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8 protocols using mannitol salt phenol red agar

1

Antimicrobial Poly(lactic acid) Granules

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Poly(lactic acid) granules were obtained from Goodfellow (Goodfellow Cambridge Limited, Huntingdon, UK). Gram-negative strain Escherichia coli KCTC 1039 (E. coli) and Gram-positive strain Staphylococcus epidermidis KCTC 13172 (S. epidermidis) used as model bacteria were purchased from KCTC (Korean Collection for Type Cultures, Jeongyep-si, Korea). Geraniol, carvacrol, dimethylformamide (DMF), sodium chloride (NaCl), mannitol salt phenol red agar, tryptic soy broth, osmium tetroxide (4% w/v), and dichloromethane (DCM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tryptic soy agar and MacConkey sorbitol agar were purchased from Becton Dickinson (BD DifcoTM, Sparks, MD, USA). Phosphate-buffered saline (PBS, pH 7.4) and LIVE/DEAD® BacLightTM Bacterial Viability Kit L-7007 (molecular probes) were supplied by Thermo Fisher Scientific (Thermo Fisher Scientific Inc., Waltham, MA, USA).
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2

Coculture of S. aureus and E. coli

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S. aureus RN450 lysogenic for phi80α and S. aureus RN450 lysogenic for phi11 were grown overnight at 37 °C in fresh brain heart infusion (BHI) medium, and E. coli BW25113 harbouring BAC-pks or empty BAC were grown overnight at 37 °C in fresh LB broth supplemented with chloramphenicol. The overnight cultures were back-diluted 1:100 into fresh BHI medium and mixed in a 1:1 ratio and incubated at 37 °C for 24 h. The cultures were plated on LB agar supplemented with Cm for E. coli colony-forming units (CFUs), and mannitol salt phenol-red agar (Sigma) for S. aureus CFUs.
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3

Wound-like Media Mono/Co-culture Assay

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Wound-like media was prepared as previously described23 (link) and 600 µl was added to tubes (glass, 6 × 50 mm). For mono-culture experiments wound-like media was inoculated with individual transposon mutants (construction described in Bacterial Strains and Culture Conditions) or the S. aureus WT strain HG003 at 5 × 104S. aureus. For co-culture experiments wound-like media was inoculated with 2.5 × 104 CFU of the S. aureus WT or individual S. aureus transposon mutants and 2.5 × 104 CFU P. aeruginosa PAO1. Cultures were incubated statically at 37°C for 6 days, and 10 µl samples removed for bacterial counts on days 2, 4, and 6. S. aureus cells were enumerated by plate counts on Mannitol Salt Phenol Red Agar (Sigma) as this medium does not permit growth of P. aeruginosa.
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4

Wound-like Media Mono/Co-culture Assay

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Wound-like media was prepared as previously described23 (link) and 600 µl was added to tubes (glass, 6 × 50 mm). For mono-culture experiments wound-like media was inoculated with individual transposon mutants (construction described in Bacterial Strains and Culture Conditions) or the S. aureus WT strain HG003 at 5 × 104S. aureus. For co-culture experiments wound-like media was inoculated with 2.5 × 104 CFU of the S. aureus WT or individual S. aureus transposon mutants and 2.5 × 104 CFU P. aeruginosa PAO1. Cultures were incubated statically at 37°C for 6 days, and 10 µl samples removed for bacterial counts on days 2, 4, and 6. S. aureus cells were enumerated by plate counts on Mannitol Salt Phenol Red Agar (Sigma) as this medium does not permit growth of P. aeruginosa.
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5

Isolation and Identification of Foodborne Staphylococci

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Ready-to-eat food samples (n = 198) including sushi, salads, fresh squeeze juices, hamburgers, beef tartar, salmon tartar obtained from 11 randomly selected bars and restaurants in Olsztyn, Poland were investigated. Isolation of the strains was performed by methods described previously [46 (link)]. Briefly, food samples (10 g) were homogenised in 90 mL buffered peptone water (Merck, Germany), incubated overnight at 37 °C and streaked on selective plates containing Mannitol Salt Phenol-red Agar (Merck, Darmstadt, Germany). Mannitol (+) colonies were differentiated into coagulase-positive and coagulase-negative with a test detecting the production of a clumping factor (Staphylase Test Kit, Oxoid, Basingstoke, UK) and production of coagulase on the RPF medium (bioMérieux, Marcy l’Etoile, France) with rabbit blood plasma and fibrinogen.
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6

Enumeration of Microbial Populations in Food

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Samples of 25 g were added to 225 mL physiological saline (0.85 NaCl%) and homogenized in a stomacher (Lab Stomacher, London, UK) for 1 min. In determining the number of lactic acid bacteria, de Man Rogosa Sharpe Agar (Merck, Darmstadt, Germany) was used and incubation was carried out at 30 °C for 48 h under anaerobic conditions (Anaerocult A, Merck, Darmstadt, Germany). Mannitol Salt Phenol Red Agar (Merck) was used for the enumeration of Micrococcus/Staphylococcus, and the plates were incubated aerobically at 30 °C for 48 h. Enterobacteriaceae were determined on Violet Red Bile Dextrose Agar (Merck), and the plates were incubated at 30 °C for 48 h under anaerobic conditions (Anaerocult A, Merck) [23 ].
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7

Screening of Microbial Enzymatic Activities

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All chemicals and growth media were purchased from commercial sources. LB broth and LB agar medium were bought from A&A Biotechnology (Gdynia; Poland). Mannitol salt phenol−red agar, Spirit blue agar, tributyrin agar, skimmed milk, starch, carboxymethylcellulose, guaiacol, X−gal (5−bromo−4−chloro−3−indolyl−β−D−galactopyranoside), Tween 80, cottonseed oil, Lugol’s solution, Congo red dye, and PBS tablets (pH 7.4) were purchased from Merck (Darmstadt, Germany). Ultrapure H2O (18.0 MΩ) was produced with the Milli−Q Advantage A10 system (Millipore, Billerica, MA, USA).
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8

Isolation and Identification of S. aureus from Street Food

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In September 2022, street food samples, including stir-fried rice noodles, sticky rice with assorted toppings, and spring rolls, were randomly purchased from street vendors in Ward 4, District 5, Ho Chi Minh City.
Isolation of S. aureus from street food samples was carried out as the method reported previously.22 First, 10 g of each sample was transferred separately to a sterile Stomacher bag and 90 mL of NaCl (Merck, Germany) 0.9% solution were added. The sample underwent homogenization in a stomacher (Seward, England) operating at 200 rpm for 5 min. Then, a 10-fold dilution of the sample was prepared using sterile NaCl (Merck, Germany) 0.9%, and 0.1 mL of various dilutions was spread onto the surface of mannitol salt phenol red agar (Merck, Germany) plates. The plates were then incubated at 35 ± 2 °C for 24 h. Yellow colonies with yellow zones were transferred to Baird-Parker agar (Merck, Germany) medium with tellurite egg yolk (Merck, Germany). Subsequently, the plates were incubated at 35 ± 2 °C for 24 h. Black, shiny, convex colonies surrounded by a lightening halo of the egg yolk were used for biochemical tests, including Gram stain, catalase test, indole test, Methyl red-Voges Proskauer test, oxidase test, test for fermentation of glucose, lactose, and sucrose, and coagulase test.
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