Scintillation fluid preparation

Figure 1.1 Schematic of a representative enzymatic assay. The reaction mixture is prepared (Mix Preparation) and the reaction can be started (Initiation) by the addition of the enzyme. During the reaction (Incubation), samples are removed at intervals labeled h, t2, and r3, and the reaction is stopped (Termination) by inactivating the enzyme. The incubation mixture is fractionated (the illustration shows a traditional chromatographic column), and the product is isolated from the substrate (Separation). In this assay, a radiochemical was used as the substrate and therefore the amount of product that formed is determined by its collection, the addition of scintillation fluid, and the measurement of radioactivity by scintillation counting (cpm Detection). The progress of the reaction is given by the amount of radioactive product recovered (Data Reduction).

D. The gel slices were treated according to B ( 1 or 2 ) or C ( 1 or 2 ). After the radioactive analysis the slices prepared according to B2 or C2 were treated overnight with 0.2 ml Soluene 350 and 0.05 ml bidistilled water. After addition of the original sampels of scintillation fluid the vials were treated as in A. (The Soluene 350 and the water can also be added to the Permablend III Scintillation fluid, but the subsequent swelling of the gel slices will then take much more time.) The Bl and Cl slices had to be rinsed twice in 3 ml toluene and soaked for at least 3 hours in this medium prior to Soluene 350 treatment. This was to ensure that only traces of butyl-PBD would remain. (Butyl-PBD reacts with Soluene 350 and produces... 

Volume of water was the abscissa and weight of NaOH was the ordinate in these diagrams. In the preparation of the samples aliquots of water from O to 3.0 ml in steps of 0.25 were added to series of vials containing lO ml scintillation fluid. Ten such series of vials were prepared from each scintillation fluid and to each series was added a fixed amount of 5% (w/v) aq.NaOH. The amounts used were O, 20 yl, 50 yl,... 

NotesTo avoid problems with alkaline chemiluminescence in freshly prepared counting samples it is advisable to prepare a stock solution of alkaline scintillation fluid (see conclusion )... 

Careful preparation of the isolated fractions for counting is of considerable importance (48,49). Generally, samples are solubilized directly into the scintillation fluid prior to counting. Commercial scintillation cocktails are available which can be used for a wide range of sample types. Samples that cannot be readily solubilized can be counted while suspended in emulsions or gels (48,49). 

We decided at the outset that we would not use a method that would require first dismembering the spore and this stricture forced us to invent the technique of /3-attenuation. The method depends on the expectation that the closer a tritium-labeled material is to the center of the spore, the less likely that the /5-particles will escape the dimensions of the spore and be scored by the surrounding scintillation fluid. It was possible to empirically prove the correctness of this assumption by preparing spores labeled with tritium-marked uracil, diaminopimelate or lysine, added late in the sporulation sequence, to selectively label the core, cortex and coat respectively. The observation of increasing attenuations of 3, 18 and 26 % as one placed the marker at positions nearer the spore center, validated the proceduie.< >... 

Another procedure for dealing with samples insoluble in counting solution is to support them on a medium such as paper strips, filter discs, glass fibre or DEAE cellulose prior to adding them to a counting vial [235-237]. As indicated earlier, use of this has been in chromatography where the spot has been cut out of the paper or scraped from the plate. Although useful for materials insoluble in scintillator fluid, self-absorption for tritiated samples may constitute a major drawback in this technique, just as it does for the suspension methods. It should be evident why the recent developments in in combustion procedures are so important in the problem of sample preparation. 

Tvanty xl H-chitin with 0.5 ml enzyme preparation was incubated at 37 C for one hr before the reaction v s stopped with 0.2 ml 10% TCA. EJ riments involving Zn toxicity contained 0.25 ml solution of ZnSO of 0.0008-0.32 mg/ml. The mixture vas filtered through glass fiber filters and collected in scintillation fluid. Soluble products of the reaction mixture are detected in a Beckman wodel LS3801. 

These two methods produce different release profiles in vitro. Figure 5 demonstrates the release kinetics of BCNU from wafers loaded with 2.5% BCNU pressed from materials produced using these two methods. The wafers containing tritiated BCNU were placed into beakers containing 200-ml aliquots of 0.1 M phosphate buffer, pH 7.4, which were placed in a shaking water bath maintained at 37 C. The shaking rate was 20 cycles/min to avoid mechanical disruption of the wafers. The supernatant fluid was sampled periodically, and the BCNU released was determined by liquid scintillation spectrometry. The BCNU was completely released from the wafers prepared by the trituration method within the first 72 hr, whereas it took just about twice as long for the BCNU to be released from wafers... 

For homogeneous sample counting the radioactive material must be soluble in the organic scintillation solvent (toluene, xylene, dioxane). Unfortunately most inorganic salts, hydrophilic substances, macromolecules (such as proteins, nucleic acid or polysaccharides) or biological tissues (muscle, bone, liver, brain) and body fluids (blood, plasma, urine, spinal fluid) are incompatible with the solubility characteristics of the liquid scintillant. To overcome these problems various useful methods for tissue preparation have been developed such as solubilisation by hydrolysis, wet oxidation, combustion. 

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