Continuous monitoring of rhizosphere respiration after labelling of plant shoots with 14CO2

The present work describes an original method to follow rate of ¹⁴CO₂ and total CO₂ production from rhizosphere respiration after plant shoots had been pulse-labelled with ¹⁴CO₂. We used a radioactivity detector equipped with a plastic cell for flow detection of beta radiation by solid scintillation counting. The radioactivity detector was coupled with an infrared gas analyser. The flow detection of ¹⁴CO₂ was compared to trapping of ¹⁴CO₂ in NaOH and counting by liquid scintillation. First, we demonstrated that NaOH (1 M) trapped 95% of the CO₂ of a gaseous sample. Then, we determined that the counting efficiency of the radioactivity flow cell was 41% of the activity of gaseous samples as determined by trapping in NaOH (1 M) and by counting by static liquid scintillation. The sensitivity of the ¹⁴CO₂- flow detection was 0.08 Bq mL⁻¹ air and the precision was 2.9% of the activity measured compared to 0.9% for NaOH trapping method. We presented two applications which illustrate the relevance of ¹⁴CO₂-flow detection to investigations using 14C to trace photoassimilates within the plant-soil system. First, we examined the kinetics of 14CO₂ production when concentrated acid is added to NaH¹⁴CO₃. This method is the most commonly used to label photoassimilates with ¹⁴C. Then, we monitored ¹⁴CO₂ activity in rhizosphere respiration of 5-week old maize cultivated in soil and whose shoots had been pulse-labelled with ¹⁴CO₂. We conclude that alkali traps should be used for a cumulative determination of ¹⁴CO₂ because they are cheap and accurate. On the other hand, we demonstrated that the flow detection of ¹⁴CO₂ had a finer temporal resolution and was consequently a relevant tool to study C dynamics in the rhizosphere at a short time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

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.     

Technique for Measuring 14CO2 Uptake by Soil Microorganisms In Situ

Uptake of ¹⁴CO₂ in soils due to algae or sulfur-oxidizing bacteria was examined by incubation of soil samples with gaseous ¹⁴CO₂ and subsequent chemical oxidation of biologically fixed radioactive isotope to ¹⁴CO₂ for detection with a liquid scintillation counting system. The ¹⁴CO₂ was added to the soil in the gas phase so that no alteration of the moisture or ionic strength of the soil occurred. Wet oxidation of radioactive organic matter was carried out in sealed ampoules, and the ¹⁴CO₂ produced was transferred to a phenethylamine-liquid scintillation counting system with a simply constructed apparatus. The technique is inexpensive and efficient and does not require elaborate traps since several possible interfering factors were found to have no harmful effects. Experiments in coal mine regions and in geothermal habitats have demonstrated the ecological applicability of this technique for measurement of CO₂ fixation by sulfur-oxidizing bacteria and soil algae.     

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.     

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.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

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 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.     

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%.     

A method for continuous measurement of export from a leaf

Export of labeled material derived by continuous photosynthesis in ¹⁴CO₂ was monitored with a Geiger-Müller detector positioned next to an exporting leaf blade. Rate of export of labeled material was calculated from the difference between rates of retention and net photosynthesis of labeled carbon for the observed leaf. Given certain conditions, including nearly constant distribution of labeled material among minor veins and various types of cells, count rate data for the source leaf can be converted to rate of export of carbon. Changes in counting efficiency resulting from changes in leaf water status can be corrected for with data from a transducer which measures leaf thickness.     

Interaction of purified DNA with plant protoplasts of different cell cycle stage: The concept of a competent phase for plant cell transformation

Summary The polycation mediated attachment of purified tritiated DNA to plant protoplasts has been measured by quantitative microautoradiography. The automated grain counting technique used, also provides information on the cell cycle stage of individual protoplasts, which circumvents the need to synchronize the plant cell population before preparation of protoplasts. With protoplasts from asynchronously dividing suspension cultures of Nicotiana syhestris (NS-1), S-phase protoplasts appear to be inefficient binders of ³H-DNA, as compared with G₁ or G₂ protoplasts. Protoplasts derived from a tumour line of Crepis capillaris (CAPT) exhibit ³H-DNA binding at all cell cycle phases, but Sphase protoplasts appear to be preferential binders. These differences are discussed with reference to cell cycle kinetics, membrane charge variation and the possibility of increasing the efficiency of genetic transformation of higher plant cells in culture.     

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.     

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.     

Non-photochemical fluorescence quenching in photosystem II antenna complexes by the reaction center cation radical

In direct experiments, rate constants of photochemical (k_P) and non-photochemical (k_P⁺) fluorescence quenching were determined in membrane fragments of photosystem II (PSII), in oxygen-evolving PSII core particles, as well as in core particles deprived of the oxygen-evolving complex. For this purpose, a new approach to the pulse fluorometry method was implemented. In the “dark” reaction center (RC) state, antenna fluorescence decay kinetics were measured under lowintensity excitation (532 nm, pulse repetition rate 1 Hz), and the emission was registered by a streak camera. To create a “closed” [P680⁺Q_A^–] RC state, a high-intensity pre-excitation pulse (pump pulse, 532 nm) of the sample was used. The time advance of the pump pulse against the measuring pulse was 8 ns. In this experimental configuration, under the pump pulse, the [P680⁺Q_A^–] state was formed in RC, whereupon antenna fluorescence kinetics was measured using a weak testing picosecond pulsed excitation light applied to the sample 8 ns after the pump pulse. The data were fitted by a two-exponential approximation. Efficiency of antenna fluorescence quenching by the photoactive RC pigment in its oxidized (P680⁺) state was found to be ～1.5 times higher than that of the neutral (P680) RC state. To verify the data obtained with a streak camera, control measurements of PSII complex fluorescence decay kinetics by the single-photon counting technique were carried out. The results support the conclusions drawn from the measurements registered with the streak camera. In this case, the fitting of fluorescence kinetics was performed in three-exponential approximation, using the value of τ₁ obtained by analyzing data registered by the streak camera. An additional third component obtained by modeling the data of single photon counting describes the P680⁺Pheo^– charge recombination. Thus, for the first time the ratio of k_P⁺/k_P = 1.5 was determined in a direct experiment. The mechanisms of higher efficiency for non-photochemical antenna fluorescence quenching by RC cation radical in comparison to that of photochemical quenching are discussed.     

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.