A review of bioluminescent ATP techniques in rapid microbiology

Use of firefly luciferase to assay adenosine triphosphate (ATP) extracted from microorganisms provides an easy means to enumerate microbes within minutes. The small amount of light produced is proportional to ATP and thus microbial number. The average bacterium contains around 10^?15 g ATP per cell. Present reagents permit detection of 10³ cells per tube. Luminometers currently on the market detect about 10^?12 g ATP. Proper extraction of ATP from the microbes is an essential part of any protocol, as is the removal of non-microbial ATP from, for example, somatic cells also present in samples. The technique may be applied to a wide range of samples, for example food and beverages and clinical samples such as urine. The ATP assay gives a global measure of microbial numbers, i.e. it is not species specific unless a species separation step is included in the protocol.     

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

The importance of having a rapid method for determining the viable biomass in activated-sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out-of-coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated-sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed-liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.     

The adenosine triphosphate content of cysts of Globodera pallida and G. rostochiensis as a possible quantitative estimate of field populations

A rapid method is described for estimating the number of juveniles of Globodera pallida and G. rostochiensis in cysts collected from field soils. The technique uses a photometer to measure the adenosine triphosphate (ATP) content of the cysts by bioluminescence. Results for ten to twelve soil samples of 100 g taken from each of four fields showed that the number of juveniles that emerged when the samples were placed in potato root diffusate was significantly related to ATP content of a second series of cyst samples from these fields. Cysts from one field gave a higher ATP content per hatched juvenile than was recorded for the remaining three fields. There are several, untested, explanations of this discrepancy, but some evidence suggests that the hatching test and not the ATP method may have given an unreliable estimate of the viable egg content of cysts from this field. Further work may result in an enhanced precision and increased rapidity in estimating field populations of cyst nematodes by this technique.     

Measurement of the adenosine triphosphate content of cysts of Globodera rostochiensis as a method for assessing the efficacy of fumigant nematicides

The measurement of the adenosine triphosphate (ATP) content of samples of 50 cysts of Globodera rostochiensis collected from 40 fields in the Netherlands has been tested as a method for determining the mortality of the nematodes achieved by fumigation of these field soils. The technique used is based on bioluminescent photometry and it has been fully described previously. The ATP assessment has been compared with results obtained by the advisory service in the Netherlands using hatching tests based on samples of 100 cysts from the same field soils. The ratio of the values before and after soil treatment showed that the methods usually provided similar estimates of mortality. The relationship between ATP and hatched juveniles was also similar from pre-treatment and post-treatment samples. The amount of ATP, calculated from these results, which is equivalent to the maximum post-treatment hatch allowed for a successful treatment in the Netherlands was used in conjunction with the mortality estimates to assess the success of the fumigation. On this basis the two methods disagreed in the overall assessment of fumigation in only five of the 40 fields. This frequency of disagreement is not significantly greater than expected from the variability of either method. The ATP technique clearly offers a reliable and relatively inexpensive method for estimating the efficacy of fumigant nematicides and is much more rapid than the alternative hatching tests or egg counts. Therefore, it could replace these in situations where the grower wanted a rapid assessment on the success, or otherwise, of his fumigation.     

Occurrence and preservation of ATP in Antarctic rocks and its implications in biomass determinations

The low temperature and moisture availability in Antarctic rocks would seem to preclude the existence of any sizeable algal biomass. The very high levels of ATP associated with endplithic algal communities have led to speculation that the measured ATP largely represents accumulated exogenous material as well as living biomass. A method was developed to selectively hydrolyze free, extracellular ATP without affecting intracellular ATP. Use of this technique on Antarctic rock samples has led to the conclusion that the ATP found in endolithic communities exclusively reflects living material and not preserved, free ATP.

From these measurements we concluded that the ATP biomass in a gram of rock is equivalent to that found in more temperate and favorable environments.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Development of a Rapid Assimilable Organic Carbon Method for Water

A rapid method for measurement of assimilable organic carbon (AOC) is proposed. The time needed to perform the assay is reduced by increasing the incubation temperature and increasing the inoculum density. The ATP luciferin-luciferase method quickly enumerates the test organisms without the need for plate count media or dilution bottles. There was no significant difference between AOC values determined with strain P17 for the ATP and plate count procedures. For strain NOX, the plate count procedure underestimated bacterial levels in some samples. Comparison of AOC values obtained by the Belleville laboratory (by the ATP technique) and the Stroud Water Research Center (by plate counts) showed that values were significantly correlated and not significantly different. The study concludes that the rapid AOC method can quickly determine the bacterial growth potential of water within 2 to 4 days.     

The ATP-bioluminescence method for a rapid evaluation of the microbial activity in the stone materials of monuments

The examination of the state of conservation of works of art in stone includes the assessment of the presence of microbiological agents on the surface of the decayed monuments. These microorganisms can accelerate, via their metabolic activity, the decay process of the stone surface. At present this assessment is made with the traditional techniques for the microbiological examination of the soil, provides results only after a delay of 30 days. A bioluminescent ATP assay should provide rapid quantitation of actively growing organisms on the surface of a stone monument, and the applicability of this technique was verified on some samples of sandstone (Pietraforte) collected from a historic building (the Strozzi Palace) in Florence. These samples were evaluated for the amount of the ATP and the total number of microorganisms. The results obtained suggest that the bioluminescent assay could be suitable for detecting and quantitating the presence of microorganisms in a sample of stone.     

Fourteen protomers compose the oligomer III of the proton-rotor in spinach chloroplast ATP synthase

Three fundamentally different chloroplast ATP synthase samples of increasing complexity were visualized by atomic force microscopy. The samples are distinguishable in respect to the isolation technique, the detergent employed, and the final subunit composition. The homo-oligomer III was isolated following SDS treatment of ATP synthase, the proton-turbine III+IV was obtained by blue-native electrophoresis, and complete CFO was isolated by anion exchange chromatography of NaSCN splitted ATP synthase. In all three ATP synthase subcomplexes 14 and only 14 circularly arranged subunits III composed the intact transmembrane rotor. Therefore, 14 protomers built the membrane-resident proton turbine. The observed stoichiometry of 14 is not a biochemical artifact or affected by natural growth variations of the spinach, as previously suggested. A correlation between the presence of subunit IV in the imaged sample and the appearance of a central protrusion in the narrower orifice of the oligomeric cylinder III14 has been observed. In contrast to current predictions, in chloroplast FO the subunit IV can be found inside the cylinder III14 and not at its periphery, at least in the reconstituted 2D arrays imaged.     

The Stiffness of the Flagella of Impaled Bull Sperm

The elastic rigidity (stiffness) of impaled motionless bull sperm flagella has been determined by a manipulatory technique which permitted direct analytical treatment of the experimental system. The effects of external ATP and ADP were measured. It was found that ATP acts as a plasticizing agent, while ADP does not. The stiffness measured for flagella in a medium without ATP was 15 times greater than the value measured with 10 mM ATP present. The rigor-like stiffness measured with no ATP present is reversible with ATP and seems to be correlated to a transition in the state of the contractile system.     

Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.     

ATP synthesis and degradation rates in the perfused rat heart. 31P-nuclear magnetic resonance double saturation transfer measurements.

A limitation of magnetization transfer techniques for studying enzyme kinetics in vivo has been the difficulty of treating systems with more than two exchanging species. This problem was addressed in the original papers describing saturation transfer. Since then, a number of approaches have been devised to study these complex situations. Here, we present a method based on the transient saturation transfer experiment in which spin-lattice relaxation time constants and reaction rates are obtained from the same magnetization transfer data. This technique is particularly suitable for biological samples. We apply the method to evaluate flux balance in the three-site linear exchange network composed of ATP, creatine phosphate, and inorganic phosphate in the isolated, perfused rat heart and show that the method yields reasonable values for the reaction velocities of ATP synthesis and degradation.     

PEI-cellulose thin-layer chromatography. Product studies of the creatine kinase and pyruvate kinase reactions

Detailed methods for polyethylenimine-cellulose thin-layer chromatographic determination of the reactants and products of the creatine kinase and pyruvate kinase reactions are presented. The technique has been developed as a highly sensitive method both for estimation of relative initial rates afforded by creatine and creatine analogs in the creatine kinase reaction and also for estimation of trace amounts of inorganic phosphate and ADP contamination in samples of ATP.     

Interstitial ATP level and degradation in control and postmyocardial infarcted rats

With the aim of estimating interstitial levels and the breakdownprocess of ATP, cardiac microdialysis was performed in the leftventricular wall of in situ control and postinfarcted as well as ofisolated, Langendorff-perfused rat hearts. With the use of abioluminescence technique for dialysate ATP measurement, the baselineinterstitial fluid ATP concentration in in situ hearts was estimated tobe 38 ± 8 nM. Regional ischemia induced an early peakincrease in interstitial fluid ATP to 373 ± 73 nM thatcorrelates with the maximal incidence of ventricular arrhythmias.During continuous infusion of individual adenine nucleotides (50 µMATP, ADP, or AMP), the dialysate samples were analyzed for adenine nucleotides, nucleosides, and bases using HPLC with ultraviolet detection. The patterns of catabolites were consistent with the majorpathway of metabolism, that is, sequential dephosphorylation catalyzedby a chain of separate ecto-nucleotidases. In in situ postinfarctedhearts as well as in perfused hearts, a reduced catabolism rate ofextracellular adenine nucleotides was observed. In conclusion, in insitu rat hearts, ATP can be released in substantial amounts in theinterstitium where it readily undergoes enzymatic degradation.Dephosphorylation occurs sequentially and faster in in situ controlhearts than in in situ postinfarcted or in perfused hearts.

     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Rate of ATP synthesis in the perfused rat liver by 31P cryo-NMR

ATP concentrations in the perfused rat liver during normoxic perfusion, transient ischemia, and recovery from transient ischemia were measured using the modified 31P cryo-NMR method (Chance, B., Nakase, Y., Bond, M., Leigh, J. S., Jr., and McDonald, G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4925-4929). Transient ischemia was induced in the perfused livers of starved rats, and multiple freeze-trapped tissue samples were taken from each liver at short intervals (15-30 s) during ischemia or following reperfusion. The freeze-trapped tissue was pulverized together with an antifreezing agent and high energy metabolites were measured by 31P NMR at 243 K after thawing. By using the cryo-NMR technique, a biochemical time resolution of 2 s could be achieved. Absolute metabolite concentrations were calculated by comparing the peak areas with internal standards mixed into the samples. Good time resolution and reliable concentration measurements provided by the cryo-NMR method enable us to estimate the ATP synthesis rate in the perfused liver during reperfusion following transient ischemia. The rate of ATP synthesis in the normoxic perfusion was 1.95 mumol/min/g wet weight; the maximal ATP synthesis rate during the recovery phase from ischemia was 5.75 mumol/min/g wet weight.     

Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.     

31P-MRS study of bio-energy recovering phenomenon

The ATP and creatine phosphate (PCr) contents in isolated guinea-pig hearts were determined by 31P-MRS measurement at 80.75 MHz using the Langendorff technique. Reperfusion of post-ischemic hearts with adenosine for 180 minutes increased ATP to 117.4% and decreased PCr to 59.8% of the preischemic value. Reperfusion without adenosine did not increase ATP and did not decrease PCr. The depressed cardiac function due to ischemia was remarkably improved in post-ischemic hearts by the increase in ATP due to adenosine. We found that the loss of ATP due to ischemia is not necessarily proportional to the extent of myocardial ischemic injury.     

New and improved molecular sexing methods for museum bird specimens

We present two new avian molecular sexing techniques for nonpasserine and passerine birds (Neognathae), which are more suitable for use with museum specimens than earlier methods. The technique for nonpasserines is based on a new primer (M5) which, in combination with the existing P8 primer, targets a smaller amplicon in the CHD1 sex-linked gene than previously. Primers targeting ATP5A1, an avian sex-linked gene not previously used for sex identification, were developed for passerines. Comprehensive testing across species demonstrated that both primer pairs sex a range of different species within their respective taxonomic groups. Rigorous evaluation of each method within species showed that these permitted sexing of specimens dating from the 1850s. For corn bunting museum specimens, the ATP5A1 method sexed 98% of 63 samples (1857-1966). The M5/P8 CHD1 method was similarly successful, sexing 90% of 384 moorhen specimens from six different museum collections (1855-2001). In contrast, the original P2/P8 CHD1 sexing method only identified the sex of less than half of 111 museum moorhen samples. In addition to dried skin samples, these methods may be useful for other types of material that yield degraded or damaged DNA, and are hence potential new sexing tools for avian conservation genetics, population management and wildlife forensics.     

The ATP pool in Escherichia coli. I. Measurement of the pool using a modified luciferase assay

A sensitive modification of the luciferase assay for ATP is described. Light output is measured using a conventional liquid-scintillation counter. ATP over the range 2 to 20 μμmoles can be assayed. The sensitivity of the method allows samples to be taken rapidly from growing cultures of bacteria without concentrating them from the medium. Chilling and anaerobiosis of growing cells before extracting with HClO₄ have been shown to cause a reduction in the ATP pool. ATP measurements in growing cultures of Escherichia coli show four different phenomena depending on culture conditions: (a) ATP production is in balance with growth. (b) ATP is consistently over-produced. (c) ATP is consistently under-produced. (d) Cyclic oscillations in ATP production are observed.

Doubling times for growth and ATP production are compared, and the ATP pool in cells grown under carbon-limiting conditions in a chemostat has been measured. Starvation experiments have shown that the ATP pool drops more rapidly anaerobically than aerobically.