Three periderm disks from each tuber (1.5 mm thick and 1 cm diameter) were cut using a cork borer from the stem, the middle and the bud end of each tuber periderm. The disks were snap frozen in liquid nitrogen and ground to powder using a mortar and pestle. Samples were extracted with 5 mL of N, N-dimethylformamide for chlorophyll analysis. All samples were stored at 4 °C in the dark for 24 hours. After centrifugation for 15 min at 2500 × g, the absorbance was measured with a spectrophotometer (Thermo Scientific Spectronic 200E) at 647 and 664 nm. Chlorophyll concentrations were determined according to [14 ], using the below equation and expressed in mg L-1 fresh weight:
Spectronic 200e
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Spectronic 200E is a spectrophotometer designed for basic UV-Vis absorbance measurements. It features a wavelength range of 190-1100 nm and has a bandwidth of 2 nm. The instrument can be used to determine the concentration of samples through absorbance readings.
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Lab products found in correlation
7 protocols using spectronic 200e
1
Periderm Chlorophyll Quantification Protocol
2
Total Protein Extraction from Cotton Fibers
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For total protein extraction from cotton fibers by following the method with some modifications reported by Dure & Chlan19 (link). Cotton fibers were carefully removed from 16DPA ovules. The dried weight of fiber was taken in a 1.5 mL tube with an addition of 5 parts of insoluble PVPP (Polyclar, AT) to each part of the dry weight of fiber (w/w); 15 mL of extraction buffer (50 mM Tris–HCl, pH 8 to 6; 2% 2-mercaptoethanol; 2% SDS) for each 100 mg of dried sample was added and homogenized. After homogenization, the mixture was incubated at 100 °C in a dry heat bath for 5 min and subjected to centrifugation to separate pellet, cell debris, and PVPP. The supernatant was shifted to the new 1.5 mL tubes, and 10 volumes of ice-chilled acetone were added and placed overnight at -20 °C for protein precipitation. The precipitated protein pellet was obtained through centrifugation of the sample mixture. The acetone supernatant obtained was discarded and re-suspended in PBS. The F-actin of the total protein was stained using FITC-phalloidin molecular probes20 (link). The Fluorescence intensity of FITC-phalloidin-stained F-actin was recorded by spectrophotometer (Thermo Scientific Spectronic 200E) at 530 nm wavelength.
3
Bacterial Growth and Enumeration
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Both S. aureus (Newman strain) and S. mitis (NCTC 12261) were grown in tryptic soy broth (TSB) medium overnight at 37 °C and 5% CO2 atmosphere in centrifuge tubes. The overnight culture was diluted 10 times and left to grow again in the same conditions for 2 h. After that, samples were centrifuged at 5000 rpm (2912 rcf) at 21 °C to obtain a pellet (Thermo Scientific™ Heraeus Multifuge X3FR Centrifuge, Waltham, MA). The supernatant was discarded, and the pellet was resuspended in PBS (OD600 = 0.6, Thermo Scientific™ Spectronic 200E, Waltham, MA) before use in the experiments. The average colony-forming unit (CFU) was measured by culturing bacteria overnight on agar plates.
4
Salmonella Enteritidis and Clostridium perfringens Inoculation in Chicks
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For the in vivo experiment, Salmonella enterica serovar Enteritidis (SE ) was prepared and administered to all chicks on day of hatch as described by Shivaramaiah and co-authors (2011 (link)). Briefly, approximate concentration of SE was quantified spectrophotometrically (Spectronic 200E, Thermo Scientific, Madison, WI), followed by serial dilutions in sterile saline to reach an approximate concentration of 104 CFU/chick. Exact concentration was retrospectively determined by serial dilution plating on TSA and determined to be 1.5 × 104 CFU/chick. To prepare CP inoculum, a single frozen aliquot of wild-type CP TXAM0108 was inoculated into individual tubes with TSB containing 0.25% sodium thioglycolate (VWR, Radnor, PA), and incubated under anaerobic conditions at 37°C for up to 24 h. Post-incubation, cells were washed 3 times in sterile saline by centrifugation at 1,800 × g for 15 min. The approximate concentration of CP was quantified spectrophotometrically, followed by serial dilutions in sterile saline for the desired inoculum of 107 CFU/chick. Exact concentration was determined retrospectively by serial dilution plating on TSA with sodium thioglycolate and determined to be 3.0 × 107 CFU/chick.
5
Genetically Engineered E. coli Strains
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E. coli strains BW25113 7636 (later referred as E. coli-WT), JW4277-1 11065 (ΔfimA, E. coli–ΔfimA), and JW4283-3 11068 (ΔfimH, E. coli–ΔfimH) (The Coli Genetic Stock Center, Yale University, New Haven, CT, USA) were grown in tryptic soy broth (TSB, Sigma-Aldrich, Oslo, Norway) medium overnight at 37 °C in centrifuge tubes and 5% CO2 atmosphere. E. coli–ΔfimA and E. coli–ΔfimH are mutant strains that lack the fimA and fimH genes, respectively. Such genes play a role in the production of fimbriae, and their parent is the E. coli–WT strain that has type 1 fimbriae [42 (link)]. The overnight culture was diluted 10 times the morning after and left to grow again in the same conditions until optical density reached OD600 = 1 (Thermo Scientific Spectronic 200E, Waltham, MA, USA). After that, samples were centrifuged at 5000 rpm at 21 °C to obtain a pellet. Medium residues were discarded and exchanged for PBS in order to obtain the same OD600 = 1. Afterwards, the pellet, in phosphate buffered saline (PBS, Lonza, Verviers, Belgium), was shaken, and the obtained solution was used later for injection. Average colony-forming units (CFUs) were measured by culturing bacteria overnight on TSB agar plates (overnight at 37 °C and 5% CO2 atmosphere). There was no observed significant difference in the number of colony forming units (CFU) at OD600 = 1 between the strains investigated.
6
Preparing Salmonella Enteritidis for Poult Study
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Salmonella Enteritidis was prepared as described by Shivaramaiah and co-authors (2011) (link). The approximate concentration of Salmonella Enteritidis was quantified spectrophotometrically (Spectronic 200E, Thermo Scientific, Madison, WI), followed by serial dilutions in sterile saline to reach a concentration of approximately 104 CFU/poult. Exact concentration was measured retrospectively by serial dilution plating on tryptic soy agar and determined to be 6 × 103 CFU/poult.
7
Quantifying Periderm Chlorophyll Content
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Three periderm disks from each tuber (1.5 mm thick and 1 cm diameter) were cut using a cork borer from the stem, the middle and the bud end of each tuber periderm. The disks were snap frozen in liquid nitrogen and ground to powder using a mortar and pestle. Samples were extracted with 5 mL of N, N-dimethylformamide for chlorophyll analysis. All samples were stored at 4 °C in the dark for 24 hours. After centrifugation for 15 min at 2500 × g, the absorbance was measured with a spectrophotometer (Thermo Scientific Spectronic 200E) at 647 and 664 nm. Chlorophyll concentrations was determined according to [14] , using the below equation and expressed in mg L -1 fresh weight:
Total chlorophyll = 17.67 (A647) + 7.12 (A664).
Total chlorophyll = 17.67 (A647) + 7.12 (A664).
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