Batch incubation experiments

In the absence of any one standardized method, many investigators have used laboratory batch incubation of wetland soils and aquatic sediments and measured ammonium production as a measure of nitrogen mineralization rates (Wang et al., 2001 White and Reddy, 2001) (Figure 8.20). These methods range from simple batch incubation experiments in which ammonium accumulation is measured over time to stable isotope dilution technique in which soil cores or anaerobic soil slurries are injected with N-labeled ammonium nitrogen. This method allows to estimate both gross and net mineralization rates (Blackburn, 1979). 

Batch culture experiments were performed to measure the rate of glucose consumption. Once the seed yeast had been incubated for exactly 48 h, the serum vials were each injected with 0.2 mL of freshly vortexed yeast. The moment of inoculation became time zero (f = 0) for the experiment. Viable cell counts taken at time zero showed the initial cell concentration to be on the order of 100,000 colony-forming units/mL. Samples were taken every 2 h over the next 12 h, except for the first 4 hours, and analyzed for glucose. 

FPU/CBU). (3-Glucosidase was added to avoid end-product inhibition owing to cellobiose accumulation. Batch hydrolysis experiments were conducted at 2% consistency of cellulose in 50 mM acetate buffer, pH 4.8, with 4 mg of tetracycline/100 mL of buffer as antibiotic. The hydrolysis incubation was performed at 45°C on a rotary shaker at 150 rpm. Samples of the supernatants were taken at assigned intervals for sugar analysis. All hydrolysis experiments were performed in duplicate, and the averages were reported. 

The first semi-high-throughput automated system to dispense crystallization trials of less than 1 jl1 was designed in 1990 to deliver batch trials imder oil (Chayen et ah, 1990). The method was named microbatch to define a microscale batch experiment. It was designed to obtain maximum information on the molecule to be crystallized while using minimal amounts of sample. In order to prevent the evaporation of such small volumes, the trials are dispensed and incubated under low density (0.87 g/cm ) paraffin oil (Fig. 3.2). The crystallization drops remain under the oil since the aqueous drops are denser than the paraffin oil. 

The system chosen to conduct CYP inhibition studies should be well characterized. This procedure requires initial time-course experiments and determination of linearity of metabolite formation with the chosen incubation time and enzyme concentration. After these experiments, the kinetic parameters (i.e., Km and Vmax) for each substrate used with six or more concentrations spanning from 1/3 to 3 A), and inhibition potencies (i.e., IC50 or K,) of typical inhibitors should be determined. This characterization does not need to be repeated for each batch or lot of test system. 

A portion of each of the three different batches used for the HPLC analysis shown in Figure 3 was sent to the National Heart, Lung and Blood Institute Sickle Cell Disease Center at Children s Hospital of Philadelphia, Philadelphia, Pennsylvania for determination of the level of antisickling activity. In these experiments, a low concentration (0.05 mg/ml) of Nicosan product was incubated with SS cells for 1 hr instead of the normal 3-4 hrs so that any differences could be seen more clearly. The levels of anti-sickling activity of the 3 different batches were nearly identical (Figure 4). 

Batch culture systems consisted of 125-mL Erlenmeyer flasks containing 50 mL MSM and 100 mg/L alachlor. The flasks were inoculated directly with 0.5 g of soil from simulated chemical spill experiments or with 1 mL aliquots of soil suspensions (1 g soil/10 mL H2O). The flasks were incubated on a rotary shaker at 25°C for up to 4 weeks with periodic sampling for microbial isolation. 

Encapsulated islets secreted insulin into the extracapsule medium (here aMEM with 50 mg/dL glucose 24 h static incubation) at a rate that appeared to be one-third that of control islets (Fig. 20) based on the number of islets added to the encapsulation syringe (typically 100-150) for up to 30 days after encapsulation. Unfortunately the capsules were opaque so that it was impossible to determine the actual number of islets in a batch of capsules until the end of an experiment. According to a vital stain (alcian blue), 70-80% of the islets (after 4 weeks) inside the capsule were intact and viable. However the yield of encapsulated islets was 30% relative to the number added to the syringe. Hence the insulin secretion values, based on the actual number of islets in the capsules, was comparable to that for the control. Encapsulated rat islets also responded to... 

Robustness and ruggedness experiments are used to demonstrate how reproducible a method is when conditions vary. An assay method protocol defines the exact steps to be followed to ensure an accurate and precise result. Unfortunately, slight-to-large deviations occur in the everyday process, which may or may not impact the result. Assay conditions like incubation temperature or exposure to light define the robustness of the assay, whereas changes to the routine, for instance, multiple analysts or different instruments, define the ruggedness. The actual batch size for a routine sample analysis run is frequently overlooked but is often an impactful ruggedness measurement that should be assessed. 

Figure 3.19 Increase with incubation time, in the Koc of imidacloprid in a sandy loam (LH AXXa) and a silt loam (LH A2) as defined by desorption batch experiments. [Reproduced with permission from M. Oi, J. Agr. Food Chem. 47, 327 (1999). Copyright 1999, American Chemical Society.]...

Crystallization. The crystallization procedure is taken from that described by Fujii et For crystallization experiments, the Sulfolobus sp. ferredoxin solution obtained from a preparative Sephadex G-50 gel filtration column (Amersham Pharmacia Biotech) is concentrated by pressure filtration through an Amicon YM3 or YMIO membrane at 4° and made to 5 mg/ml in 0.5 M Tris-maleate-NaOH buffer, pH 5.0, containing 1% 2-methyl-2,4-pentanediol. Crystals suitable for X-ray diffraction analysis are obtained by a batch method performed under aerobic conditions. Fine-powdered ammonium sulfate is slowly added to 300 p.1 of 5 mg/ml protein solution until the turbidity is observed to persist (1.9-2.1 M). The crystallization solution is stored at 37° in an incubator. Dark brown crystals with appropriate dimensions of 0.3 x 0.3 x 0.5 mm are obtained in 3-5 weeks. Fujii etaO reported that reproducibility of the crystallization is enhanced by seeding a drop of the mother liquor containing microcrystals into the crystallization solution just before the crystallization begins. 

In a similar experiment, Proell et al. (39) incubated 18-[ C] trans-9 C18 l in batch cultures of mixed ruminal microorganisms. After 48 h of incubation, enrichment was observed in stearic acid and all trans Cl8 1 isomers having double bond positions between carbons 6 through 16. No conversion of elaidic acid to either stearic acid or any other monoene was observed in the absence of ruminal bacteria, suggesting that the isomerization was enzymatic rather than nonenzymatic double bond migration. Opposite results were reported by Kemp et al. (28) where some cis to trans isomerization was seen in incubations without bacteria. 

The sensor shelf lifetime has been investigated in a series of experiments. Batches with uniformly fabricated sensors were kept in defined conditions in refrigerator at 4 °C, at room temperature of 20 C and in an incubating box at 40 C. At particular time intervals sensors from those batches were tested with a model OPC (trichlorfon) to determine the sensitivity of the sensor response to the inhibitor. Having in mind that the sensors are disposable, the shelf lifetime of the electrodes made by this technology was determined as the period of time witliin which the sensor sensitivity obtained from such a test does not fall outside the deviation range of the intra-batch... 

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