To isolate the LAB, each sample (15 g) was ground aseptically, suspended in Ringer’s solution (135 mL) (Sigma–Aldrich, Milan, Italy), and homogenized in a stomacher (BagMixer 400; Interscience, Saint Nom, France) for 2 min. at maximum speed. The homogenized solution was then serially diluted. Decimal dilutions were plated on de Man, Rogosa, and Sharpe (MRS) agar (Oxoid, Milan, Italy) and incubated under anaerobic conditions at 37 °C for 48 h. After incubation, the colonies (10–15 colonies for each sample) were randomly selected and plated on the MRS agar until pure cultures, identified by colony morphology, were formed. All of the isolates then underwent Gram staining and catalase reactions. Only Gram-positive and catalase-negative samples were selected as LAB and stored in MicrobankTM vials (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80 °C for further analysis.
Mrs agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Germany, Italy, China, Spain
MRS agar is a growth medium used for the isolation and cultivation of lactic acid bacteria, particularly Lactobacillus species. It provides essential nutrients and growth factors required by these microorganisms. The formulation is based on de Man, Rogosa, and Sharpe's (MRS) original recipe.
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Lab products found in correlation
Mrs broth, by Thermo Fisher Scientific (30 mentions)
M17 agar, by Thermo Fisher Scientific (6 mentions)
Anaerogen, by Thermo Fisher Scientific (4 mentions)
De man, by Thermo Fisher Scientific (3 mentions)
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Macconkey agar, by Thermo Fisher Scientific (3 mentions)
Plate count agar, by Thermo Fisher Scientific (3 mentions)
Nutrient broth, by Thermo Fisher Scientific (2 mentions)
Columbia agar, by Thermo Fisher Scientific (2 mentions)
Sabouraud agar, by Biomaxima (2 mentions)
Saline solution, by 3M (2 mentions)
Bacterial genomic dna extraction kit, by Qiagen (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lb agar, by Thermo Fisher Scientific (2 mentions)
Bagmixer 400, by Interscience (2 mentions)
Sabouraud agar, by Thermo Fisher Scientific (2 mentions)
Anaerocult a, by Merck Group (2 mentions)
Plate count agar pca, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Anaerobic system, by Thermo Fisher Scientific (2 mentions)
162 protocols using mrs agar
1
Isolation and Identification of Lactic Acid Bacteria
2
Isolation of Lactic Acid Bacteria from Tropical Fruits
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Sampling was conducted in Tambrauw forest area, West Papua, Indonesia on April 2016. Plants specimens were identified and deposited in Herbarium Bogoriense (BO), Indonesian Institute of Sciences, Bogor, Indonesia. Fruit samples were picked from the tree and cut to small pieces (0.4 cm x 0.4 cm x 0.4 cm) with a 70% ethanolsterilized knife. The pieces of fruits were swiftly placed on 5 ml of MRS agar (Oxoid) in a 15 ml "corning" tube with or without the addition of 7 ml of sterilized cooking oil. The addition of oil on the surface of the medium was performed to reduce the amount of air oxygen that could dissolve in the MRS agar media thus inhibiting the growth of LAB in the field. Media containing pieces of fruits were left for about 10 days in the field before further isolation in the laboratory. LAB isolation was carried out by serial dilution (up to 10 8 dilution series) with sterilized 0.85% NaCl solution and poured onto MRS agar (Oxoid) containing 1% CaCO3 in petri dish. Incubation was conducted at 37 0 C for 24 hours. The formation of LAB colonies was indicated by a clear zone around the colony on MRS agar containing CaCO3. Selected colonies were transferred to a new medium for purification and the purified isolates were then preserved both in slanted agar and in the glycerol medium at -80 0 C deep freezer.
3
Preparation and Culture of Lactobacillus plantarum
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Dehydrated peptone, de Man, Rogosa & Sharpe (MRS) broth and MRS agar (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were prepared according to label instructions prior to sterilization. Powdered SPI (ProFam 646, Archer Daniels Midland Company, Chicago, IL, USA) and Powdered WPI (ISOPURE, The Isopure company, Downers Grove, IL, USA) were dissolved in reverse osmosis water to yield 9% suspensions that were stored at 4 °C for 24 h prior to any UHPH or culture suspension in order to ensure full hydration of the powders and provide a sufficiently low inlet temperature during processing. A pure culture sample of Lactobacillus plantarum, (NRRL B-1927 aka ATCC 10241) originally isolated from sauerkraut and frequently identified as a probiotic [19 (link),20 (link),21 (link)], was obtained from the USDA-ARS Culture collection (Peoria, IL, USA) and used to propagate all cultures used in this study.
4
Profiling Gut Microbiome in Tumor Mice
5
Cultivation and Characterization of Lactobacillus acidophilus LA14
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Lactobacillus acidophilus LA14 was supplied by DuPont. The strain was cultured anaerobically in MRS agar (Thermo Fisher, Shanghai, China) for 48 h at 37°C, identified by 16S ribosomal DNA sequencing, and stored at −80°C for further use. Before the experiment, all the strains were cultured in MRS broth (Thermo Fisher) at 37°C for 24 h. Cells were collected by centrifugation at 8,000 × g and 4°C for 10 min, resuspended in phosphate buffer to a final concentration of 3 × 109 CFU/ml, and used for further animal experiments (9 (link)).
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6
Microbiological Assessment of Meat Samples
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From each storage condition and time point, 10 g of each sample including the meat surface area was collected under aseptic conditions in triplicates and was mixed with 90 g peptone solution (0.1% (w/v) peptone, 0.9% (w/v) NaCl) in stomacher filter bags. This solution was homogenized for 60 s (Seward Stomacher 400); further decimal dilutions were made in the peptone solution (ISO 6887-1, 1999). TVC was determined using plate count agar (PCA, Thermo Scientific OXOID) (ISO 4833-2, 2013); LAB count was determined using MRS agar (Thermo Scientific OXOID) (ISO 15214, 1998). One set of plates was incubated at 30°C for 3 days and another set at 4°C for 14 days.
7
Enumerating Probiotic Microorganisms from Lyophilized Products
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Microorganisms from lyophilized probiotic products stored at 4 °C were inoculated onto de Man-Rogosa-Sharpe (MRS) broth (Thermo Scientific™, Waltham, MA, USA) containing 0.05% l -cysteine-HCl (Sigma-Aldrich, St. Louis, MO, USA). Viable microorganisms were determined by plating serial 10-fold dilutions onto MRS-agar (Thermo Scientific™) containing 0.05% l -cysteine-HCl. Tests were performed in duplicate. Colonies were enumerated after incubation of plates at 37 °C for 48 h under anaerobic conditions in Anaerogen system (Thermo Scientific™). Individual strains from Blend 2 were provided by Mendes S.A.-Via Giacometti, 1, 6900 Lugano, Switzerland).
8
Isolation and Cultivation of Lactobacillus Plantarum
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Pasteurized, grade-A fat-free skim milk was obtained from a local supermarket (The Kroger Company, Cincinnati, OH, USA), MRS broth and MRS agar from Thermo Fisher Scientific (Waltham, MA, USA), Butterfield’s phosphate buffer from Hardy Diagnostics (Santa Maria, CA, USA). A well-preserved culture sample of LP (ATCC 10241) was generously provided by the U.S. Department of Agriculture Agricultural Research Service [57 ] culture collection (Peoria, IL, USA). The rest of chemicals were analytical grade and were obtained from Sigma Aldrich (St. Louis, MO, USA).
9
Profiling Gut Microbiome in Tumor Mice
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10
Quantifying Gut Bacterial Loads
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To measure LAB, Salmonella enterica, and E. coli loads, fecal samples (200 g) were collected monthly from each pen at five random locations and immediately analyzed. Each fecal sample (10 g) was weighed and placed in a stomacher bag containing 90 mL of sterile saline (0.9%) at a dilution of 1:10. Fecal samples were then plated on Difco MRS agar (Difco, Detroit, MI, USA), DifcoTM SS agar (Becton, Dickinson and Company, Sparks, MD, USA), and DifcoTM Violet Red Bile agar (Becton, Dickinson and Company, Sparks, MD, USA). The MRS agar plates were incubated in a CO2 incubator (Thermo Scientific, Waltham, MA, USA) at 30 °C for 48 h, whereas the SS agar and Violet Red Bile agar plates were incubated for 48 h at 37 °C in an aerobic incubator (Johnsam Corp., Boocheon, Republic of Korea). Visible colonies from the plates were counted, and the number of CFU/g of fecal extract at weeks 0, 30, and 60 was calculated. Microbiological data were transformed to log10.
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