Lactobacillus casei DGDG (Sofar S.p.A., Milan, Italy), Lactobacillus paracasei LPC-S01 (Sofar S.p.A., Milan, Italy), Lactobacillus acidophilus LA14 (Danisco S.p.A. Milan, Italy), Bifidobacterium lactis BL04 (Danisco S.p.A. Milan, Italy), Streptococcus thermophilus ST21 (Danisco S.p.A., Milan, Italy) were tested. Lyophilized L. casei, L. paracasei, L. acidophilus and B. lactis were suspended in saline solution and seeded on DeMan-Rogosa-Sharpe (MRS) agar (Oxoid S.p.A.,Milan, Italy) with 0.05% L-cysteine, incubated in jar for 24 h at 37 °C in an anaerobic atmosphere (AnaeroGen GasPack, Oxoid S.p.A., Milan, Italy). Lyophilized S. thermophilus was suspended in saline solution and seeded onto blood agar containing 5% horse blood (HB) (Kima S.r.L., Padova, Italy), incubated in jars for 24 h at 37 °C under microaerobic conditions (CO2Gen GasPack, Oxoid S.p.A., Milan, Italy). To confirm bacterial species identification, matrix-assisted laser desorption ionization-time of flight mass spectrometry (Maldi-Tof microflex, Bruker Daltonics S.R.L., Macerata, Italy – SciLsLabSoftware, 3D version 2016b (Bruker Daltonics S.r.L, Macerata, Italy) was performed [34 (link)].
De man rogosa sharpe agar
Manufactured by Thermo Fisher Scientific
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De Man Rogosa Sharpe (MRS) agar is a microbiological culture medium used for the selective isolation and enumeration of lactic acid bacteria. It provides the necessary nutrients and growth factors to support the growth of Lactobacillus and other lactic acid-producing microorganisms.
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Lab products found in correlation
Anaerogen gas pack, by Thermo Fisher Scientific (1 mentions)
Microflex maldi tof, by Bruker (1 mentions)
Malt extract agar, by Thermo Fisher Scientific (1 mentions)
Violet red bile glucose agar vrbga, by Thermo Fisher Scientific (1 mentions)
Api 50 chl, by bioMérieux (1 mentions)
Mannitol salt phenol red agar, by Thermo Fisher Scientific (1 mentions)
Potato dextrose agar (pda), by Thermo Fisher Scientific (1 mentions)
Plate count agar, by Thermo Fisher Scientific (1 mentions)
Violet red bile dextrose agar, by Merck Group (1 mentions)
Anaerobic jar, by Thermo Fisher Scientific (1 mentions)
Anaerogen gaspak system, by Thermo Fisher Scientific (1 mentions)
12 protocols using de man rogosa sharpe agar
1
Identification of Probiotic Bacterial Strains
2
Anaerobic Bacterial Culture on MRS Agar
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The tested strains were cultured in anaerobic conditions at 37 °C for 48 h on de Man-Rogosa-Sharpe MRS agar (Oxoid).
3
Co-culture of Probiotic L. reuteri and Candida
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Two strains of the probiotic L. reuteri (DSM 17938 and ATCC PTA 5289; Biogaia, Stockholm, Sweden) were used in this study. The Candida strains used were six laboratory reference strains from the Culture Collection, University of Gothenburg, Sweden: C. albicans CCUG 46390; C. dubliniensis CCUG 48722; C. glabrata CCUG 63819; C. krusei CCUG 56126; C. parapsilosis CCUG 56136, and C. tropicalis CCUG 47037. In addition, six clinically isolated strains from humans were included, generously provided by the Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark. Prior to the in vitro studies, the clinical strains were characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry [22 (link)] to confirm their identity as C. albicans CBS 562 NT, C. dubliniensis 41_3 ZZMK, C. glabrata CBS 863, C. krusei (Issatchenkia orientalis RV 491), C. parapsilosis 26 PBS, and C. tropicalis DSM 7524.
The lactobacilli were initially cultured on de Man Rogosa Sharpe (MRS) agar (Oxoid Ltd., Basingstoke, Hampshire, UK) for 24 h in an anaerobic chamber at 37°C (10% H2, 5% CO2, and 85% N2). The Candida strains were cultured on BD Difco™ Sabouraud Maltose (DSM) agar (Becton, Dickinson and Company, Sparks, MD) for 24 h in ambient air at 37°C.
The lactobacilli were initially cultured on de Man Rogosa Sharpe (MRS) agar (Oxoid Ltd., Basingstoke, Hampshire, UK) for 24 h in an anaerobic chamber at 37°C (10% H2, 5% CO2, and 85% N2). The Candida strains were cultured on BD Difco™ Sabouraud Maltose (DSM) agar (Becton, Dickinson and Company, Sparks, MD) for 24 h in ambient air at 37°C.
4
Cultivation of S. cerevisiae and L. plantarum
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S. cerevisiae strain BM-SC17426 and L. plantarum strain BM-LP14723 were maintained as glycerol stock stored at −80°C. A small amount of S. cerevisiae stock was spread onto yeast peptone dextrose (YPD) agar and incubated at 30°C for 48 h to obtain single colonies. A single colony of S. cerevisiae was transferred to 2 mL of YPD broth and incubated at 30°C with shaking at 200 rpm overnight prior to further experiments. A small amount of L. plantarum stock was spread onto De Man Rogosa Sharpe (MRS) agar (Oxoid) and incubated at 37°C for 48 h to obtain single colonies. A single colony of L. plantarum was transferred to 2 mL of MRS broth and incubated at 37°C with shaking at 200 rpm overnight prior to further experiments.
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5
Cultivation of S. cerevisiae and L. plantarum
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S. cerevisiae strain BM-SC17426 and L. plantarum strain BM-LP14723 were maintained as glycerol stock stored at −80°C. A small amount of S. cerevisiae stock was spread onto yeast peptone dextrose (YPD) agar and incubated at 30°C for 48 h to obtain single colonies. A single colony of S. cerevisiae was transferred to 2 mL of YPD broth and incubated at 30°C with shaking at 200 rpm overnight prior to further experiments. A small amount of L. plantarum stock was spread onto De Man Rogosa Sharpe (MRS) agar (Oxoid) and incubated at 37°C for 48 h to obtain single colonies. A single colony of L. plantarum was transferred to 2 mL of MRS broth and incubated at 37°C with shaking at 200 rpm overnight prior to further experiments.
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6
Silage Microbial and Organic Acid Analysis
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Microbial groups were counted by using 25 g of fresh silage sampled from each silo; at each sample, 225 mL of phosphate buffer solution was added to obtain a dilution of 10 -1 (Kung, 1996) . Several dilutions were performed aiming to obtain dilutions varying from 10 -1 to 10 -7 , and immediately plated in duplicate on sterile Petri dishes. Lactic acid bacteria (LAB) were determined on De Man-Rogosa-Sharpe (MRS) agar (Oxoid) after incubation at 32 °C for 48 h. Enterobacteria was determined on Violet Red Bile Glucose Agar (VRBGA; Oxoid) incubated at 37 °C for 24 h. Yeasts and mold were determined on malt extract agar (Oxoid) after incubation at 32 °C for 48 h. Only dishes with between 30 and 300 CFU were counted. The results were transformed into log x to achieve normal distribution.
Organic acids were determined from the aqueous extract of silages using the methodology described by Kung (1996) . This method consists in an aliquot of 25 g fresh silage mixed in 225 mL deionized water for 1 min, then filtered on a filter paper, acidified with 50% sulfuric acid, and centrifuged for 15 min at 10,000 rpm. Lactic acid (LA), propionic acid (PA), and butyric acid (BA) were determined using a high-performance liquid chromatography (HPLC) of Shimadzu-BIORAD brand, SPD-10 model, C18 column, and reverse phase at a wavelength of 210 nm.
Organic acids were determined from the aqueous extract of silages using the methodology described by Kung (1996) . This method consists in an aliquot of 25 g fresh silage mixed in 225 mL deionized water for 1 min, then filtered on a filter paper, acidified with 50% sulfuric acid, and centrifuged for 15 min at 10,000 rpm. Lactic acid (LA), propionic acid (PA), and butyric acid (BA) were determined using a high-performance liquid chromatography (HPLC) of Shimadzu-BIORAD brand, SPD-10 model, C18 column, and reverse phase at a wavelength of 210 nm.
7
Isolation and Identification of Lactobacillus and Pathogenic Bacteria from Chicken
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Lactobacillus spp. have been isolated from fresh droppings of healthy laying hens (Withe Leghorn, 40 weeks of age) kept on litter. All birds were provided with free access to clean water and a standard feed. Bacteria were grown on De Man-Rogosa-Sharpe (MRS) agar (Oxoid) in anaerobic conditions at 37 o C for 48 h. The isolates were identified based on Gram's stain morphology, catalase reaction, and biochemical properties tested in API 50CHL (bioMerieux). Pathogenic bacteria: Escherichia coli, Salmonella Enteritidis and Clostridium perfringens have been isolated from chicken internal organs in the Microbiological Diagnostic Laboratory, Faculty of Veterinary Medicine, Warsaw Agriculture University.
8
Microbial Enumeration in Avian Jejunum and Ileum
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The microbial counts of jejunum and ileum were obtained as previously described24 (link). Approximately 1 g of jejunal and ileal digesta was obtained from all birds and serially diluted 10-fold (from 10−1 to 10−7) with sterile physiological saline solution (0.9% NaCl) and subsequently homogenised for 3 min by using an ultra-turrax. Dilutions were then plated onto selective agar medium for enumeration of the target bacterial groups. Escherichia coli were grown on MacConkey agar (Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China). Lactobacilli were cultivated using de Man–Rogosa–Sharpe agar (Oxoid Ltd., Hampshire, UK). Lactobacillus plates were incubated anaerobically, whereas E. coli plates were incubated aerobically at 37 °C for 24 h. Bacteria were enumerated by visual counting of colonies by using the best replicate set from dilutions that resulted in 30 to 300 colonies per plate. The microbial enumerations of jejunum and ileum were expressed as base-10 logarithm colony-forming units per gram of jejunum and ileum digesta.
9
Quantifying Gut Bacterial Populations
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Frozen cecal digesta samples were thawed at room temperature. Approximately 1 g of cecal digesta was taken from each sample and serially diluted 10-fold (from 10−1 to 10−7) with sterile physiological saline solution (0.9% NaCl) and subsequently homogenized for 3 min using an ultra-turrax. Dilutions were then plated onto selective agar medium for enumeration of target bacterial groups. Escherichia coli were grown on MacConkey agar (Beijing Aoboxing Bio-tech Co., Ltd., Beijing, China). Lactobacilli were cultivated using de Man, Rogosa, Sharpe agar (Oxoid Ltd., Hampshire, UK). Plates for Lactobacillus were incubated anaerobically, while plates for Escherichia coli were incubated aerobically. All plates were incubated at 37°C for 24 h. Bacteria were enumerated by visual count of colonies, using the best replicate set from dilutions that resulted in 30 to 300 colonies per plate. The microbial enumerations of cecal digesta were expressed as base-10 logarithm colony-forming units per gram of cecal digesta.
10
Microbiological Analysis of Food Samples
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Sample solutions for microbiological analysis were prepared by the homogenization of 25 g sample with 225 mL physiological saline (0.85% NaCl) in a Stomacher (Laboratory Blender Stomacher 400, Seward, England) for two minutes. Incubation temperature and time for other microbiological analyses were as follows: For counting total aerobic mesophilic bacteria (TAMB), Plate Count Agar (Oxoid) was used. Colonies were counted at the end of incubation for 48 h at 30℃ under aerobic conditions. Lactic acid bacteria (LAB) were incubated at 30℃ anaerobically for 2 d by using De Man Rogosa Sharpe Agar (Oxoid). Micrococcus/Staphylococcus (M/S) were counted using Mannitol Salt Phenol-Red Agar (Oxoid) incubated aerobically at 30℃ for 2 d. Mould-yeast (M-Y) were incubated at 25℃ aerobically for 5 d by using Potato Dextrose Agar (Oxoid). Enterobacteriaceae was incubated on Violet Red Bile Dextrose Agar (Merck) at 30℃ anaerobically for 2 d.
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