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.     

Increased scintillation counting of 3H-amino acids bound to transfer RNA.

Approximately a threefold higher tritium scintillation counting efficiency was observed for the ³H-labeled amino acids covalently bound to tRNA, compared to the nonbound ³H-amino acids used as counting efficiency standards.     

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.     

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.     

Liquid scintillation counting of aqueous solutions of potassium salts in a Triton X-100/toluene scintillant

The use of a toluene/Triton X-100 scintillant for counting ¹⁴C in aqueous solutions of potassium salts including potassium hydroxide has been investigated. Suitable conditions for the counting of CO₂ entrapped in potassium hydroxide are described. Quench correction by automatic external standard channels ratio procedures has been found to be of value with optimised conditions of counting.     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

Determining liver retention of transuranium elements in living beagles

Summary A method has been developed whereby the liver content of photon-emitting transuranium elements can be determined in living beagles by a combination of total-body and partial-body counting. Calibration of the system was accomplished through the photon counting of intact dogs and also of the parts of the same animals following autopsy. A determination of the calibration factors for²⁵²Cf,²⁴⁷Cf,²⁴³Cm,²³⁷Pu, and²⁴¹Am has been made. The special case of²⁵²Cf was treated in which a significant fraction of the high energy fission gamma-ray spectrum penetrates the Pb shield employed in partial-body counting, and methods were developed which allow for this effect in the calculation of liver content. Some uniformity of response in the system was evident for all of the emitters which were considered. It is proposed that similar techniques could be applied in the determination of selected organ radioactivity in other species including man.Supported by ERDA Contract E (11-1)-119.     

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.     

Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

The use of a triton X-100-based scintillant for counting fractions from density gradients

The properties of an aqueous scintillation mixture containing butyl-PBD as the sole scintillant and using Triton X-100 as emulsifier are described. This counting mixture, which is considerably cheaper than other published mixtures for aqueous samples, is shown to perform extremely satisfactorily with polysomes and RNA labeled by prior injection of [¹⁴C]orotic acid. When, however, this counting mixture is used with ³H-labeled samples, the density gradient solutes sucrose and cesium chloride are shown to quench the counting of RNA and polysomes but not of toluene or orotic acid.     

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.     

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.     

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%.     

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%.     

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.     

Liquid scintillation counting of 14C- and 3H-labeled samples on solid supports: a general solution of the problem

The data presented demonstrate the potential of surfactant-fortified scintillation cocktails in overcoming many of the problems encountered in the quantitation of radioactivity on solid supports. Using a broad range of representative ionic and polar biochemical compounds, the in vial elution and quantitative recovery of ³H and ¹⁴C-labeled components from a wide assortment of common solid support media has been demonstrated. This methodology in combination with zero elution counting systems and in some circumstances gelled suspension counting should combine to overcome most of the problems associated with determination of isotopic activity on such solid support media.     

Liquid scintillation counting of aqueous samples using Triton-containing scintillants

Markedly unstable count rates were observed using a toluene-Triton (2:1, $\text{v}\text{v}$) scintillant during counting of water-soluble radioactive compounds when < 5% ($\text{v}\text{v}$) water was present, because of the separation of phases. Efficiency correction in these instances could not be made by using ³H₂O as internal standard, because under the same conditions count rates with tritiated water were stable. Increasing water to ≥6% stabilized the count rates. With toluene-Triton (2:1, $\text{v}\text{v}$) scintillant, the water level should preferably be maintained between either 6 and 12 or 18 and 24% for ¹⁴C- and ³H-labeled compounds for counting at 6°C or at ambient temperature (but only between 6 and 12% for ³H counting at room temperature). With a “Tritosol” (Anal. Biochem.63, 555 (1975) modified to contain 35 ml of ethylene glycol, 140 ml of ethanol, 250 ml of Triton X-100, 575 ml of xylene, 3 g of PPO, and ±200 mg of POPOP, water levels of up to 23% were acceptable for ¹⁴C and ³H for counting at room temperature or at 6°C. Within these limitations, with the toluene-Triton or with the modified Tritosol as scintillant, both polar and apolar radioactive compounds exhibited similar efficiencies and gave quench-correction curves, based on the external standard ratio, that were linear for both ¹⁴C and ³H-labeled compounds.     

An improved scintillation cocktail of high-solubilizing power

A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for ³H (47%) and ¹⁴C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

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.