Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays

A several-fold greater counting efficiency is observed for protein labeled with [³H]leucine than for free [³H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for ¹⁴C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.     

Use of Nitrifier Activity Measurements To Estimate the Efficiency of Viable Nitrifier Counts in Soils and Sediments

A procedure for estimating the efficiency of the most-probable number (MPN) technique for counting ammonium-oxidizing bacteria was tested on sediments and soils collected from Delaware Inlet, Nelson, New Zealand. The procedure involved estimating the nitrifier populations required to produce observed activities and comparing these estimates with the MPN-countable populations. MPN counts ranged between 0.15 × 10³ to 3.0 × 10³ cells g⁻¹ in sediments and between 4.4 × 10³ to 19 × 10³ cells g⁻¹ in soils. These counts were only 0.1 to 5.0% of the estimated populations that would be required to produce the observed activity. Similar efficiency calculations were made for data already in the literature, and these calculations gave much higher percentages. Thus, we concluded that for the soils and sediments we studied, the MPN counting technique greatly underestimated the populations present and that the efficiency calculation could be used as a counting efficiency index.     

The effect of sample composition and vial type on Ĉerenkov counting in a liquid scintillation counter

The effect of sample vial type and sample composition on the ?erenkov count rate detected from ³²P and ³⁶Cl was studied using a liquid scintillation counter. When counting was done in the noncoincident mode, glass vials allowed higher counting efficiency than plastic vials. In the coincident mode light scattering caused by polyethylene and polyproplyene vials allowed higher counting efficiency than glass vials. Highest coincident counting efficiency was from plastic minivials in a glass carrier vial. Increased solute concentration in samples caused increased counting efficiency due to changes in the refractive index of the solution. This can cause significant counting efficiency changes with no sample channel ratio change in density gradient fractions. The use of wavelength shifters is shown to be inappropriate when the sample pH varies, as this can change the fluorescent properties of the shifters and thereby the observed count rate.     

A comparison of instrument performance for six liquid scintillation counters

The counting characteristics of six liquid scintillation counters are compared. Each instrument was tested to determine the figure of merit ($\text{E}{}^2\text{B}$), the change in percentage counting efficiency and external standard ratio over sample volumes from 1 to 20 ml, the reproducibility of chemical quench curves over sample volumes from 5 to 20 ml, and the effects of increasing sample activity on the actual and calculated counting efficiency. Tests were performed using both ³H and ¹⁴C. The results show that differences in counting efficiency are more important than maximum sensitivity ($\text{E}{}^2\text{B}$). All instruments demonstrated the same response to changes in sample volume. The external standard ratio varied with sample volume in all instruments except one. Quench curves were essentially volume independent, with two exceptions. This study demonstrates that periodic performance tests must be conducted to assure that these instruments are operating in an efficient and reproducible manner.     

Tritosol: A new scintillation cocktail based on Triton X-100

A scintillation cocktail consisting of 3.0 g PPO, 257 ml Triton X-100, 106 ml ethanol, 37 ml ethylene glycol, and 600 ml xylene is described. A linear relationship between counting efficiency and the external standard ratio could be demonstrated over a wide range of quenching. The counting efficiencies (unquenched) for³H are about 47%, for¹⁴C about 87%, and for⁴⁵Ca about 80%.     

Counting of p32 in plant tissues using the Cerenkov effect

Summary A procedure for counting p³² in plant tissues is presented. The method, based on the use of Cerenkov radiation, involves practically no sample preparation. Plant tissue are placed into vials containing water or hexane and counted with a liquid scintillation counter. Counts obtained, using this procedure were found to be linearily related to that obtained with a G.M. tube. The counting efficiency was, however, higher with the proposed method. The use of hexane is advantageous if leakage of p³² from the tissue is possible, or when higher counting efficiency is desireable. The use of different liquids may also enable a discriminative count of different beta emitters. As suggested recently⁸ use of wavelength shifter may further increase efficiency of counting Cerenkov radiation.     

The determination of the radioactivity of 14C samples adsorbed on glass vials by direct liquid scintillation counting

A method for obtaining the true activity and counting efficiency of a ¹⁴C sample partially or completely adsorbed on the walls of a counting vial by liquid scintillation counting is presented.     

Liquid scintillation counting of polyacrylamide gels crosslinked with N,N′-methylene-bis-acrylamide and N,N′-diallyltartardiamide

Tritium (³H)-labeled material within polyacrylamide gel slices is commonly quantified by four general liquid scintillation counting methods: combustion, gel solubilization, selective solubilization of modified crosslinked gels, and elution. Of these four methods examined in this study, only combustion ensured complete recovery of ³H label; however, a less expensive and more convenient elution method yielding both a high recovery and cocktail counting efficiency was the use of Soluene-350 with 0.55% Permablend III in toluene. This particular solubilizer-cocktail system eliminates almost all chemiluminescence as does combustion.     

High-efficiency liquid-scintillation counting of 14C-labelled material in aqueous solution and determination of specific activity of labelled proteins

1. Inexpensive scintillation mixtures are described which enable the detection of as little as 40μμc of ¹⁴C in aqueous solution with an efficiency of counting of over 80%. 2. A rapid method for the counting of alkaline, acidic and neutral aqueous solutions of up to 1ml. volume is described. Ethanol or 2-ethoxyethanol is used as blending agent. 3. The scintillation counting of alkaline solutions is applied to the accurate determination of the specific activity of ¹⁴C-labelled proteins from plant tissues. 4. Attention has been paid to the importance of a standardized washing procedure for the removal of all traces of radioactive material from glassware.     

Counting efficiency in the radioassay of 3H- and 14C-labeled lipids adsorbed on silica gel by liquid scintillation

The effect of silica gel on the recovery of radioactivity from ¹⁴C- and ³H-labeled lipids by liquid scintillation is analyzed. In the most unfavorable ease, when counting with a toluenefluor solution directly added to the vials containing the adserbed lipid, drastic reductions in the counting efficiency were found, which depended on the amount of silica gel, sample activity, and chemical nature of the lipid. For certain lipids like phosphatidylcholine, these effects were not completely overcome even by introducing water and detergents in the counting system. This paper intends to draw attention to the fact that these factors should be especially taken into account when comparing different lipids from thin-layer chromatograms.     

A convenient, efficient, and inexpensive method for scintillation counting of polyacrylamide gel slices after electrophoresis

A method is described for analyzing the radioactivity of ³H-labeled RNA after separation by gel electrophoresis. The gels were soaked in 10% acetic acid and were sliced. The gel slices were dehydrated in alcohol, then saturated with toluene, and finally permeated with a toluene-based scintillation fluid containing butyl-PBD. The radioactivity of RNA was then analyzed in situ in the gel slices using a liquid scintillator. The counting efficiency of this technique is high, about 58%. This is even slightly better than the counting efficiency attained after solubilization of the RNA in Soluene 350 (about 55%). With the same fluor, Permablend III, the counting efficiencies of the two techniques are alike.     

Liquid scintillation counting of radioactive monomeric and polymeric carbohydrates on paper chromatograms

A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol.The water elution-Aquasol procedure allows water-soluble substances (¹⁴C- and ³H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.     

A water-miscible, nonhazardous liquid scintillation cocktail

The optimal way to count aqueous samples by liquid scintillation counting is in a homogeneous solution. Technical limitations have previously made this difficult. Triton X-100 is a water-miscible liquid scintillant which counts ¹⁴C with 80% efficiency and ³H with 17% efficiency. It has a high flash point (over 300°F), is nonvolatile, and does not cause swelling or leaching when used in polyethylene vials. Liquid-scintillation counting cocktail using Triton X-100 as the sole scintillant (i.e., no toluene or xylene) does not have to be disposed of as a hazardous waste. The large aqueous sample capacity of a miscible cocktail, its safety, and ease of disposal make its use highly attractive for many applications.     

Improved Double-Vial Radiorespirometric Technique for Mineralization of 14C-Labeled Substrates

New procedures which simplify sample preparation and improve counting efficiency were developed for double-vial radiorespirometry. Under certain conditions, efficiency of fluor-NaOH-impregnated wicks was not stable, but was adversely affected by water vapor, NaOH concentration, and CO₂ loading. Glass fiber wick material treated with a methanolic fluor-NaOH solution showed improved ¹⁴C counting efficiency (58%) compared with previous methods.     

Variable self-absorption of (C14)glycogen

With this paper we hope to warn other investigators of the difficulty in counting [C¹⁴]glycogen in liquid scintillators. The pulse height shift encountered makes it impossible to determine the counting efficiency in the C¹⁴-channel by use of external or internal standards. The efficiency is variable from one isolate of glycogen to another and the use of standards to determine efficiencies can give errors up to 50% of the value found with hydrolyzed glycogen. Hydrolysis of glycogen obviates this problem.     

Čerenkov Counting of 24Na in Suspension Cultures — an Economical Use of Scintillation Apparatus for Studying Ion Exchange

The feasibility of using ?erenkov radiation to estimate ²⁴Na in the study of ion exchange in cell suspension cultures of Acerpseudoplatanus has been investigated. Some practical procedures for preparing samples and counting have been proposed according to the particular need of the experiment and the desired degree of counting efficiency.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.     

The role of light in soybean seed filling metabolism

Soybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐¹³C₆]‐glucose, [U‐¹³C₅]‐glutamine, the combination of [U‐¹⁴C₁₂]‐sucrose + [U‐¹⁴C₆]‐glucose + [U‐¹⁴C₅]‐glutamine + [U‐¹⁴C₄]‐asparagine, or ¹⁴CO₂ labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO₂, O₂) were established by a combination of gas chromatography‐mass spectrometry, ¹³C‐ and ¹H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.     

Use of a simple gamma flow cell to monitor chromatography column effluents containing 75Se.

Scintillation flow cells provide a convenient means of monitoring column effluents for radioactively labeled compounds. The use of flow cells to monitor effluents containing ³H, ¹⁴C, and other β-emitting isotopes is well established (1) and such cells are readily available as accessories for use with liquid scintillation counters. β-Flow cells have occasionally been used to monitor γ emitters [e.g., Martin et al. (2)] but the counting efficiency is extremely low even for weak γ emitters such as ⁷⁵Se, due to the limited mass of scintillant available to absorb the radiation.Spencer et al. (3) described a simple γ flow cell consisting of a coiled polyethylene tube inserted in to the well crystal of a γ counter, but no information on counting efficiency was given. Although spiral plastic γ cells have been manufactured commercially (e.g., Nuclear Enterprises Ltd., NE702¹) they are not offered as a standard accessory for most common makes of γ counter and do not appear to be readily available from commercial sources.This communication describes a simple, low cost γ flow cell constructed for use in a study on selenium transport in higher plants, which involved the separation of ⁷⁵Se-labeled constituents in a large number of samples of xylem sap.     

Extraction of aluminum from fly-ash by commercial and microbiologically-produced citric acid

Summary Aluminum extraction from two aluminum-rich fly-ashes by commercial and microbiologically-produced citric acids was tested. Up to 12% Al₂O₃ of the total was extracted by a 21 hrs¹ shaking treatment at 60° C. Extraction efficiency is considerably affected by extracting acid concentration and extraction temperature. The extraction efficiency of microbiologically-produced citric acids was only slightly lower than that of commercial citric acid of equal molarity.