For the detection of kanamycin A in cleaned waste water, a water sample was taken in January 2013 at the drain of a vertical flow type of constructed wetlands and was experimentally spiked with the pharmaceutical. The real water was filtered by using a vacuum filtration unit (0.22 μm, Sartorius, Göttingen, Germany) and subsequently mixed in equal parts with selection buffer that was beforehand spiked with different amounts of kanamycin A. The pH of the resulting solution was about 7.6. This solution was the measuring sample added in the MTP assay (see description in Section 2.4.1) to the wells. All other steps, like washing steps and fluorescence detection, were performed in selection buffer.
Vacuum filtration unit
Manufactured by Sartorius
Sourced in Germany
The Vacuum filtration unit is a laboratory equipment designed for the separation of solid and liquid components in a sample through the process of vacuum filtration. The core function of this device is to create a negative pressure differential to draw the liquid portion of the sample through a filtration membrane, leaving the solid particles behind. This allows for the efficient collection and isolation of the desired components in the sample.
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Lab products found in correlation
4 protocols using vacuum filtration unit
1
Kanamycin A Detection in Wastewater
2
Establishing Grapevine Cell Cultures
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Establishment and maintenance of suspension cell cultures are described in Zottini et al. [19] (link). Briefly, grapevine cell lines were obtained from leaf disk explants incubated in solid B5F medium. After several subculture cycles, aliquots of callus were inoculated into liquid B5F medium. Every week, 2 mL of suspension cell cultures were transferred to Erlenmeyer flasks (250 mL) filled with 50 mL liquid B5F medium. The suspension cultures were maintained in a climate growth chamber at 25±1°C on an orbital shaker (80 rpm) under a long photoperiod (16 h light and 8 h dark). To determine fresh weight, intact cells were separated from culture medium and cell debris through a vacuum filtration unit (Sartorius, Florence, Italy).
3
Bacterial Metabolite Extraction and LC-MS Analysis
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Bacteria were cultivated as described above, if not indicated otherwise,
until the beginning of the stationary growth phase as described with
modifications.15 (link) Briefly, a 0.22 μM
cellulose-acetate membrane filter (Ø 47 mm, Sartorius) was washed
three times with 10 mL of ultrapure water, each using a vacuum filtration
unit (Sartorius), followed by the addition of 10 mL of cell culture
per OD600 unit and washing of the cells three times with
10 mL of ultrapure water. The filter with cells was transferred immediately
to a bottle containing 20 mL of extraction mixture chilled to −20
°C consisting of acetonitrile, methanol, water, and formic acid
(60:20:19.9:0.1 (v/v), LC–MS grade, VWR). Cells were disrupted
by three freeze–thaw cycles, that is, for 1 h at −70
°C, followed by 5 min of sonication in an ultrasonic bath (Bandelin).
The extracts were transferred to glass flasks and lyophilized at 1
mbar and −90 °C, followed by reconstitution in a total
of 1 mL of water (LC–MS grade, VWR). The extracts were prepared
for LC–MS measurement by centrifugation for 20 min at 17,000g and room temperature to remove cell debris, followed by
transfer of the supernatant to a fresh tube and centrifugation for
20 min at 17,000g and room temperature. Extracts
were stored at −20 °C until measurement.
until the beginning of the stationary growth phase as described with
modifications.15 (link) Briefly, a 0.22 μM
cellulose-acetate membrane filter (Ø 47 mm, Sartorius) was washed
three times with 10 mL of ultrapure water, each using a vacuum filtration
unit (Sartorius), followed by the addition of 10 mL of cell culture
per OD600 unit and washing of the cells three times with
10 mL of ultrapure water. The filter with cells was transferred immediately
to a bottle containing 20 mL of extraction mixture chilled to −20
°C consisting of acetonitrile, methanol, water, and formic acid
(60:20:19.9:0.1 (v/v), LC–MS grade, VWR). Cells were disrupted
by three freeze–thaw cycles, that is, for 1 h at −70
°C, followed by 5 min of sonication in an ultrasonic bath (Bandelin).
The extracts were transferred to glass flasks and lyophilized at 1
mbar and −90 °C, followed by reconstitution in a total
of 1 mL of water (LC–MS grade, VWR). The extracts were prepared
for LC–MS measurement by centrifugation for 20 min at 17,000g and room temperature to remove cell debris, followed by
transfer of the supernatant to a fresh tube and centrifugation for
20 min at 17,000g and room temperature. Extracts
were stored at −20 °C until measurement.
4
Enumeration of E. coli in Contaminated Sediment
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On sampling days, approximately 3 g of contaminated mix (faecal streambed sediment) was randomly sampled from all replicates of each treatment using a sterile spatula after the removal of the overlying water using a pipette. To enumerate the E. coli present in the sediment, each 3 g sample was transferred to 27 mL of sterile river water in a 50 mL centrifuge tube and vortex-mixed for 30 s to ensure homogeneity prior to subsequent 1:10 serial dilution in PBS. Subsequently, 1 mL of each serially diluted sample was pipetted on to a 0.45 µm cellulose acetate membrane and washed through a vacuum-filtration unit (Sartorius Stedim Biotech., Goettingen, Germany) with ~20 mL of sterile PBS to ensure the capture of between 20 and 200 colony-forming units (CFU). To determine presumptive E. coli, the membranes were aseptically transferred to a Petri dish containing MLGA (CM1031, Oxoid, Basingstoke, UK), inverted, and incubated at 37 °C (±0.2 °C) for 18–24 h. The remaining sediment (~12 g) was used to determine gravimetric water content by drying at 100 °C for 48 h.
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