Nasal swab storage medium (100 μL) was inoculated onto BHI agar supplemented with 0.1% Tween-80 +/− 0.005% wt/vol lithium mupirocin (Sigma-Aldrich). Oral swab storage medium (100 μL) was inoculated onto BHI agar supplemented with 0.1% Tween-80 and Mitis-Salivarius agar (BD). All isolation plates were incubated for 5 days at 37°C. Colonies of distinct morphotype (≥2 colonies per plate) were selected from each sample and passaged on the medium from which isolates were obtained at 37°C. Each isolate was inoculated in 3 mL of BHI supplemented with 0.1% Tween-80 and grown overnight at 37°C with shaking. All isolates were stored at −80°C in 20% (vol/vol) glycerol. Bacterial isolates were identified by sequencing the 16S rRNA gene. Briefly, 1 μL of each bacterial isolate liquid culture was used as the template for PCR amplification of the 16S rRNA gene using the universal 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) primers [83 (link)]. Amplification of PCR products was verified using electrophoresis with Tris-acetate-EDTA gels (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA disodium salt, 1% [wt/vol] agarose). PCR products were cleaned and underwent Sanger sequencing using the 27F primer at Functional Biosciences (Madison, WI). Isolates were identified to the genus level using the eHOMD 16S rRNA sequence identifier tool [73 (link)].
Lithium mupirocin
Manufactured by Merck Group
Sourced in Germany
Lithium mupirocin is a pharmaceutical compound used as a laboratory reagent. It is a salt formed by the combination of lithium and the antibiotic mupirocin. The core function of lithium mupirocin is to serve as a research tool in various scientific studies and experiments, though its specific applications may vary depending on the research context.
Automatically generated - may contain errors
4 protocols using lithium mupirocin
1
Bacterial Isolation and Identification from Nasal and Oral Swabs
2
Microbial Enumeration in Dairy Products
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The counts of microorganims were determined during storage at 4°C (1 st , 7 th , 14 th and 21 st days). One gram of sample was diluted with 9 mL of sterile 0.1% (w/v) peptone water (Oxoid, Basingstoke, UK), mixed with a vortex and subsequently serially diluted. The spread plate method was used to evalute of microbial counts. M17 agar (Merck, Germany) was used for enumeration of S. thermophilus at 37°C for 48 hours under aerobic conditions (Rybka and Kailasapathy, 1996) (link). MRS agar (Oxoid CM 361, Thermo-Fisher Scientific Inc., USA) was acidified with HCl to reach 5.2 pH value. It was used to determine the count of L. delbrueckii ssp. bulgaricus and incubated anaerobically at 37°C for 72 hours (Dave and Shah, 1996) (link). The counts of L. paracasei ssp. paracasei were determined with using Vancomysin added MRS agar and incubated anaerobically at 37°C for 72 hours. MRS agar was supplemented with cysteine chloride and lithium mupirocin (69732, Sigma-Aldrich) to determine the count of Bifidobacterium strains. The incubation was carried out anaerobically (5% CO2 atmosphere) at 37°C for 72 hours (Tharmaraj and Shah, 2003) (link). After the incubation, plates were counted and results were expressed as log CFU/g.
3
Enumeration and Identification of Probiotic Strains
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A series of 10-fold dilutions of the test beverages or ileal fluids was prepared with sterilized PBS and spread on lactitol-LBS vancomycin (LLV) agar medium19 (link) (for isolation of LcS) or TOS-MUP agar medium (for isolation of BbrY; TOS-Propionate agar base (Yakult Pharmaceutical Ind. [cat. no. 8-MJ54]) supplemented with lithium mupirocin (Merck KGaA [cat. no. 1. 00045.0010]) solution [50 mg/l]), respectively. The agar plates were incubated aerobically (for LLV agar plate) or anaerobically (for TOS-MUP agar plate) at 37°C for 72 h. Each type of colony was picked up and identified as LcS or BbrY by using strain-specific PCR-based assays.29 (link),52 (link) The number of LcS or BbrY per milliliter of ileal fluid was estimated from the number of colonies that were identified as LcS or BbrY. The survival rate of ingested strain from ileal fluid was calculated by using the following equation:
4
Culturable Bacterial Diversity in Phyllosphere
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MRS-Agar (deMan-Rogosa-Sharpe-Agar, MERCK, Germany), which favors the growth of lactic acid bacteria, was used to cultivate LABs, and TOS-Propionate agar with lithium mupirocin (MERCK, Germany) was applied to cultivate bifidobacteria. Bacterial washes from phyllosphere plant samples (as described in total cultivable bacterial numbers detection part) and their first three diluted samples (10−1, 10−2 und 10−3) were spread in triplicates on media. They were incubated in anaerobic jars (MERCK, Germany) with Oxoid AnaeroGen satchets (Thermo Scientific, Schwerte, Germany) for generating a microaerophilic atmosphere in the jars for 72 h at 37 °C. All emerging colonies were counted and collected for protein pattern composition analysis using the MALDI-TOF biotyper approach. No colonies were detected on TOS-agar.
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