Fig 1.
Flow cytometric analysis of B coagulans MTCC 5856 spores.
10 mg of dry spores were suspended in 1ml of PBS and activated for 30 min at 75°C, followed by fluorescent staining as described in the methods section. Data represents mean± standard deviation of at least 3 independent experiments. A: Gating strategy for SYTOBC ™ staining- The microbeads and the stained spores are gated separately based on unstained control B: Gating strategy for CFDA and PI staining. The dot plot represents a double staining for both CFDA and PI. The quadrant 4(Q4) represents live cells, Q1- dead cells and Q2 damaged cells. The double positive cells were not considered for enumerating the live spores. C: Representative dot plot for the FCM analysis of Live and Dead spores D: Table showing the calculation of viable spores.
Fig 2.
A: Spore suspension was diluted and each dilution was analysed by FCM as described earlier. Quantitative analysis is given in the table. B: FCM analysis of spores, vegetative cells and a mixture of spores and vegetative cells and killed cells.
Fig 3.
The spore suspension (15 X109/ml) was incubated in 0.1M acetate buffer (pH 2) for 24 hours, or incubated at 140°C for 5 minutes followed by washing and suspension in Nutrient Broth (NB) and incubated at 37°C for 60 minutes. The viable spores were enumerated by both FCM and plate count as described earlier. Data represents mean± standard deviation of at least 3 independent experiments.
Fig 4.
Spore viability in commercial preparations.
Viability of B coagulans spores in tablets (A) capsules(B) and Orange juice(C): Total count and live spore count were determined by FCM and plating method. Data represents mean± standard deviation of 3 independent experiments.
Fig 5.
Comparative stability of probiotics.
Bacterial count was assessed by flow cytometry as described earlier. The probiotic formulations A—C were purchased from the US market, while probiotic D was purchased from India. All the commercial products were stored as per the manufacturer’s instructions and used before their expiry date. Probiotic A: Commercial tablet of Bifidobacterium infantis, Probiotic B:Commercial tablet of Lactobacillus rhamnosus GG, Probiotic C:Commercial tablet of Lactobacillus acidophilus, Probiotic D:Commercial preparation of Lactobacillus casei and Probiotic E:Commercial tablet of B coagulans MTCC 5856. CFDA positive cells were taken as live cells. Data represents mean± standard deviation of 3 independent experiments.