Preparation of 14C-labeled algal samples for liquid scintillation counting

Four commonly used techniques for preparation of ¹⁴C-labeled algal samples on membranes for liquid scintillation counting were compared and a simple technique for apparent net assimilation measurement from aqueous samples was introduced. All four techniques yielded similar radioactivities from the test cultures and are thus suitable for measurements of ¹⁴C algal samples. The possibly carcinogenic solvent dioxane was not necessary with PCS scintillation cocktail for dissolving radioactivity from algae on filters.     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Flow scintillation counting of 14C-labeled microalgal photosynthetic pigments

Photopigment radiolabcling, a useful method for measuring thein situ carbon-specific growth rates of microalgae, is basedon the determination of synthesis rates of chemosystematic (i.e.specific for microalgal phylogenetic groups) chlorophylls andcarotenoids using photosynthetically assimilated ¹⁴C as a radiotracer.The reliability of this method depends on accurate measurementsof the ¹⁴C-specific activity of individual photopigments. Typically,photopigments are separated by high-performance liquid chromatography(HPLC) with fraction collection of individual peaks, followedby further purification and standard scintillation counting.To simplify analyses, we evaluated in-line flow scintillationcounting to determine its applicability and reliability formeasuring the activity of radio-labeled photopigments. Incubationswere conducted using both pure cultures and natural phyto-planktonsamples. The radiochemical purity of photopigments was determinedby extract acidification (10% HC1) to transform chlorophyllsinto degradation products. Purity was also checked by comparingabsorbance spectra with purified standards. Although ¹⁴C-labeledcolorless compounds are a common feature in radiograms, thesecompounds do not co-elute with photopigments using our HPLCprotocol. Flow scintillation counting, coupled with a highlyselective HPLC protocol, provides an efficient, reliable andfeasible alternative to fraction collection/repurification methodsfor measuring the ¹⁴C-specific activity of microalgal photosyntheticpigments.     

Bleaching of chlorophylls in alcohol extracts with benzoyl peroxide for liquid scintillation counting of C-labeled compounds

Benzoyl peroxide in toluene was used to bleach chlorophylls in alcohol extracts from plants fed with ¹⁴CO₂ for improved direct measurement by liquid seintillation counting. The 0.5% benzoyl peroxide was better than the saturated solution due to less color quenching. Increased counts occurred with all five common scintillation systems when the leaf extracts were bleached with the 0.5% benzoyl peroxide. However, the relatively colorless root extracts of ¹⁴C-labeled photosynthates in alcohol did not require the addition of benzoyl peroxide for decolorization. The addition of 0.5 ml of benzoyl peroxide solution (0.5%) to 0.5 ml of a solution of 10 ¹⁴C-labeled compounds did not induce the degradation of these compounds. In contrast, sodium hypochlorite at an equal concentration caused considerable losses of radioactivity, with an overall average of 30% reduction of the 10 ¹⁴C-labeled compounds examined.     

An improved method for the liquid scintillation counting of 14CO2

A method has been described which permits the determination of the specific activity of ¹⁴CO₂ with an accuracy limited only by the accuracy of the liquid scintillation counter. Its principal idea is the selective conversion of CO₂ to nonvolatile organic compounds, such as benzoic acid. While this method is applicable quite generally it has been applied with particular success in connection with the Schmidt degradation of samples as small as 0.2 mmole where contamination of the evolved CO₂ is serious due to the necessarily large excess of reactants.     

Scintillation counting of 14C-labeled soluble and insoluble compounds in plant tissue

A method is described for the liquid scintillation counting of 14C in plant tissues. Samples are fixed, in the scintillation vial, in a solution of ethanol and acetic acid (3:1) and decolorized with commercial bleach before the addition of scintillation liquid. The method was compared to other techniques of tissue oxidation or digestion and found to be equally effective at least with thin tissue samples. The technique is simple, rapid, and inexpensive and does not result in loss of 14C.     

Triple-label beta liquid scintillation counting.

The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.     

Liquid scintillation counting of 14C-labeled hemin and its application to the biosynthesis of heme in bone marrow

A rapid procedure is described for the liquid scintillation counting of ¹⁴C-labeled hemin isolated after incubation of bone marrow with radioactively labeled glycine-2-¹⁴C. The method has been applied for studies on the biosynthesis of heme in bone marrow of several animal species.