All the bacterial species (Clostridium perfringens, Bacillus cereus, Serratia marcescens, Pseudomonas aeruginosa and Alcaligenes faecalis) used in the screening of antimicrobial activities were obtained from the Department of Microbiology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia (UPM), Malaysia. Vibrio parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila were obtained from the Department of Aquaculture, Faculty of Agriculture, UPM, Malaysia. All bacterial strains (PD9, B7, PU1, BP1 and L9) that belonged to the B. amyloliquefaciens operational group isolated from stingless bee, Heterotrigona itama, products [18 (link)] were obtained from the Enzyme and Microbial Technology Research Centre (EMTech, UPM, Malaysia). MRSA strains (ATCC 33742, ATCC 700699, ATCC 25922 and ATCC 33741) and MSSA strain ATCC 12228 were obtained from the Department of Biomedical Science, Faculty of Medicine and Health Science, UPM, Malaysia. Cultures were grown in 10 ml of nutrient broth (Merck, Darmstadt, Germany) at 37°C with shaking at 200 rpm for 24 h. For long-term storage, cultures were preserved in nutrient broth using 20% (v/v) glycerol at -80°C.
Nutrient broth
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom, Romania, India, Malaysia, Canada, Italy
Nutrient broth is a type of general-purpose culture medium used in microbiology laboratories. Its core function is to provide essential nutrients for the growth and cultivation of a wide range of microorganisms, such as bacteria, yeast, and fungi.
Automatically generated - may contain errors
Lab products found in correlation
Nutrient agar, by Merck Group (50 mentions)
Mueller hinton agar (mha), by Merck Group (13 mentions)
Hydrochloric acid (hcl), by Merck Group (11 mentions)
Glycerol, by Merck Group (10 mentions)
Methanol, by Merck Group (10 mentions)
Sodium chloride (nacl), by Merck Group (10 mentions)
Sodium hydroxide (naoh), by Merck Group (10 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (9 mentions)
Ethanol, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Merck Group (7 mentions)
Brain heart infusion broth, by Merck Group (7 mentions)
Gallic acid, by Merck Group (6 mentions)
Muller hinton agar, by Merck Group (6 mentions)
Phosphate buffered saline (pbs), by Merck Group (5 mentions)
Macconkey agar, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Quercetin, by Merck Group (5 mentions)
Potato dextrose agar (pda), by Merck Group (5 mentions)
Ascorbic acid, by Merck Group (5 mentions)
Hydrogen peroxide, by Merck Group (5 mentions)
240 protocols using nutrient broth
1
Bacterial Species Screening for Antimicrobial Activities
2
Antimicrobial Potency of Bacterial CFS against MRSA
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MIC was determined by using the microplate dilution method with slight modifications [28 (link)]. The CFS used in the experiment was obtained by growing strain PD9 alone as well as co-culture of strain PD9 and MRSA ATCC 33742 in nutrient broth (Merck, Germany) for 20 h. In the strain PD9 alone flask, 1 ml of strain PD9 culture at final cell density 103 cfu/ml was inoculated into 99 ml nutrient broth. In the co-culture flask, 1 ml of strain PD9 and 1 ml of MRSA ATCC 33742 at final cell density 103 cfu/ml were inoculated into 98 ml nutrient broth. The culture was centrifuged at 8 000 x g for 10 min to obtained the CFS. The CFS was serially diluted with nutrient broth by two-fold dilution (2−1 to 2−10). Then, 5 μl of MRSA ATCC 33742 cell suspension (standardized to 107 cfu/ml) was deposited into each well. The 96-well plates were incubated aerobically at 37°C overnight. The absorbance was measured at 600 nm by using a microplate reader (BioTek Instrument, USA). The minimum dilution showing no growth indicated the MIC value of strain PD9 CFS against MRSA ATCC 33742.
3
Quantifying Indole Acetic Acid Production
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Indole acetic acid production was quantitatively measured according to Gordon and Weber (1951) (link). Bacterial cultures were grown in test tubes, each containing 5 mL nutrient broth (Merck Millipore, UK) amended with 0.1% (w/v) tryptophan, incubated at 28 ± 2°C for 24 h. nutrient broth was used instead of yeast extract mannitol medium (YEM) as described in Gordon and Weber (1951) (link) method. Then, the cultures medium was centrifuged at 10,000 rpm for 10 min. The total of 1 mL supernatant was mixed with 2 mL of Salkowski reagent. Tubes were incubated in dark at room temperature for 25 min. The development of pink colour indicates high production of IAA and the intensity of pink colour was read at 530 nm wavelength. The concentration of IAA produced, then, was extrapolated from the standard curve (Gordon & Weber 1951 (link)).
4
Isolation and Identification of E. coli from Dairy Products in Iran
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Overall 600 dairy products including 200 yoghurt, 200 doogh and 200 kashk samples were purchased from supermarkets and retailers in various parts of Iran at summer of 2012. All of these dairy products were made traditionally by native people and after collection were kept under refrigeration in plastic bags. Samples were transported under refrigeration (at 4-6°C) in thermal boxes containing ice packs. All samples were diluted in phosphate buffered saline (PBS, Merck, Germany). A 25 g portion of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for 2 min, using a Stomacher lab blender and incubated at 37°C for 24 h. A 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for 24 h. One loop of each tube was streaked on MacConkey agar (Merck, Germany). Such colonies were confirmed as E. coli using standard biochemical tests (e.g., Indole, Methyl red, Voges-Proskauer and Citrate utilization tests). Colonies were confirmed as E. coli by PCR [26 (link)]. E. coli isolates were stored in Tryptic Soy Broth (TSB, Merck, Germany) containing 20% glycerol at -70°C for further characterization.
5
Isolation and Identification of E. coli
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Three milliliters of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for two minutes at normal speed, using a Stomacher lab blender and incubated at 37°C for 24 hours. A 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for 24 hours. One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli from each sample was streaked on a separate eosin methylene blue agar (EMB agar) plate (Merck, Germmany) and incubated at 37°C for 24 hours. A metallic green colony from each plate with a typical E. coli morphology was selected and examined by biochemical tests, including hydrogen sulfide, citrate, urease and indol based on the API test system (BioMerieux, Marcy L’Etoile, France) (23 ).
6
Pediatric UTI Urine Sample Analysis
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This cross sectional study was performed from October to December 2012. A total of 100 urine samples were collected from patients with UTIs. All of patients were younger than 3 years. All samples were collected from the hospitalized pediatrics of Baqiyatallah Governmental Hospital in Tehran, Iran. Midstream urine was collected in sterile condition to decrease potential bacterial, cellular and artifactual contamination. All samples were immediately transferred to the Biotechnology and Microbiology Research Center of the Islamic Azad University at 4°C. Totally, 3 mL of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for 2 min at normal speed, using a Stomacher lab blender and incubated at 37 °C for 24h. One milliliter sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37 °C for 24h. One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli was streaked on Eosin Methylene Blue agar (EMB agar) plate (Merck, Germany) and incubated at 37 °C for 24h. A metallic green colony from each plate with typical E. coli morphology was selected and examined by biochemical tests, including hydrogen sulfide, citrate, urease and indole.
7
Wheat Flour Characterization by AACC Methods
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Wheat flours (Null, Star and Whole) were purchased from a local milling factory (Zahedi, Gorgan, Iran) and assessed in terms of chemical properties using AACC standard methods (44-19 moisture content, 46-10 protein (N×5.70), 38-12 wet gluten and 08-01 ash content) (AACC, 2010 ). Based on these assessments, Null, Star and Whole flours had in order: 67.5, 76, and 92% extraction rate; 14.2, 13.80, and 8.10% moisture content; 8.5, 10.90, and 12.25% protein; 23.4, 25.85, and 26.40% wet gluten; 0.45, 0.75, and 1.55% ash content; and 2.4, 3.5, and 4.1% acidity. Active dry yeast extract containing Saccharomyces cerevisiae was acquired from the Iran-Mellas company (Iran). All chemical reagents and microbial media (MRS broth, MRS agar and Nutrient broth) were purchased from Merck (Germany).
8
Antimicrobial Screening of Lactic Acid Bacteria
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Pediococcus pentosaceus subsp. aureus ATCC 25923, Escherichia coli O157: H7 and Bacillus cereus ATCC 10876, were used as indicator strains. These were grown in Nutrient Agar (NA) at 37°C. Antimicrobial activity of LAB against E. coli O157 and B. cereus was determined by the agar well diffusion assay and against S. aureus subsp. aureus by disc diffusion assay. Isolates were grown in MRS and M17 broth (Merck, Germany) at 37°C for 24 hours. Each indicator bacteria was grown in nutrient broth (Merck, Germany) at 37°C for 24 hours. Furthermore, 100 μL of this broth culture of pathogenic bacteria was cultured on Muller-Hinton Agar (MHA) (Merck, Germany). The resulting supernatants of overnight culture of representative isolates were neutralized to pH = 6.5 - 7.0, centrifuged at 14000 rpm for five min, and filtered through a 0.20 μm pore membrane (Millipore, USA). Next, wells of 6 mm in diameter were created in these agar plates and filled with 50 μL of each isolates supernatant (11 (link)). Sterile discs (6 mm in diameter) were soaked with culture filtrate of each isolate and placed on a 100 mm plate for the disc diffusion assay (12 (link)).
9
Seaweed-Derived Alginate Biopolymer
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Sodium alginate derived from seaweed and CaCO3 were purchased from a local food ingredient supplier (Katki Dunyasi, Turkey). Calyces of roselle (Bagdat Baharat, Turkey), glucono delta-lactone (GDL, Smart Kimya, Turkey), Nutrient broth (Merck, Germany), bacteriological agar (Himedia, India), and glycerol (Sigma-Aldrich, Germany) were utilized in the present study. Distilled water was used throughout the experiments.
10
Microdilution Assay for Fungal MIC
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The microdilution method described by Nchu et al. (2010) was used with minor modifications in determination of the minimum inhibitory concentration (MIC) of the extracts. S. aethiopicus aerial and rhizome extracts were diluted into acetone to obtain a starting concentration of 6 mg/ml. The starting concentration was diluted two fold in each successive serial dilution. The Fusarium oxysporum f sp.glycines strain (UPFC no. 21) was obtained from the Phytomedicine Programme, University of Pretoria. The fungus strain was originally isolated by C. Cronje from roots of a maize plant in Delmas, Gauteng. F. oxysporum was sub-cultured from stock agar plates and grown in Nutrient Broth (Merck, South Africa) for four hours. The fungal culture (100 ml) was added to each well of the 96-well microplates (105 cells/ml). Amphotericin b (160 μg/mL) was prepared as stock solution in acetone and served as a positive control and acetone was used as a negative control. Forty micro litre (40 μl) of 0.2 mg/ml of p-iodonitrotetrazolium chloride (INT) (Sigma) dissolved in sterile distilled water was added to each microplate well, sealed in a plastic bag and incubated at 37 °C and 100% RH. The MIC values were recorded after 12 and 18 hours. The antifungal bioassay (MIC) consisted of three replicates per treatment and per watering regime.
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