Isolation of Lactobacillus reuteri and Lactobacillus rhamnosus from Probiotic Blend using Lactobacillus MRS Agar, A laboratory-Based Study.

 

Lactobacillus reuteri and Lactobacillus rhamnosus are two distinct species of Gram-positive, rod-shaped, lactic acid-producing bacteria that function as powerful probiotics. They naturally inhabit the human gastrointestinal and urogenital tracts, where they help maintain a healthy microbial balance, fight off pathogens, and support immune health.
In recent times, the use of probiotics has been increasing due to the high health concerns of people. Most manufacturers use Lactobacillus reuteri and Lactobacillus rhamnosus in a combined formulation during production of probiotics. Moreover, the method of test, culture medium, dilution medium, incubation temperature and time are also the same for both organisms, which makes it a complicated job for a microbiologist to distinguish these species from each other.

Test Method
a. Materials and Equipment
1. Lactobacillus MRS Agar
2. Normal Saline pH 7.10 (as diluent)
3. Probiotic Blend (From an approved vendor)
4. Weighing Balance
5. Hot Plate
6. Autoclave
7. Biosafety Cabinet
8. Incubator
9. Colony counter
10.0.04% Acitidone solution

b. Sample Preparation
One gram probiotic blend sample was dissolved in 100 ml Normal Saline pH 7.10 (10² Dilution) and the sample was incubated at 30°C for 30 minutes on Water Bath.. Further, serial dilution was done using a 1 ml sample of 9 ml normal saline pH 7.10 up to a dilution of 10¹⁰ Dilution and 10⁸, 10⁹ and 10¹⁰ dilutions were used as test dilution.

c. Media preparation (Lactobacillus MRS Agar, HI Media)
13.43 gm of medium was weighed and heat dissolved in 200ml purified water on a hot plate at 100°C. The medium was autoclaved at 121°C, 15psi for 15 minutes. After autoclaving, the media was cooled to about 40-50°C and 1 ml of 0.04% Actidione solution was added to sterile media and mixed well.
Note:
-200 ml of Lactobacillus MRS Agar was prepared during the study.
- Actidione solution was used to inhibit growth of yeast and mold on Lactobacillus MRS Agar from a Probiotic blend.
Sample analysis using Pour Plate Method
1 ml of sample from each dilution (10⁸, 10⁹ and 10¹⁰) was piped in triplicate on pre-sterilized Petri plate and about 20ml of Lactobacillus MRS Agar was poured aseptically onto Petri plates under a biosafety safety cabinet. The Petri plates were gently rotated in circular motion and allowed to solidify at room temperature. After solidification, the plates were incubated at 37°C for 48 hours.

Observation.
After incubation, plates were observed using a colony counter.

 Fig: Growth of Lactobacillus reuteri and Lactobacillus rhamnosus on Lactobacillus MRS Agar

Colony appearance
a. White circular colony; Lactobacillus rhamnosus
b. Convex light brown colony: Lactobacillus reuteri

Conclusion
Lactobacillus MRS Agar was used to isolate the organism from a probiotic blend containing Lactobacillus reuteri and Lactobacillus rhamnosus. Use of the 0.04% Actidione solution was found to be highly effective for inhibiting growth of yeast and mold in Lactobacillus MRS Agar. The colonies of both Lactobacillus reuteri and Lactobacillus rhamnosus were found clearly separable from each other despite being the same species