The frozen bacterial suspensions with ACE, CAS, or GUA as well as the frozen positive (FOS) and negative (NEC) controls were subjected to freeze-drying at −55 ± 2 °C, with a vacuum pressure of <138 μHG and a freeze-drying rate of 1 mm/h, for approximately 40 h using a benchtop freeze dryer (LIOTOP®, Model L-101, São Carlos-SP, Brazil). Just after freeze-drying, the viable cells of the tested strains were enumerated. For this, the freeze-dried strains in the different treatments were rehydrated in sterile distilled water (30 ± 0.5 °C) for 15 min. Serial dilutions were subsequently performed using sterile saline solution (NaCl 8.5 g/L) and dispensed onto MRS agar plates (HiMedia, Mumbai, India) using a microdrop inoculation technique [29 (link)]. After an incubation period of 48 h at 37 °C under anaerobic conditions (Anaerobic System Anaerogen, Oxoid), the visible colonies were enumerated and the results expressed as log CFU/g. The detection limit of the assays for viable cell counts was 1 log CFU/g.
Mrs agar plates
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MRS agar plates are a type of culture media used for the isolation and enumeration of lactobacilli in food, dairy, and other samples. The plates are formulated according to the Man, Rogosa, and Sharpe (MRS) recipe, providing a selective environment for the growth of Lactobacillus species.
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9 protocols using mrs agar plates
1
Freeze-Drying Efficacy of Bacterial Strains
2
Isolation and Characterization of Probiotic Bacteria from Tilapia and Red Paree
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O. niloticus (tilapia) and Nemipterus japonicas (red paree) were procured from Mindhora River, Bardoli. The fish samples were selected at random, collected in pre-sterilized polyethylene bags containing the habitat water, and transported to a laboratory. Fishes were washed with sterile distilled water to remove any undesired dust particles and dissected to open the gastrointestinal tract under laminar airflow conditions. The gastrointestinal tract was homogenized using sterile distilled water and centrifuged at 10,000 rpm for 10 min. After centrifugation, the supernatant was serially diluted and spread-plated on MRS agar plates (HiMedia). The plates were incubated at 37 °C for 24 h. The obtained isolates were further screened for their probiotic properties and four strains were selected for detailed characterization. The pure cultures were maintained on MRS agar slants at 4 °C for further study.
3
Antimicrobial Effects of Common Drugs on Lactobacilli
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The agar well diffusion assay was used to study the effect of commercially available drugs on growth of lactobacilli isolates. The various tablet formulations used in this study were–paracetamol(500 mg/ml), diclofenac (10 mg/ml), nimugesic (20 mg/ml), ibuprofen (120 mg/ml), cetrizine HCl (2 mg/ml), and lansoprazole(4 mg/ml).
The lactobacilli were grown at 37°C for 18 h in MRS broth. Following the incubation period, 100 μl of culture was spread onto MRS agar plates (HiMedia). Using sterile well-borer, wells were cut onto agar plates. An aliquot (100 μl) of various drugs were poured in wells and plates were placed at 4°C for diffusion. After 4 h, plates were incubated at 37°C overnight. The diameter of zone of clearance was noted in millimeters.
The lactobacilli were grown at 37°C for 18 h in MRS broth. Following the incubation period, 100 μl of culture was spread onto MRS agar plates (HiMedia). Using sterile well-borer, wells were cut onto agar plates. An aliquot (100 μl) of various drugs were poured in wells and plates were placed at 4°C for diffusion. After 4 h, plates were incubated at 37°C overnight. The diameter of zone of clearance was noted in millimeters.
4
Bile Tolerance of Bacillus coagulans MTCC 5856
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Bile tolerance of B. coagulans MTCC 5856 cells was determined by the method described earlier (Gilliland et al. 1984 (link); Hyronimus et al. 2000 (link)). Briefly, overnight grown B. coagulans MTCC 5856 in MRS broth (20 μL corresponding to 2 × 106 cfu mL−1) was spotted onto MRS agar plates containing oxgall bile (0.1–1 % w/v) (HiMedia, Mumbai, India). Plates were incubated at 37 °C for 5 days. The minimal inhibitory concentration (MIC) of bile for B. coagulans MTCC 5856 was determined as the lowest concentration totally inhibiting the growth of spots as judged from visual examination of spots. MRS broth (HiMedia) was inoculated with approximately 106 cfu mL−1 of B. coagulans MTCC 5856 overnight grown culture and then supplemented with 0.3 (w/v) and 0.5 % (w/v) oxgall. Samples were incubated for 24 h at 37 °C with shaking at 120 rpm. Growth in control (no bile) and test cultures (0.3 and 0.5 % oxgall) was monitored hourly by measuring absorbance at 600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan). In same set of experiment, the viable count of B. coagulans MTCC 5856 was determined in triplicate on glucose yeast extract agar (HiMedia) at 0 and 24 h by pour plate method (Majeed et al. 2016 ).
5
Lactobacillus Vaginal Infection Treatment
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During the treatment phase of the study, both L. acidophilus (LA-5®) and L. johnsonii (B-2178) strains were subcultured daily on MRS agar plates (Himedia, India) anaerobically using Anaerogas Pack (Himedia, India) at 37°C for 48 h to obtain separate colonies. Suspensions were prepared daily by suspending separate colonies of both strains in 0.9% saline solution and suspensions were adjusted to 6 × 108 CFU/mL. After confirming the establishment of VVC by microscopic examination on the 6th day of infection, 500 μL of L. acidophilus (LA-5®) and L. johnsonii (B-2178) suspensions were administered intravaginally to the animals in Groups 4 and 5, respectively, once daily for 7 days. In parallel, the animals in Groups 1–3 received an intravaginal injection of 500 μL of sterile saline (Li et al., 2019 (link)).
6
Isolation of Lactic Acid Bacteria from Fermented Cacao
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For the isolation process of LAB from 24 to 72 h of fermented cacao, a mixture of 50 grams of cacao seeds and pulp was prepared with 450 mL of water containing 0.1% peptone. The viable bacterial cell count was determined using the pour-plate technique. The rinse water underwent serial dilution using 0.1% peptone water. Subsequently, 100 μL of the diluted samples were inoculated onto deMan, Rogosa, and Sharpe (MRS) agar plates (Himedia, Mumbai, India). The plates were then incubated at 37°C for 24–48 h in anaerobic conditions. The visible colonies were counted, and the cell counts were converted to colony-forming units per milliliter (CFU/mL).
7
Isolation and Characterization of Lactic Acid Bacteria from Fecal Samples
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The collected fecal samples were cleaned and processed within a Class II biosafety cabinet (Biobase, China) using surgical blades to extract the superficial portion of the feces, subsequently placing them in Petri dishes for each of the three designated groups. One gram of fecal surface was taken and diluted in 50 ml of sterile saline solution (NaCl 0.9%). Subsequently, 1 ml of the prepared solution underwent serial dilutions to a value of 10-4. One hundred microliters of each dilution were spread on Man, rogosa, and sharpe (MRS) agar plates (Hi-Media, India), a medium specific for lactic acid bacteria. These plates were then incubated under microaerophilic conditions at 37°C for 24 hours.
Bacterial colonies obtained were further purified through sub-culturing on MRS agar plates, followed by an additional incubation at 37°C for 20 hours under microaerophilic conditions to ensure the attainment of pure colonies. Finally, 10–12 colonies were selected from each group (designated as G1, G3, and G4) for subsequent evaluations.
Bacterial colonies obtained were further purified through sub-culturing on MRS agar plates, followed by an additional incubation at 37°C for 20 hours under microaerophilic conditions to ensure the attainment of pure colonies. Finally, 10–12 colonies were selected from each group (designated as G1, G3, and G4) for subsequent evaluations.
8
Quantification of Gut Bacteria
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In the contents of the small intestine and caecum were analysed to determine the number of lactic acid bacteria and enterobacteria using the plate count method after a 10-fold dilution in saline. MRS agar plates (HiMedia, Karnataka, India) that had been anaerobically incubated (GasPak system, Becton Dickinson, San Diego, CA, USA) for 48 h at 37 • C were used to determine the number of lactic acid bacteria. Enterobacteria were counted on Endo agar plates (HiMedia, Karnataka, India) after a 24 h incubation period at 37 • C under aerobic conditions. The bacterial counts are expressed in log 10 of colony forming units per gram of content (log 10 cfu•g -1 ) ± standard deviation.
9
Isolation and Identification of Lactic Acid Bacteria from Milk
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Forty samples, 20 each of fresh cow and goat milk, were randomly obtained from different locations in Jashore, Bangladesh, in sterile corked plastic tubes followed by immediate storage in a 4°C icebox, transportation to the laboratory, and examination for the presence of LAB upon arrival. To identify LAB, 10 mL of each sample was enriched in 40 mL of de Man, Rogosa, and Sharpe (MRS) broth (Hi-Media, Mumbai, India; (Sharifi Yazdi et al., 2017) (link), and stirred overnight in a shaking incubator at 37°C (Wisd Laboratory Instruments, Seoul, South Korea) under aerobic conditions. All tubes with visible turbidity were further cultured on MRS agar plates (Hi-Media) followed by incubation for 24 to 72 h at 37°C under aerobic conditions. Then, individual colonies from each plate were selected and purified through 3 successive transfers on MRS agar. Finally, the pure isolates were characterized as LAB by Gram staining, cell morphology, catalase test, and coagulase reaction according to standard procedures (Sharpe, 1979) , wherein gram-positive, catalase-and coagulase-negative isolates were selected before being stored at -20°C in MRS broth plus 28% glycerol (El Soda et al., 2003) (link).
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