all lactobacilli showed this color and remained white, the color of the colonies was not

Discussion

The isolation of lactobacilli from faeces is generally difficult, due to the presence of competing micro-organisms, like bifidobacteria, streptococci and enterococci, which may be present in higher numbers than lactobacilli. Most media used for the isolation of lactobacilli also support the growth of these competing micro-organisms 9, 10. The results presented above indicate that LAMVAB does not support the growth of these bacteria, whereas growth of lactobacilli is unaffected.

LAMVAB is easy to

Acknowledgements

The authors gratefully acknowledge the financial support for this study, which is part of a multi-donor funded research programme on the role of non-digestible oligosaccharides in food and feed. Also, we would like to thank the staff of Burger's Zoo, Arnhem, for their help with sampling the animal faeces.

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LAMVAB—A new selective medium for the isolation of lactobacilli from faeces

Abstract

Introduction

Section snippets

LAMVAB

Growth of different intestinal bacteria on LAMVAB

Discussion

Acknowledgements

Adv. Food Nut. Res.

J. Microbiol. Methods

J. Infect.

J. Dairy Sci.

Int. Dairy J.

Die darmflora von mensch und tier

Goldschmidt Informiert

Influence of two infant formulas and human milk on the development of the faecal flora in newborn infants

Acta Paediatr.

Bacterial faecal flora in healthy women of different ages

Microbiol. Ecol. Health Dis.

Die faekalflora bei menschen. V. Mitteilung: Die schwangkungen in der zusammensetzung der faekalflora gesunder erwachsener

Zbl. Bakt. Hyg., I. Abt. Orig.

Effect of diet on human fecal flora: comparison of Japanese and American diets

Am. J. Clin. Nutr.