Extraction Of Adenosine Triphosphate Research Articles

The critical adenosine triphosphate (ATP) concentration in treated ballast water

Measuring Adenosine triphosphate (ATP) provides a proxy to check compliance with IMO's ballast water D-2 standard: <10 cells mL−1 in the 10–50 μm size class. Measured with standard boiling techniques the ATP concentration in aquatic eukaryotic microorganisms is 0.6 mol m−3. Model calculations with 10–50 μm spherical organisms show their ATP content is 0.2 to 20 pg cell−1, in line with several cell lysis data. However, at 10 cells mL−1, these ATP contents lead to a maximum of only 2 to 200 pg ATP mL−1, at least 7.5× below a D-2 test kit 1500 pg ATP mL−1 upper limit. Different cell shape and ATP extraction scenarios to reach 1500 pg ATP mL−1 are discussed but remain improbable. Because cell lysis data are inconclusive, and a novel phosphoric acid-benzalkonium chloride method indicates up to 3× higher ATP concentrations, an independent test kit validation and a comparison of all three techniques are recommended.

The Contamination Degree of Characteristic Fungi in Jet Fuel Detected by ATP Bioluminescence

Adenosine Triphosphate (ATP) lysate of the characteristic fungi in jet fuel was screened and three fluorescein-luciferase systems were compared in the paper. The correlation between ATP bioluminescence and traditional plate count for measuring the number of microorganism was also investigated and then a method of detecting the contamination degree of the characteristic fungi of jet fuel by ATP bioluminescence were initially established. The results show that the effect of ATP extraction taking surfactant Benzyldimethylhexadecylammonium chloride (BAC) as the microbial lysate was optimal and the optimal concentration and the action time were 0.15% and 30s respectively; The fluorescein-luciferase system after screening has a good detection limit, up to 10-15mol ATP; using ATP bioluminescence and traditional plate method to measure the microbial quantity, they have good correlation. Utilizing ATP bioluminescence to detect the contamination degree of the characteristic fungi in jet fuel can shorten the test time to 10min, which is suitable for rapid detection and has good application prospects.

Direct measurement of ATP in seawater and application of ATP to monitor bacterial growth potential in SWRO pre-treatment systems

The use of adenosine triphosphate (ATP) to monitor bacterial growth potential of seawater is currently not possible as ATP cannot be accurately measured at low concentration in seawater using commercially available luciferase-based ATP detection. The limitation is due to interference of salt with the luciferin–luciferase reaction, which inhibits light production. This research demonstrates that new reagents developed for (i) ATP extraction from microbial cells and (ii) ATP detection in seawater are able to reliably detect Microbial ATP as low as 0.3 ng L–1 in seawater. The luminescence signal of the new detection reagent is significantly higher (>20 times) than the luminescence signal of the freshwater reagent, when applied in seawater. ATP can now be used to monitor bacterial growth potential (BGP) through pre-treatment trains of seawater reverse osmosis (SWRO) plants. The level of detection of the new BGP test is significantly lower than the estimated threshold value required to prevent biofouling in SWRO systems. The new reagents have been used to monitor Microbial ATP in coastal North Sea water. Moreover, Microbial ATP has been applied to monitor the bacterial growth potential (using indigenous bacteria) through the pre-treatment train of an SWRO desalination plant. A significant reduction (>55%) of the bacterial growth potential was found through the dual media filtration with 4.5 mg-Fe(III) L–1 coagulant. Overall, the new reagents can detect low Microbial ATP concentrations in seawater and can be used to monitor bacterial growth potential in seawater desalination plants.

Improvements of ATP Assay as a Substitute for the CFU Method in Estimating Viable Cell Count for BCG/rBCG Vaccine Preparations

Bacillus Calmette–Guerin (BCG) and recombinant BCG (rBCG) vaccines can be genetically traced back to a live attenuated strain of Mycobacterium bovis. As organism viability is essential for the stimulation of a protective immune response, monitoring the count of viable organisms is an integral part of vaccine quality control. The colonyforming unit (CFU) test has been the conventional assay for determining BCG viability, and is a widely accepted surrogate for BCG potency. CFU analysis, however, is problematic and time consuming. The slowness and high variability of CFU test results are the main driving forces for manufacturers and control laboratories to look for a rapid, more reproducible alternative viable count assay. A modified adenosine triphosphate (ATP) luminescence assay was developed by Statens Serum Institut and was promoted by the WHO as an alternative viable count assay. However, certain conditions during the processes of sample preparation and ATP extraction have to be established before the ATP assay can meet the requirements of robustness and reproducibility. This study is focused on identifying the conditions necessary for a reliable process of ATP analysis for BCG/rBCG preparations. Using our improved ATP assay protocol, we demonstrated that the correlation coefficient between CFU count and ATP concentration of BCG/rBCG vaccines was high (R2=0.83 for accelerated stability samples and R2>0.97 for all other preparations). The ATP luminescence assay is a rapid, sensitive, reliable, strain-non-specific method in quantification of the viability of live attenuated mycobacterial vaccine preparations.

ATPを指標とした医療現場における洗浄プロセス評価

We have reported the qualification result of Adenosine Tri-phosphate (ATP) extraction as an alternative cleaning process assessment method described in Guideline for Sterilization Assurance in Clinical Setting published by Japanese Society of Medical Instrumentation (JSMI) in 2010 at JSMI Annual Conference 2012. This study result indicated the possibility of that ATP extraction method is practical in clinical setting and can be an alternate to protein mass measurement because of the evaluation result regarding the correlation study result between ATP and protein measurements. In this article, additional study result of cleaning process assessment is reported using clinically-used surgical instruments and artificial soil devices at clinical site. ATP extraction and conventional ATP swabbing as well as protein extraction was performed after two different cleaning processes to evaluate the effectiveness of these cleaning processes for determining which cleaning process to take. It is observed that ATP swabbing is not appropriate for surgical instrument having such narrow lumen and/or space where swab cannot access to, because high ATP value was detected by extraction method from a surgical instrument after sampling with swab for ATP measurement. ATP measurement result from standardized artificial soil devices after one cleaning process was from 66 to 478 relative light unit (RLU) while ATP measurement result after another cleaning process was from 30 to 73 RLU (p value = 0.025). However, such statistical analysis was not achieved for protein measurement as detected protein level was under limitation for all protein measurements. ATP extract method is practical cleaning process assessment method at clinical setting, and this method has quantitative capability as well as excellent sensitivity compared to protein measurement method. In addition to these advantages, this extraction method can be applied to more devices which have physically-complex design and lumen devices compared to conventional swabbing method.

An integrated analytical approach for assessing the biological status of the soil microbial community

An integrated multicriteria analytical procedure for rapid, cost-effective characterisation of the biological status of soil bacterial community was developped. Commercially-available, light emission-based bioassays were selected for measuring cell density, activity, and diversity. All but Terminal Restriction Fragment Length Polymorphism (T-RFLP) were designed for multiwell-plate formats and high-throughput screening potential. Adenosine Tri Phosphate (ATP) was measured using a bioluminescence assay. Dehydrogenase activity (DHA) was measured on growing cells. Kinetic measurements of the formation of a coloured formazan derivative was used after nutrient broth addition to estimate initial cell concentrations by reference to Escherichia coli added as internal standard. Compared to conventional ATP and DHA determinations in soils, the procedures described here do not require extraction of ATP or formazan derivative from the soil matrix. Metabolic diversity was characterised using the Biolog™ system. T-RFLP was chosen for assessing bacterial community structure. The bioassays were performed on microbial preparations obtained after either direct dilution of soil suspensions or prior density-gradient separation of microbial cells from the soil matrix. Dilution maintains the original structure of native dominant microbial communities. Density-gradient separation of microbial cells is highly selective, drastically modifying metabolic (CLP Profiles) and species (T-RFLP patterns) diversity, as well as activity parameters.

An ATP-based method for monitoring the microbiological drinking water quality in a distribution network

The titration of adenosine triphosphate (ATP) by bioluminescence permits rapid evaluation of the quantity of viable micro-organisms present in a water sample. During two sampling campaigns, Société Anonyme de Gestion des Eaux de Paris (SAGEP) tested a new extraction and titration system of bacterial ATP in the Paris drinking water distribution network. As far as the entire set of results of analyses of water in the network is concerned there is a linear relationship between log [ATP] and log(HPC-R2A/ml). Furthermore, as regards the drinking water originating from treatment of surface waters, some of the results obtained indicate a slight change as regards the Paris network in the microbiological quality. This is certainly linked to the distance travelled from the production location as well as to a reservoir effect observed on a site. Conversely, no change is apparent with regard to waters of underground origin. Lastly, despite changes in temperature and chlorine residual, no significant influence has been observed, essentially because of the very low density of culturable bacteria.

Effect of chloroform on the extracting efficiency of adenosine tri-phosphate (ATP) from the cells of Mycobacterium leprae

Effect of chloroform on the extraction of ATP from the cells of Mycobacterium leprae was investigated. The results demonstrate that the yields of ATP from the resting cells of M. leprae were much more higher when 100mM Tris-EDTA was added to the procedure than that of the routine method. The amounts of ATP extracted from the cells of M. leprae incubated either in 10% calf serum-buffer (pH 7) or 10% calf serum-2% glycerin-Dubos medium at 30 degrees C gradually increased with time of incubation when chloroform and 100mM-Tris-EDTA were used for extraction of ATP, whereas no increase and periodical decrease of ATP were observed when Tris-EDTA only without chloroform was used in the procedure of ATP extraction. Therefore, it is noted that the use of chloroform and 100mM-Tris EDTA is indispensable for extraction of ATP from the cells of M. leprae.

Influence of paraquat on the extraction of adenosine triphosphate from soil by trichloracetic acid

Il est possible de mesurer l'ATP dans les sols en utilisant un reactif d'extraction contenant seulement de l'acide trichloracetique et des phosphates. Cependant, l'inclusion de paraquat dans le reactif d'extraction ameliore la precision des mesures

The determination of fungal biomass using adenosine triphosphate

Growth of Trichoderma reesei in batch culture with glucose as the carbon source was followed by measuring culture absorbance, culture dry weight, mycelial protein, and adenosine triphosphate (ATP) content of the biomass. All methods except extraction of mycelial protein gave measurements which resulted in similar specific growth rates. During exponential growth the ratio of ATP per unit weight was 6.8 μmol ATP/g dry wt. During autolysis the half-life of the ATP content of the biomass was much shorter than the half-life of mycelial protein or culture dry weight. The addition of holocellulose to the culture did not affect the efficiency of the ATP extraction and detection procedures employed, and therefore ATP measurements can be utilized to estimate fungal biomass in cellulose fermentations. However, the presence of a clay caused a considerable decrease in the amount of ATP recovered from fungal biomass.

Adenosine Triphosphate (ATP) Determination of Green Leaves by Heat Treatment Extraction in Higher Plants

The effects of leaf disc size, extraction solvents and the time of heating and cooling on the ATP extraction from the green leaves as well as the optimal MgSO4 concentration and pH on the luciferin-luciferase enzyme reaction system with ATP were investigated. Leaf discs were prepared by using the leaf disc-punch from the fully expanded top-leaf blade of rice (Oryza sativa L.) and soybean (Glycine max Merr, ) plants grown in the glass house or in the field in pot. These discs with buffer solution or distilled water (D.W.) were dipped in the boiling water for several minutes and then cooled for several hours with mild shaking under the cold condition (3°C) to extract ATP from leaves as a disc. ATP in the extract was determined from the luminescence intensity induced by the enzyme reaction between ATP in the extract and luciferin-luciferase in the FLE-50 (Sigma Chem. Co.). Luminescence intensity was measured by ATP photometer (Type 2000, SAI Tech. Co. USA). Following results were obtained: 1. In this experiment, 15 ml of 13.33 mM MgSO4 were added to FLE-50, which contained 6.67 mM MgSO4 as a kit, to give 20 mM MgSO4 as a optimal concentration for enzyme reaction (Fig. 1). 2. Luciferin-luciferase reaction with ATP was inhibited by potassium-phosphate buffer and CaCl2, but not by HEPES- and Tris-buffer and D. W. (Table 1). Then, 50 mM HEPES buffer (pH 7.2), which optimal pH of this enzyme reaction was 7.2-7.4 as same as explained in the FLE-50 kit, was used as the buffer of extraction and determination of ATP in the leaf discs. 3. The effective extraction of ATP from the discs by the boiling water extraction method was obtained by using the discs with 2.0-3.0mm diameter in size with HEPES buffer (pH 7.2, 5 pieces/2.5 ml), heating them for 0.5-1.0 min in the boiling water and subsequent cooling for about 5 hr at 3°C (Fig. 2 and Fig. 3). 4. The boiling water extraction method was compared with the cold perchlorate extraction. The data obtained by the boiling water method were consistently higher than by perchlorate extraction method (Table 3). 5. Each variety in rice and soybean plants has different ATP contents within the limits of experiment. The ranking of these varietal differences in each plant did not change between experiments except Shin No. 2, one of soybean variety (Table 4). 6. There were no clear correlationships between ATP and chlorophyll contents in each plant (Fig. 4). These results mentioned above were discussed with reference to the availability of the boiling water extraction method for ATP extraction from green leaves.

Microassay of adenine nucleotides in intima and media of the aortic wall

Combining a quantitative histochemistry method with the Firefly luciferase method we found that adenosine-triphosphate (ATP) could be accurately measured in 100 to 200 μg of aortic tissue. Trichloracetic acid solution was used for extraction of ATP and by so doing, weighing at the freezing point or neutralization by (potassium hydroxide) KOH in case of perchloric acid could be eliminated. Simultaneous assay of cyclic nucleotides was thus feasible. Contents of ATP and ADP in intima and media of aortic wall of rabbits, cows, and pigs and levels of these nucleotides in atherosclerotic lesions of aorta of rabbits fed a 1% cholesterol containig diet for 15 weeks were measured. Levels of adenine nucleotides in atherosclerotic lesions of rabbits were low compared to levels in intima of the aortic wall. Acquisition of these data is vital for monitoring changes in levels of adenine nucleotides in relation to pathological changes in tissues.

An improved method for determination of adenosine triphosphate (ATP) in soil

Different methods for extraction of ATP from soil were examined. The methods were compared with respect to the efficiency of extraction of ATP from soil, and the content of ATP in the extracts was measured by the luciferin-luciferase system. The following extraction procedure was found to be the most efficient: Shaking of the soil with sulphuric acid, followed by filtration, cation exchange of the extract on Na + resin, and adjustment to pH 9.8 with ethanolamine. The method is simpler, faster and more convenient for processing large numbers of samples, than the earlier-described acid extraction methods. Furthermore, ATP levels of 5ngATPg −1 dry wt soil can be measured and results can be reproduced within ± 5%. The respiration rates of the soils were also measured, and a high degree of correlation was found between carbon dioxide production and ATP levels determined by extraction with sulphuric acid or dimethylsulfoxid.

An improved method for the determination of adenosinetriphosphate in environmental samples

An improved method has been developed for the analysis of the adenosinetriphosphate (ATP) content of environmental samples with the luciferin-luciferase assay. Factors affecting the recovery and stability of the ATP as well as the enzymatic reaction were optimized. The most versitile method utilizes procedure which can be used for natural waters, sediments and sludges. This involves homogenization at room temperature in 10 −2 M trisodium phosphate, pH 11.7, containing 23% chloroform. This method is more effective than other methods tested and results in minimal interference with the enzymatic reaction. It also provides improved ATP recovery and long term stability of extracted ATP. Another method that can be used on natural waters and sludges is by boiling for 5 min in 10 −2 M glycine buffer, 5 × 10 −3 M Mg-EDTA, pH 10, which resulted in complete extraction and no hydrolytic loss of ATP. However, it is not satisfactory for sediments containing high humic acid substances as well as high metal loadings. In addition to the direct comparison of endogenous ATP extraction by various methods, actual recoveries of spiked radioactive ATP was measured. ATP adsorption, hydrolysis and recoveries in each method were determined by the use of [8- 14C] ATP and [γ- 32P] ATP.

The extraction and measurement of adenosine triphosphate from marine sediments1

A technique has been developed, using boiling sodium bicarbonate buffer, to extract adenosine triphosphate (ATP) from marine sediments and has been tested on a variety of sediments, including those with high organic content, clay, and carbonate. Recovery of ATP, as measured by the addition of bacteria of known ATP content to sediment, varied from 64–100%. The technique also was as effective as the conventional Tris buffer for extraction of ATP from both pure cultures of bacteria grown in broth and natural seawater samples.

Quantitative Extraction of Adenosine Triphosphate from Cultivable and Host-Grown Microbes: Calculation of Adenosine Triphosphate Pools

Existing data on adenosine triphosphate (ATP) pools in microbes are deficient for two reasons: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei , and Mycobacterium lepraemurium . Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time ( E. coli , 3 min; M. phlei , 5 min; M. lepraemurium , 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.

Quantitative Extraction of Adenosine Triphosphate from Cultivable and Host-Grown Microbes: Calculation of Adenosine Triphosphate Pools

EXISTING DATA ON ADENOSINE TRIPHOSPHATE (ATP) POOLS IN MICROBES ARE DEFICIENT FOR TWO REASONS: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.