CLIAMS:
1. A media for selective enumeration and differential detection of lactic acid
bacteria (LAB), the media comprising a basal composition, characterized in that the media comprising:
• bromocresol green as pH indicator in a range of 0.005 grams by weight
to 0.04 grams by weight; and
• 2, 3, 5 triphenyl tetrazolium chloride as a chromogenic redox indicator
in a range of 0.05 grams by weight to 0.5 grams by weight.

2. A method for preparing a media for selective enumeration and differential
detection of lactic acid bacteria, the method comprising the steps of:
(a) preparing a basal composition;
(b) adding bromocresol green as pH indicator to the basal composition of
step (a);
(c) adjusting pH of the basal composition of step (b) in a range of 6-5 to
7.8 using any of an acid and alkali solution;
(d) sterilizing the basal composition of step (c) at a temperature of about
1210C for about 15 lbs pressure for about 15 minutes; and
(e) adding separately sterilized 2, 3, 5 triphenyl tetrazolium chloride in a
range of 0.05 grams by weight to 0.5 grams by weight to the sterilized basal
composition of step (d) to form the media.

3. The media as claimed in claims 1 and 2 has pH in a range of 6.5 to 7.8.

4. The media as claimed in claims 1 and 2, wherein the basal composition

comprises dextrose in a range of 18 grams by weight to 22 grams by weight, proteose peptone in a range of 8 grams by weight to 12 grams by weight, beef extract in a range of 8 grams by weight to 12 grams by weight, yeast extract in a range of 3 grams by weight to 7 grams by weight, polysorbate 80 in a range of 0.5 mili litre to 2 mili litre, ammonium citrate in a range of 1 gram by weight to 3 grams by weight, sodium acetate in a range of 3 grams by weight to 7 grams by weight, magnesium sulphate in a range of 0.05 grams by weight to 0.5 grams by weight, manganese sulphate in a range of 0.02 grams by weight to 0.07 grams by weight, dipotassium phosphate as a buffer in a range of 1 gram by weight to 3 grams by weight, agar as a gelling agent in a range of 20 grams by weight to 30 grams by weight and deionized water quantity sufficient to make 1000 ml.

5. The media as claimed in claims 1 and 2 is highly selective and permits
identification, isolation, detection/differentiation as well as identification of LAB species obtained from any one of a pure culture sample and a mixed culture sample/consortium bacteria when present in defined combinations.

6. The media as claimed in claims 1 and 2 discriminates between different
genera of bacterial species selected from but not restricted to all the members of lactic acid bacteria including Lactobacillus, Bifidobacterium, Saccharomyces and Enterococcus based on the colony morphology.

7. The media as claimed in claims 1 and 2 effectively discriminates between L.
acidophilus and L. rhamnosus even when present in the mixed culture sample.

8. The media as claimed in claims 1 and 2 effectively discriminates between L.
acidophilus, L. rhamnosus and Enterococcus species when present in a defined combination in the mixed culture sample.
9. The media as claimed in claims 1 and 2 inhibits growth of Gram negative
bacteria selected from Escherichia coli, Salmonella species and Pseudomonas aeruginosa.

10. The media as claimed in claims 1 and 2 inhibits growth of Staphylococcus
aureus strain.
,TagSPECI:
Media for Selective Enumeration and Differential Detection of Lactic Acid Bacteria

Field of the invention

The present invention relates to a culture media, and more particularly, to a media for selective enumeration and differential detection of species of commercially important genera such as lactic acid bacteria and a method of preparation thereof.

Background of the invention

Lactic acid bacteria (herein after ‘LAB’) constitute a group of Gram-positive, non-sporulating, cocci or rods. The members of LAB group produce lactic acid as the major end product during the fermentation of carbohydrates. The lactic acid group of bacteria is reported to be associated with food fermentation, and also with themucosal surfaces of humans and animals (Axelsson 2004). The genera included in this group include: Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella.

The genus Bifidobacterium is historically also considered to belong to the LAB group. Bifidobacterium is phylogenetically unrelated to LAB. The members of this genus have a unique mode of sugar fermentation and their catabolic products are essentially lactic and acetic acids (Morelli 2001). LAB group of microorganisms find wide commercial applications in food industry as starter cultures in fermented food and feed products, in bio preservation and as probiotics. The members of LAB are a part of natural microbiota in mammals. Being ubiquitous, LAB can be isolated from many plant and animal sources as well as from fermented food products like dairy, meat, vegetables, fruits, and beverages.

The current identification techniques of LAB rely on morphological, physiological, biochemical as well as genetic criteria. With the advent of molecular tools, DNA-based methods are widely used for identification and typing of LAB isolates. 16S rRNA gene sequencing is one of the most commonly used techniques for identification of LAB species. However, this technique often fails to discriminate between phylogenetically closely related LAB species or between subspecies (Temmerman et al., 2004) and the molecular tools used in such techniques are expensive. Also, phenotypic tests are subject to distinctions, making data interpretation difficult. Similarly, selective isolation of LAB is hindered due to interference of non-lactic acid bacteria and Gram negative bacteria which are present in the same ecosystem.

Literature survey reveals that a few media have been developed for differential growth of lactic acid bacteria. These include Rogosa agar (Rogosa et al., 1951), MRS (de Man et al., 1960) and LAMVAB (Hartemink et al., 1997). However, these media are observed to support growth of almost all LAB species and are not selective in nature. Very few studies have been conducted on selective media for differentiation between closely related LAB species. The available selective media are based on the tolerance of LAB to low pH (Coeuretet al., 2003; Dave and Shah, 1996), the replacement of glucose by other sugars, tolerance to antibiotics, bile and inorganic salts (Dave and Shah, 1996). In addition to medium composition, temperature (Champagne et al., 1997) and oxygen (Shah, 2000) have also been observed to selectively enumerate the cultures.

Triphenyl-tetrazolium chloride (TTC) has been incorporated in media to distinguish between species viz. Streptococcus (Turner et al., 1963). Similarly Nagasaki et al., (1992) developed a basal media with TTC to test sugar utilization profile in lactobacilli. The authors reported that on fermentation, lactic acid is produced which leads to development of white or light-red colonies since TTC reduction is inhibited by acidic conditions. Bacteria which do not utilize the sugar form red colonies due to TTC reduction (Nagasaki et al., 1992).

A modified media M-RTLV was reported (Sakai et al., 2010) wherein rhamnose along with TTC was incorporated in basal media supplemented with vancomycin and metronidazole. The media was able to differentiate between L. rhamnosus and L. casei strains, however, strain variation was observed amongst strains for utilization of rhamnose. A modified MRS medium was developed by Lee and Lee (2008) to enumerate lactic acid bacteria along with differentiation of each LAB in a mixed culture. However, the study was restricted to differentiating between few species of Lactobacillus and Bifidobacteria. Due to common nutritional requirements and also, due to the strain variability in the nutritional requirements, the formulation of selective media LAB has been a challenge.

Accordingly, there is a need of a media for selective isolation, enumeration and differentiation of lactic acid bacteria that overcomes the drawbacks of the prior art.

Objects of the invention

An object of the present invention is to provide a media for selective enumeration and differential detection of lactic acid bacteria (LAB).

Another object of the present invention is to provide a media that is not complex, consumes less time and offers a good cell recovery for LAB.

Yet another object of the present invention is to provide differentiation of LAB at genus level as well as species level.

Still another object of the present invention is to provide selective enumeration and differentiation of LAB from pure as well as mixed culture samples/ consortia.

Further object of the present invention is to provide a mixture of nutrients and an indicator mixture for differentiation of LAB.

Furthermore object of the present invention is to provide a method for preparation of the media.

Summary of the invention

Accordingly, the present invention provides a media for selective enumeration and differentiation of lactic acid bacteria. The media is highly selective and permits identification, isolation, detection/differentiation as well as identification of bacterial species such as lactic acid bacteria at genus and species levels. The lactic acid bacteria obtained from any one of a pure culture sample and a mixed culture sample/consortium bacteria when present in defined combinations.

The media comprises a basal composition. Further, the media comprises bromocresol green as pH indicator in a range 0.005 gram by weight to 0.04 grams by weight. Furthermore, the media comprises 2, 3, 5 triphenyl tetrazolium chloride as a chromogenic redox indicator in a range of 0.05 grams by weight to 0.5 grams by weight. Typically, the media has pH in a range of 6.5 to 7.8.

In another aspect, the present invention discloses designing the media for differentiation of lactic acid bacteria.

Brief description of the drawings

The objectives and advantages of the present invention will become apparent from the following description read in accordance with the accompanying drawings wherein,

Figure 1 shows a flowchart for a method of preparation of a media for selective enumeration and differential detection of lactic acid bacteria, in accordance with the present invention;

Figures 2a - 2e show colony morphology for lactic acid bacteria from different genera on the media, in accordance with the present invention;

Figure 2f shows colony morphology for a mixed sample containing lactic acid bacteria belonging to different genera, in accordance with the present invention;

Figures 3a - 3g show colony morphology for different species of lactic acid bacteria belonging to same genus such as Lactobacillus species on the media, in accordance with the present invention; and

Figures 3h - 3k respectively show colony morphology for first, second, third and fourth consortia containing different species of lactic acid bacteria, in accordance with the present invention.

Detail description of the invention

The foregoing objects of the present invention are accomplished and the problems and shortcomings associated with the prior art, techniques and approaches are overcome by the present invention as described below in the preferred embodiments.

The present invention provides a media for selective enumeration and differential detection of lactic acid bacteria. The media of the present invention comprises a novel mixture of basal ingredients/nutrients along with a unique indicator mixture for selective enumeration and differentiation of lactic acid bacteria at genus level as well as species level. The media of the present invention allows identification and differentiation of different genera and species of lactic acid bacteria including commercially important genus of Lactobacillus and even yeast from mixed samples/consortia. The present invention also provides a method for preparation of the media.

Accordingly, in one aspect the present invention provides a media for selective enumeration and differential detection of bacteria such as lactic acid bacteria (herein after ‘LAB’). The media comprises a basal composition. The basal composition comprises a carbohydrate source such as dextrose in a range of 18 grams by weight to 22 grams by weight. The carbohydrate source is fermented by the LAB to obtain energy.

Further, the basal composition comprises a source of nitrogen and carbon such as proteose peptone in a range of 8 grams by weight to 12 grams by weight, beef extract in a range of 8 grams by weight to 12 grams by weight and yeast extract in a range of 3 grams by weight to 7 grams by weight.

Furthermore, the basal composition comprises a source of fatty acids such as polysorbate 80 in a range of 0.5 mili litre to 2 mili litres. Moreover, the basal composition comprises ammonium citrate in a range of 1 gram by weight to 3 grams by weight and sodium acetate in a range 3 grams by weight to 7 grams by weight. Ammonium citrate and sodium acetate selectively stimulate the growth of LAB and inhibits growth of other organisms.

Further, the basal composition comprises magnesium sulphate in a range of 0.05 grams by weight to 0.5 grams by weight and manganese sulphate in a range of 0.02 grams by weight to 0.07 grams by weight, both serving as essential ions for multiplication of probiotics. Furthermore, the basal composition comprises a buffering agent such as dipotassium phosphate in a range of 1 gram by weight to 3 grams by weight. Moreover, the basal composition comprises a gelling agent such as agar in a range of 20 grams by weight to 30 grams by weight. In an embodiment, 2.5% of agar is used.

Apart from the basal composition, the media also comprises a unique indicator mixture of a pH indicator and a chromogenic redox indicator. The media comprises bromocresol green as pH indicator in a range of 0.005 gram by weight to 0.04 grams by weight and 2,3,5 triphenyl tetrazolium chloride (herein after ‘TTC’) as chromogenic redox indicator in a range of 0.05 grams by weight to 0.5 grams by weight. In an embodiment, 0.002% of bromocresol green and 0.01% of TTC are used.

The presence of the indicator mixture results in easy differentiation between colonies of each LAB species based on ability of LAB species to reduce tetrazolium salt and produce organic acid. The degree of reduction and acid production results in appearance of different color tonalities for colonies of each species that are easily distinguished by naked eye.

The below table depicts media components and role of each component:

Table 1: Media components and their role
Media component Role
Dextrose Fermentable carbohydrate and energy source
Proteose peptone, Beef extract, Yeast extract Nitrogenous and carbonaceous compound
Polysorbate 80 Source of fatty acids
Ammonium citrate, Sodium acetate Stimulates LAB, inhibits other bacteria
Magnesium sulphate, Manganese sulphate Essential ions for multiplication of LAB
Dipotassium phosphate Buffering agent
Bromocresol green pH indicator
2,3,5 triphenyl tetrazolium chloride (TTC) Chromogenic redox indicator

The media is highly selective for LAB and hence, used for selective enumeration, differential detection, isolation and identification of LAB obtained from a pure culture and even when the LAB are present in a consortia/mixed sample in defined combinations. In an embodiment, the LABs are commensal microbes. In another embodiment, the LABs are probiotic organisms. The LAB are selected from a group consisting of but not limiting to Lactobacillus species including but not limited to L. acidophilus DSMZ 20079 T, L. acidophilus SM7, L. casei JCM 1134 T, L. fermentum DSMZ 20052 T, L. plantarum DSMZ 20174T, L. rhamnosus SM3, L. rhamnosus DSMZ 20022; Bifidobacterium animalis sub species lactis; Saccharomyces boulardii; Enterococcus faecium, Streptococcus species and the like.

Since the environmental niche of LAB is quite varied, there are chances of a non LAB growing during isolation. Hence, the media of the present invention, besides investigating for growth of the LAB is also investigated for inhibiting the growth of potential contaminants/ non-lactic acid bacteria such as Gram negative bacteria which constitute the common intestinal micro flora such as Escherichia coli, Salmonella species and Pseudomonas species as well as members of the Gram positive clade such as Staphylococcus aureus. Similarly, non-prokaryotic fungi namely, members of the genus Saccharomyces (yeast) were also studied. The media inhibits growth of Staphylococcus aureus and several Gram negative bacteria such as Escherichia coli, Salmonella species, and Pseudomonas species.

In another aspect, the present invention provides a method (100) for preparation of the media as illustrated in figure 1. The method (100) starts at step (10). At step (20), the method (100) includes preparing a basal composition by mixing dextrose in a range of 18 grams by weight to 22 grams by weight, magnesium sulphate in a range of 0.05 grams by weight to 0.5 grams by weight, manganese sulphate in a range of 0.02 grams by weight to 0.07 grams by weight, dipotassium phosphate in a range of 1 gram by weight to 3 grams by weight, agar in a range of 20 grams by weight to 30 grams by weight in deionized water sufficient to make 1 litre of the media.

At step (30), the method (100) includes adding bromocresol green in a range of 0.005 gram by weight to 0.04 grams by weight to the basal composition of step (20). Bromocresol green acts as pH indicator.

At step (40), the method (100) includes adjusting the pH of the basal composition of step (30) in a range of 6.5-7.8 using an acid or alkali solution. More particularly, pH of the media is 6.5. At step (50), the method (100) includes sterilizing the basal composition of step (40). The sterilization is done by autoclaving at 1210C at 15 lbs pressure for 15 minutes.

At step (60), the method (100) includes adding to the sterilized basal composition of step (50) a separately sterilized solution of chromogenic redox indicator, for example TTC in a range of 0.05 grams by weight to 0.5 grams by weight. TTC is sterilized separately by any of a cooker sterilization and a filter sterilization process. In an embodiment, TTC addition is done to the sterilized basal media to achieve a concentration of 0.0l% to form the media of the present invention. The method (100) ends at step (70). The media is to be used within a certain period for example within 7 days.

Examples

The media of the present invention will be described in more details below by means of examples. The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1: Method of preparation of media

The media of the present invention with components as described in Table 1 above was prepared as follows:

A basal composition was prepared by dissolving agar (25g/l), proteose peptone (10 g/l), beef extract (10 g/l), yeast extract (5 g/l), dextrose (20 g/l), polysorbate 80 (1ml/l), ammonium citrate (2 g/l), sodium acetate (5 g/l), magnesium sulphate (0.1 g/l), manganese sulphate (0.050 g/l), dipotassium phosphate (2 g/l) in distilled/ deionized water sufficient to make 1 litre of the media.

Then bromocresol green (0.02g/l) was added to the basal composition and the pH of the basal media was adjusted to 6.5 prior to sterilization. The basal media was sterilized by autoclaving at 1210C and 15 lbs pressure for 15 minutes. After sterilization, sterile TTC solution (0. l g/l) was added to the sterilized basal media thereby forming the media of the present invention.

Table 2 shows the composition of the media of the present invention:
Table 2: Media composition
S. No. Ingredient Quantity (g/l)
1 Basal Composition
a Proteose peptone 10 g
b Beef extract 10 g
c Yeast extract 5 g
d Dextrose 20 g
e Polysorbate 80 1 ml
f Ammonium citrate 2 g
g Sodium acetate 5 g
h Magnesium sulphate 0.1 g
i Manganese sulphate 0.050 g
j Dipotassium phosphate 2 g
2 Bromocresol green 0.02g
3 TTC 0. l g

Example 2:

The media described above was poured into petriplates. The petriplates were dried at room temperature for inoculation with a pure culture sample as well as a mixed culture sample of various microorganisms (as listed in table II below). The inoculation was done by spread plate method. Briefly, serial dilutions were prepared in appropriate diluents and 0.1 ml was used to perform spread plate. The dilutions were plated so as to obtain well isolated colonies (CFU/ml). The inoculated plates were incubated at respective temperatures for a predetermined period of time for colony formation that occurs as a result of carbohydrate utilization. The media was incubated at 370C under anaerobic conditions for effective differentiation between species of Lactobacillus. The media was incubated at 370C under anaerobic conditions for effective differentiation between the genera: Lactobacillus, Bifidobacterium and Enterococcus. The media was incubated at 370C under aerobic conditions for optimum growth of Saccharomyces spp.

The color tonalities and morphology of colony (as shown in figures 2-3) for each species and each genus differs based on the level of carbohydrate utilization and acid production and thus was used as parameter for differentiation.

The below table shows various microbes used in the present investigation:

Table 3: Microbial strains used in the following examples

Microorganisms used in the investigation Origin of investigated strains
L. acidophilus DSMZ 20079 T DSMZ 20079 T
L. acidophilus SM7 HTBS-SM7
L. casei JCM 1134 T JCM I 134 T
L. fermentum DSMZ 20052 T DSMZ 20052 T
L. plantarum DSMZ 20174 T DSMZ 20174 T
L.rhamnosus DSMZ 20022 DSMZ 20022
Bifidobacterium animalis subsp. lactis HTBS - C24
Saccharomyces boulardii HTBS-MSPI2
Enterococcus faecium HTBS-C52
Escherichia coli NCIM 2065
Salmonella spp. NCIM 2257
Staphylococcus aureus NCIM 2079
Pseudomonas spp. NCIM 2200

*T-Type strains procured from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, JCM-Japan Collection of Microorganisms and NCIM-National Collection of Industrial Microorganisms; HTBS- Cultures isolated and identified at Hi-Tech Biosciences Laboratory.

Example 2.1: Differentiation between lactic acid bacteria (LAB) from different genera when present in a pure culture sample and when present in a mixed culture sample
In this experiment, the ability of the media of example 1 to allow growth and differentiation of LAB from different genera was investigated. The industrially important LAB from genera Lactobacillus acidophilus DSMZ 20079 T, Lactobacillus rhamnosus DSMZ 20022, Bifidobacterium animalis subsp. lactis HTBS, Saccharomyces boulardii HTBS-MSPI2 and Enterococcus faecium HTBS-C52 were used as test organisms.

The pure cultures of each test organism as well as mixed culture containing a defined combination of test organisms were inoculated by spread plate method on the media, wherein the dilutions were spread onto the surface of the medium. The dilution was selected in order to achieve well isolated colonies. The mixed culture contained a defined combination of Lactobacillus acidophilus DSMZ 20079 T, Lactobacillus rhamnosus DSMZ 20022 and Enterococcus faecium HTBS-C52.

The plates were incubated at 370C under strict anaerobic conditions for 96 hours. The plates inoculated with the Saccharomyces culture were incubated at 300C under aerobic conditions for optimum growth of Saccharomyces spp. The plate inoculated with the mixed culture was incubated at 370C under strict anaerobic conditions for 96 hours.

The plates were observed after incubation and the colonial characteristics for each of the organisms tested are shown in table 4 and figures 2a-2f.

The below table shows colony characteristics of various microbes used in the present investigation:

Table 4: Colony characteristics of the test organisms after incubation
Test Organism Characteristics of the colony
Lactobacillus acidophilus DSMZ 20079 T 0.5-1 mm, Dark red flat colonies with fuzzy margin
Lactobacillus rhamnosus DSMZ 20022 2-4 mm, Light green circular colonies with regular margin
Bifidobacterium animalis subsp. lactis HTBS <0.5 mm, pin-point colonies, reddish in color and circular
Saccharomyces boulardii HTBS-MSPI2 0.5-1 mm, Small circular, dull pink colonies
Enterococcus faecium HTBS-C52 0.5-1 mm, Small circular red colonies with convex elevation

As apparent from the table 4, the media selectively allows growth of Lactobacillus acidophilus DSMZ 20079 T (figure 2a), Lactobacillus rhamnosus DSMZ 20022 (figure 2b), Bifidobacterium animalis subspecies lactis HTBS (figure 2c), Saccharomyces boulardii HTBS-MSPI2 (figure 2d) and Enterococcus faecium HTBS-C52 (figure 2e) that exhibited different colony morphologies and color tonalities thus proving the capability of the media in growing and differentiating LAB belonging to different genera. The media is also capable of promoting growth of Lactobacillus species (acidophilus and rhamnosus) and Enterococcus species when present in a mixed sample as shown in figure 2f. The appearance of different colony color tonalities aids in differentiation between different LABs when present in said combinations.

Example 2.2: Differentiation between different lactic acid bacterial (LAB) species from same genus when present in a pure culture sample and when present in a mixed culture sample
In this experiment, the ability of the media of example 1 to allow growth and differentiation of different Lactobacilli species was investigated. The organisms that were tested include L. acidophilus DSMZ 20079 T, L. acidophilus SM7, L. rhamnosus SM3, L. rhamnosus DSMZ 20022, L. casei JCM 1134 T, L. fermentum DSMZ 20052 T, L. plantarum DSMZ 20174 T.

The pure cultures of each test organism as well as mixed culture containing a defined combination of test organisms were inoculated by spread plate method on the media, wherein the dilutions were spread onto the surface of the medium. The dilution was selected in order to achieve isolated colonies. A total of four mixed culture/consortia were investigated which are as follows: a first consortium of L. rhamnosus DSMZ and L. acidophilus DSMZ; a second consortium of L. rhamnosus SM3 and L. acidophilus SM7; a third consortium of L. rhamnosus DSMZ, L. acidophilus DSMZ and L. plantarum DSMZ; and a fourth consortium of L. rhamnosus DSMZ, L. acidophilus DSMZ and L. fermentum DSMZ. The plates were incubated at 37 0C under strict anaerobic conditions for 96 hours.

The plates were observed after incubation and the colonial characteristics for each of the organisms tested are shown in table 5 and figures 3a-3k.

The below table shows colony characteristics of test organisms after incubation:

Table 5: Colony characteristics of the test organisms after incubation
Strain Colony Characteristics
L. acidophilus DSMZ 0.5-1 mm, Dark red flat colonies with fuzzy margin
L. acidophilus HTBS-SM7 0.5-1 mm, Dark red flat colonies with irregular margin
L. rhamnosus DSMZ 2-4 mm, Light green circular colonies with regular margin
L. rhamnosus HTBS-SM3 2-4 mm, Light green circular colonies with regular margin
L. casei JCM <1 mm, Small pinkish red circular colonies with convex elevation
L. plantarum DSMZ 2-4 mm, Light green colonies with dark center, convex elevation
L. fermentum DSMZ 2-4 mm, Dark pink/purple circular colonies with slight convex elevation

As apparent from table 5, the media was found to be effective in rapid detection, differentiation, isolation as well as identification of species of Lactobacillus. The colony morphology and color of L. acidophilus DSMZ (figure 3a); L. acidophilus HTBS-SM7 (figure 3b), L. rhamnosus DSMZ (figure 3c), L. rhamnosus HTBS-SM3 (figure 3d), L. casei JCM (figure 3e), L. plantarum DSMZ (figure 3f) and L. fermentum DSMZ (figure 3g) are different for each species of Lactobacillus. Further, the media effectively allowed differentiation between the Lactobacillus species even when present in a consortium in defined combinations as shown in figures 3h-3k. Figure 3h shows colony morphology for each Lactobacillus species present in the first consortium, figure 3i shows colony morphology for each Lactobacillus species present in the second consortium, figure 3j shows colony morphology for each Lactobacillus species present in the third consortium and figure 3h shows colony morphology for each Lactobacillus species present in the fourth consortium.

Example 2.3: Growth of other Gram positive bacteria on the media

This experiment was conducted to determine whether the media allows growth of Staphylococcus aureus NCIM 2257. The method of inoculation was spread plate technique. Staphylococcus aureus culture was inoculated on media and the plates incubated at 370C under anaerobic as well as aerobic conditions. After incubation, colonies for Staphylococcus aureus strain were not observed. Hence, the media did not support growth of Staphylococcus aureus.

Example 2.4: Growth of other Gram negative bacteria on the media

This experiment was conducted to determine whether the media allows growth of Escherichia coli NCIM2O65, Salmonella species NClM2257 and Pseudomonas species NCIM 2200. The method of inoculation was spread plate technique. The media were inoculated with individual cultures of the above mentioned organisms and the plates were incubated at 370C under anaerobic as well as aerobic conditions. After incubation, colonies were not observed proving that the media did not support growth of the Gram negative bacteria tested.

Advantages of the invention

1. The media of the present invention includes a novel combination of the pH indicator and the chromogenic redox indicator that allows easy and effective discrimination between different LABs even when present in defined combinations in a consortium based on the colony morphology.

2. The presence of the indicator mixture in the media of the present invention results in easy differentiation between colonies of each LAB species based on ability of LAB species to reduce tetrazolium salt and produce organic acid. The degree of reduction and acid production results in appearance of different color tonalities for colonies of each species that are easily distinguished by naked eye.

3. The media of the present invention allows rapid and effective detection/ differentiation as well as identification and isolation of LAB both at genus as well as species levels.

4. The media of the present invention differentiates between micro-organisms belonging to LAB group (which includes Lactobacillus/ Enterococcus, Bifidobacterium, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus and Weisella) from other non-lactic acid bacilli.

5. The media of the present invention differentiates between species of LAB. This encompasses species of Lactobacillus such as (but not limited to) L. acidophilus, L. rhamnosus, L. casei, L. plantarum, L. fermentum and species of Bifidobacterium such as B. animalis and B. longum when present in a consortium.

6. The media of the present invention is useful in differential enumeration of industrially important LAB such as probiotic LAB and also probiotic based products from food and drug industries during their quality assurance testing.

7. The media of the present invention is a highly selective in permitting selective identification/differentiation of most of the LAB species and does not permit growth of Staphylococcus aureus strain and several Gram negative bacteria such as Escherichia coli, Salmonella species and Pseudomonas species.

8. The media of the present invention is useful in isolating LAB from different sources which include dairy/ silage/ fruits/ fermented food products without any interference from Gram negative bacteria.

The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the present invention.