Whirl pak sampling bags

Milk samples were drawn under the aseptic conditions from healthy dairy cows and cows suffering from mastitis in Qalyoubia Governorate immediately before routine daily milking. The udder of each cow was washed with a diluted solution of povidone-iodine (El-Nile Co., Egypt) in warm water, dried with paper towels, and teats were swabbed with 70% ethyl alcohol and allowed to air dry for 1 min. Milk samples (150 mL) were drawn aseptically from each quarter directly into labeled with Whirl-pak sampling bags (28 g, Merck Darmstadt, Germany); these were placed in iced insulated boxes. Samples were examined for Mycoplasma by the traditional golden isolation method and polymerase chain reaction (PCR) test [19 ].
For chemical and pesticide analysis, samples were packaged in glass tubes (Merck). All samples were maintained at −10°C until analyzed.

The River Erpe is a lowland stream located east of Berlin, Germany. It is a receiving river, highly impacted by the municipal wastewater from the WWTP Münchehofe that accounts for 60–80% of its total discharge. The sampling site is located approximately 0.7 km downstream of the effluent discharge site at Heidemühle (latitude, 52.478647; longitude, 13.635146). The site is characterized by fine-sandy siliceous sediment as determined in a previous field study [19 (link)]. Sediment and water samples were collected from the site in September 2015. Surface sediment samples (0–5 cm depth) were collected from several spots on the streambed with a flat hand shovel and stored in sterile Whirl-Pak sampling bags (Merck, Darmstadt, Germany). Surface water samples were obtained from the same site and stored in sterile 2-L screw-mouth Duran^® bottles (Merck, Darmstadt, Germany). Freshly collected sediment and water samples were transferred to the laboratory at 4 °C, manually homogenized, and an aliquot stored at −80 °C for subsequent nucleic acid extraction. The remaining fresh sediment was stored at 4 °C and used to set up microcosm incubation experiments within two weeks.

Mice lungs were collected at 4 or 14 days post-infection (dpi), halved and fixed in 10% neutral buffered formalin (Sigma Aldrich) for at least 16 h before they were dehydrated, embedded in paraffin and prepared into 5 micron sections. Lung sections were stained with Haematoxylin and Eosin (H&E), and examined for the presence of histopathological abnormalities.
The remaining lung halves collected 4 dpi were immersed into 1 ml of PBS, homogenized in whirl-pak sampling bags (Sigma Aldrich), transferred into microfuge tubes and centrifuged at 6000x g for 10 min at 4°C. Supernatant fractions were then transferred into fresh tubes and stored at −80°C. To perform virus plaque assays, supernatants were thawed, serially diluted with PBS and applied onto MDCK cells at 90% confluency for 1 h at 37°C to allow virus adsorption. Cells were subsequently rinsed with pre-warmed PBS and cultured in MEM (Thermo Fisher Scientific) supplemented with 1.2% Avicel® (FMC Biopolymer) and 2.5 µg/ml trypsin (Thermo Fisher Scientific). After 2 days of incubation, cells were fixed with 10% neutral buffered formalin for at least 16 h before plaques were visualized and quantified by staining with 0.1% crystal violet solution (Sigma Aldrich).