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The middle samples were fixed in chloroform calcium for 90 min at room temperature and then stored in buffer (10 ml of distilled water, 1 g of CaCl2, 0.121 M cacodylate) at 4°C until further processing.
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Nasopharyngeal and middle ear samples were collected for quantitative microbiology.
Micro-SXRF mapping of the middle waste shale samples were done to determine the spatial elemental composition.
Middle ear fluid samples were collected either by tympanocentesis or spontaneous otorrhea swab sampling method.
All middle ear effusion samples were kept at room temperature and transported to the research laboratory within 2 h.
Middle ear effusion samples were categorized as AOM or OME by the surgeon at the time of tympanostomy tube placement.
In the OGTT performed during follow-up visits starting from the middle of 1996, samples were also taken for 30-min insulin and glucose and for 60-min glucose measurements (Supplementary Fig. 1).
To describe the clinical, epidemiological and antimicrobial resistance patterns of Costa Rican children with otitis media caused by Streptococcus pneumoniae serotype 3. Middle ear fluid samples were obtained from Costa Rican children with otitis media who participated in various antimicrobial clinical trials between 1992 and 2007.
Nevertheless, only 14% were in their twenties, 36.8% were younger than 40 years of age, and 63.2% were 40 years old or over, which indicates that the majority of samples were middle-aged or past middle-age.
These patients were treated with CAPD, and the middle-stage peritoneal dialysate samples were collected after a year.
Small sections (~1 cm of length) from the middle part of the intestine samples were taken and homogenized in 1 ml of Trizol reagent (Invitrogen, Carlsbad, USA) with sterile KONTES® pellet pestle driven by a cordless motor (Fisher Scientific, New Hampshire, USA).
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