Adenylate Energy Charge: A Method for the Determination of Viable Cell Mass in Dental Plaque Samples* CHRISTOPHER W. KEMP National Caries Program, Etiology Section, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205
The biochemical function, adenylate energy charge (AEC), correlates with the viable count of S. mutans. AEC may be used to estimate the percent viable fraction of bacteria in dental plaque samples. An interactive computer program designed to process the AEC data is described. J Dent Res 68(D):2192-2197, November 1979
Introduction.
(AEC) was used. The AEC is the ratio of the usable phosphate bond energy to the total adenine nucleotides, ATP, ADP (adenosine diphosphate), and AMP (adenosine monophosphate), and is calculated by the expression: AEC=(ATP+1/2ADP) / (ATP+ADP+AMP). The AEC is a more stable and reproducible value than the measurement of ATP alone, is relatively independent of the phase of bacterial growth, 2-1 5 and has a numerical limit below which an individual cell cannot be recovered as a viable unit.1 3 The derivation of AEC and its use to describe the regulation of intracellular metabolism has been reported by Atkinson,1 2 but its potential use to quantitate living cells has been largely ignored. This paper describes
A detailed knowledge of the microbial composition of dental plaque is of prime importance in understanding the pathogenesis of dental caries. The microflora is complex1 2,3 and, although particular components of the flora may be identified and quantitated,3,4 the procedures entailed are so tedious that only limited numbers of samples may be processed. If meaningful data are to be obtained from epidemiologic studies the methods for AEC determination of of the relationship of suspected odontopaths pure cultures of bacteria and dental plaque, to dental caries, it will be necessary to pro- the use of AEC to estimate the viable cess numbers of samples in excess of those fraction of bacterial cells in dental plaque, which can be handled by conventional and the computer program which I have developed to handle the data obtained. microbiological methods. Recently, a method has been developed Materials and methods. which circumvents the need to culture Plaque samples were obtained from large numbers of plaque samples on bacteriological media.5 It has been demonstrated three female Rhesus monkeys (MTlacaca that viable cell mass may be estimated mulatta). The procedure for sample collecfrom extractable adenosine triphosphate tion and extraction has been previously (ATP),5-10 and our laboratory has applied described.5 this technique to estimate the viable cell Streptococcus mutans 6715-14 (Brathall mass in samples of dental plaque.5 However, serotype g) and Escherichia coli K12, used because the specific ATP content in pure in this study, were from a stock culture cultures of bacteria depends on the phase collection routinely maintained in the of growth,1 1 estimation of viable bacterial laboratory. Starvation cultures of S. mutans and units in dental plaque by extractable ATP may be inaccurate. To overcome this E. coli were prepared from a 500 ml batch problem, the adenylate energy charge culture of organisms grown overnight in Brain Heart Infusion Broth* with a
*This paper was the winner in the Predoctoral Candidate category of the 1979 Hatton Awards
*Difco Laboratories, Detroit, MI Competition. 2192 Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
VoL 58, Special Issue D
AEC & VIABLE CELL MASS IN PLAQUE
N2:C02 (95%:5%) atmosphere at 370C. The cells were aseptically harvested at 40C using a Sorvall** RC-5 centrifuge at 1 6,300xg for 20 min., the supernatant liquid discarded, and the cell sediment resuspended to approximately 50 ml in 0.01 M phosphate buffered saline, pH 7.0. The washed cell suspension was transferred to a sterile flask and incubated aerobically in a rotary shaker+ at 370C and 100 rpm. A direct particle count of a 1: 100 dilution of the cell suspension was obtained with the aid of a Petroff-Hausserl counting chamber under phase contrast microscopy. Aliquots (3 ml) of the washed cell suspension were removed at various time intervals for viable cell and adenine nucleotide analysis. A portion of sample for the adenine nucleotide analysis was frozen immediately in a dry ice-ethanol bath and stored at -700C until extracted as described below. For the viable cell determinations, serial tenfold dilutions were made from 1 ml of the remaining sample, and 0.1 ml aliquots of the appropriate dilution were inoculated onto five replicate plates of Mitis Salivarius Agar* without tellurite (S. mutans) or Eosin Methylene Blue Agar* (E. coli). Viable counts were obtained after the plates were incubated in a N2 :CO2 (95%:5%) atmosphere at 370C for 72 hours. Extraction of the adenine nucleotides was carried out in 0.02 M trishydroxymethylaminomethane* -HC 1 (Tris-HC 1) buffer, pH 7.75, as previously described,5 except that an ethylene glycol bath at 1 100C was used to heat the extraction solution. Internal standards of ATP,+* ADPi* and AMP were included in the extraction mixtures for the AEC calculation of a pure culture. The luciferase used to measure ATP in the plaque determinations was prepared as previously described.5 The ATP quantitation in the pure culture experiments utilized luciferase purified by gel filtration and ion exchange chromatography, according to the method of Beny and Dolivo.16 **E. I. DuPont DeNemours and Co., Inc., Newton, CT +New Brunswick Scientific Co., New Brunswick, NJ tC. A. Hausser and Son, Philadelphia, PA *Sigma Chemical Co., St. Louis, MO *Difco Laboratories, Detroit, MI
2193
Purification removes adenine nucleotides present in the crude enzyme preparation, thus the inherent background of the reaction is decreased. In addition, these chromatographic procedures remove nucleotide diphosphokinase and adenylate kinase which can synthesize ATP from other nucleotides present in the sample extract. Thus, purification of the luciferase markedly increases the sensitivity of the analysis. In order to quantitate the ATP present in the sample extracts, the light emitted from the reaction of ATP with firefly luciferin-luciferase was analyzed using an Aminco Chem-Glo photometer * equipped with an integrator timer.+* Specifically, a 6x50 mm disposable culture tube containing 25 ,ul of 0.6 M glycylglycine§ with 0.24 M MgSO4 pH 7.4, 25 ,l of 0.06 M KH2PO4 with 0.024 M MgSO4 pH 7.4, 10 ,l of 0.3 mg/ ml luciferin, and 400 ,l of sample or a dilution of ATP standard was mixed on a vortex agitator and placed in the sample chamber of the photometer. Luciferase enzyme (100 pl) was injected through a light-tight septum into the sample tube using a 5 ml gas-tight syringe¶ housed in a repeating dispenser.T A switch attached to the dispenser activated the integrator-timer, a 5 sec. portion of the light emission peak was integrated, and the data were recorded manually. The ADP and AMP present in the sample were measured using the luciferase reaction following enzymatic conversion to ATP by pyruvate kinase* and myokinase' as described by Adam.17 For the synthesis of ATP from ADP, 0.4 ml of a sample containing at least 2.5 picomoles of adenine nucleotides was added to the following: 0.55 ml of 0.1 M Tris-HCl, pH 7.75, containing 2.4 mM MgSO4, 0.09 M KCI, and 0.09 mM ethylenediaminetetraacetic acid' (EDTA), 33 pl 0.02 M phosphoenolpyruvate' and 10 units of pyruvate kinase in a volume of 20 ,l. The final volume of the mixture was 1 ml. The mixture was incubated at 240C for 15 min., frozen
#American
Instrument Co., Silver Spring, MD § Calbiochem, La Jolla, CA ¶ Hamilton Co., Reno, NV
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
21 94
JDent Res November 1979
KEMP
in a dry ice-ethanol bath, and kept frozen until analyzed (within two hrs.). ATP was obtained from AMP in the same manner but with the addition of 6 units of myokinase* in a volume of 10 ,ul to the mixture. Dilutions of ATP used for the standard curve were also incubated to compensate for losses or gains in concentration during the synthesis. Results from preliminary experiments showed that the synthesis was quantitative and that the net recovery as ATP for ADP and AMP was 99.3% and 96.2%, respectively. The ATP content of the standard solution was assayed as previously described. 6 ADP and AMP standard solutions were quantitated with pyruvate kinase, myokinase, and lactic dehydrogenase* by the method of Adam.1 7 A computer program was written in Basic language to assist in the analysis of data collected from the experiments described above. The program is designed to work on a Digital§ PDP-8, PDP-11, or PDP-1 0 system, with parameters such as the number of standards, samples, replicates, the dilution factor, blank values for standards, and sample values being entered into the program through a direct interactive process. Raw data collected from the adenine nucleotide analysis were entered in numerical form with the use of data statements. The data collected from the standard curve were subjected to a linear regression analysis and the slope, intercept, and correlation coefficient of the best-fit line were computed and included in the printout (Table 1). The slope of the line was used to convert the sample data into picomoles of ATP which was then normalized to pmol ATP/ml. The AEC of the sample was then calculated and the data were listed in tabular form to indicate the sample number, pmol ATP/ml, pmol ADP/ml, pmol AMP/ml and the AEC value (e. g., Table 2). 1
Results and discussion. The linearity and sensitivity of the luciferase assay for ATP using purified enzyme is illustrated in Figure 1. Using the methods described above, it is possible to quantitate 10 14moI of ATP (10 femtomoles). This degree of sensitivity is essential for the accurate analysis of the low levels of ATP found in dental plaque samples from in-
'Sigma Chemical Co., St. Louis, MO § Digital
Equipment Corp., Maynard, MA
TABLE 1 SAMPLE PRINTOUT OF THE COMPUTER PROGRAM USED TO CALCULATE AEC VALUES
AEC Enter data values for standards and samples on lines 940-1000. INTERACTIVE SECTION OF PROGRAM* how many standards did you run ?5 how many replicates are there ?3 how many samples did you run ?3 what is the dilution factor ?66.667 what are the blank values ?0,80,85t the blank for today is 82.5
LINEAR REGRESSION RESULTS FOR THE STANDARD CURVE std.# b 1 2 3 4 5
pmol ATP 0
0.55 1.1 5.5 11 55
avg.
light units-blk. 0
11.5 43.5 192.5 360.5 1765.5
slope = 3.12435E-2 pmol ATP/light unit 7-intercept = 6.44196 correlation coefficient = 0.999938 RESULTS FROM DATA ANALYSIS OF SAMPLES what are the blank sample values ?78,80,81 the sample blank for today is 79.6667
sample#pmol ATP pmol ADP pmol AMP AEC
is 2s SEE TABLE 2 FOR NUMERICAL RESULTS 3s
*The first five lines of this section require direct input from the operator. tThis illustrates the ability to correct for missing sample values as discussed in Results and Discussion.
dividual teeth and pure cultures of organisms containing small numbers of viable cells. The analysis of the ATP pool has been used to estimate the viable fraction of pure cultures of microorganisms and dental plaque samples.5 Figure 2 shows the relationship between the ATP pool of E. coli K 12 and viable count during a period of
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
2195
AEC & VIABLE CELL MASS INPLAQUE
Vol. 58, Special Issue D
TABLE 2
ADENINE NUCLEOTIDE CONCENTRATIONS AND AEC VALUES FOR DENTAL PLAQUE SAMPLES OBTAINED FROM MONKEYS Sample # 1 2 3
pmol ATP/ml
pmol ADP/ml
pmol AMP/ml
AEC
252.0 254.1 81.2
1693.4 1918.3 1635.1
377.0 168.7 0
0.473 0.518 0.523
prolonged starvation. Within the initial 6.5 hrs. of starvation the concentration of ATP drops sharply as the viable count rises slightly. There appears to be only a minimal relationship between the ATP pool and viable count until the cell suspension has reached a steady state (30.75 hrs of starvation). After this time, a definite correlation between viable count and the concentration of the ATP pool is observed. Although the ATP level falls close to baseline values in the period between 73.0 and 170.0 hrs., these values are well above the limit of ATP detection with purified luciferase. E. coli was chosen for this experiment because the organisms' simple nutritional requirements allow growth on the products of the dead cells in the suspension. This is evident in the periodic oscillation in the number of viable cells and amount of extractable ATP. The organisms in dental plaque are probably comparable to the portion of Figure 2 in which the viable count of E. coli and the quantity of extractable ATP are directly related to each other; however, this might not always be the case. A plaque sample could be exposed to some localized nutrient source, markedly affecting the ATP pool while only slightly altering the AEC value. Thus, interpretations using AEC should be much less equivocal than those based on raw ATP measurements. An important corollary of the ideas originally proposed by Atkinson,12 and TABLE 3 VIABLE COUNT AND A.E.C. VALUES FOR S. MUTANS 6715-14 DURING PROLONGED STARVATION
Elapsed Time (hr.)
0"i 4.25 22.5
Viable Count x10-9 8.30 6.74 0.74
A E C
0.084 0.046 0.014
documented by data obtained from procaryotic and eucaryotic cells,1 3 was a minimum AEC value (0.45) that a cell must maintain in order to remain viable. We have obtained values for dental plaque samples corresponding to a mass of totally viable organisms from individual animals and different specific sites (Table 2). The ATP concentration is substantially lower in all cases than the ADP concentration, indicating that the cells are in a resting state without any readily available energy source. This is in marked contrast to the high ATP pool found in the E. coli suspension during the initial phase of starvation. Table 2 also illustrates that the AEC value is independent of the concentration of ATP, as evidenced by the fact that the ATP level in sample 3 was the lowest of the samples assayed. However, the AEC for sample 3 was similar to the other AEC values. The relationship of AEC and viable count was studied using a washed cell suspension of S. mutans 6715-14. The cells underwent starvation conditions identical to those used with E. coli K12, and AEC was calculated at three points during a period of 22.5 hours (Table 3). After 22.5 hrs. 8.9% of the zero time viable count and 16.3% of the zero time AEC value was recovered, the AEC decreasing concomitantly with decreasing viable count. The AEC values at zero time are substantially below those expected for a totally viable population. When the viable and direct counts of the suspension of cells were compared at the start of the experiment, it was found that only 50 percent of the direct count was recovered as colonyforming units, resulting in a substantial reduction in the AEC by the nonviable fraction of the population. This effect is compounded by the fact that one-third of the population enumerated in the direct count were pairs or chains of organisms, each of which would produce a colony-
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
2196
JDentRes November 1979
KEMP
-6_~ 9-
S
0
-3°
2 s s s _ _*
C) v v
O
5 PICOMOLES ATP
_
*
*
D~~~
120 OF STARVATION (Nis.
80
DURATION
Fig. 1. - Standard curve for the analysis of ATP with the luciferin-luciferase reaction using purified luciferase. All points represent the average of three individual determinations.
Fig. 2. - Viable counts (closed circles) and extractable ATP (closed diamonds) of a washed cell suspension of E. coli K12 during prolonged starvation.
forming unit if only one of the cells were viable. Automatic data processing methods are essential for the numerical calculations which are necessary in this work. All of the ATP standards were assayed in triplicate at each concentration, and the other nucleotide phosphates were converted to ATP by enzyme-catalyzed reactions before subsequent analysis with luciferase. Internal standards are used during the incremental phosphorylations to account for any possible loss. The combination of data obtained for ATP standards, in addition to the adenine nucleotide concentrations of the samples (with and without internal standards), greatly increases the amount of numerical computations involved in the AEC calculation. One valuable aspect of the computer program described is its ability to sense an incomplete set of replicates and make adjustments for the calculation of mean values. In addition to the use of this program in the calculation of AEC, it is clear that, with minor modifications, the program would be applicable to any analysis in which the standards are linearly related to the type of reading, as is the case with the majority of the analyses which are commonly used in the laboratory.
tion. The computation of AEC is applicable to the analysis of dental plaque, and the samples that were analyzed had AEC values consistent with a totally viable cell mass. AEC has been related to, and the function may be used as, a direct measure of the fraction of viable cells in a population. Processing large numbers of samples requires the use of automatic data analysis methods, without which the necessary numerical calculations would limit the number of samples which could be analyzed. AEC analysis, combined with automatic data processing methods, should permit the reliable analysis of the viable cell mass for large numbers of dental plaque samples, and is applicable to epidemiologic studies and other studies where large numbers of samples are required. Another obvious application is in screening the effectiveness and persistence of various anti-plaque agents. The level of sensitivity obtainable with the purified luciferase is such that AEC calculations can be made from very small plaque samples, such as those obtained from rodents.
Conclusion. Adenylate energy charge is a more suitable function for the analysis of viable cells than extractable ATP because it is independent of the physiologic state of the constituent organisms in the popula-
Acknowledgments. This work was sponsored and supervised by Dr. Stanley A. Robrish of the National Caries Program. I also wish to acknowledge the assistance of Drs. J. P. Carlos, W. H. Bowen, D. B. Mirth, and M. F. Cole of the Caries Program, also Dr. K. R. Wagner of the Neurological and Communicative Disorders and Stroke Institute.
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
VoL 58,
Special Issue D
AEC & VIABLE CELL MASS IN PLAQUE
REFERENCES 1.
9.
GIBBONS, R. J.; SOCRANSKY, S. S.; ARAUJO, W. C.; and VAN HOUTE, J.: Studies of the Predominant Cultivable Microbiota of Dental Plaque. Arch. Oral Biol.
2.
3.
4.
5.
6.
7.
8.
9:365-370, 1964. LOESCHE, W. J.; HOCKETT, R. N.; and SYED, S. A.: Predominant Cultivable Flora of Tooth Surface Plaque Removed from Institutionalized Subjects. Arch. Oral Biol. 17:1311-1325, 1972. BOWDEN, G. H.; HARDIE, J. M.; and SLACK, G. L.: Microbial Variations in Approximal Dental Plaque. Caries Res. 9:253-277, 1975. GORDON, N. F.; STUTMAN, M.; and LOESCHE, W. J.: Improved Isolation of Anaerobic Bacteria from the Gingival Crevice Area of Man. Appl. Microbiol. 21:10461050, 1971. ROBRISH, S. A.; KEMP, C. W.; and BOWEN, W. H.: Use of Extractable Adenosine Triphosphate to Estimate the Viable Cell Mass in Dental Plaque Samples Obtained from Monkeys. Appl. Environ. Microbiol. 35: 743-749, 1978. FORSBERG, C. W., and LAM, K.: Use of Adenosine 5'-triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents. Appl. Environ. Microbiol. 33:528-
537, 1977. LEE, C. C.; HARRIS, R. F.; WILLIAMS, J. D. H.; ARMSTRONG, D. E.; and SYERS, J. K.: Adenosine Triphosphate in Lake Sediments. I. Determinations. Soil Sci. Proc. 35:82-86, 1971. LEE, C. C.; HARRIS, R. F.; WILLIAMS, J. D. H.; SYERS, J. K.; and ARMSTRONG, D. E.: Adenosine Triphosphate in Lake Sediments. II. Origin and Significance. Soil Sci. Proc. 35:86-91, 1971.
10.
11.
12.
13.
14.
15.
16.
17.
2197
LEVIN, G. V.; CHEN, C. S.; and DAVIS, G.: Development of the Firefly Bioluminescent Assay for the Rapid Quantitative Detection of Microbial Contamination in Water. Aerospace Medical Research Laboratory Document no. TR6771. Defense Documentation Center, Alexandria, Va., 1967. PATTERSON, J. W.; BREZONIK, P. L.; and PUTNAM, H. D.: Measurement and Significance of Adenosine Triphosphate in Activated Sludge. Environ. Sci. Technol. 4:569-575, 1970. FORREST, W. W.: Adenosine Triphosphate Pool During the Growth Cycle in Streptococcus faecalis. J. Bacteriol. 90:1013-1018, 1965. ATKINSON, D. E.: The Energy Charge of the Adenylate Pool as a Regulatory Parameter. Interaction with Feedback Modifiers. Biochemistry 7:40304034, 1968. CHAPMAN, A. G.; FALL, A.; and ATKINSON, D. E.: Adenylate Energy Charge in Escherichia coli During Growth and Starvation. J. of Bacteriol. 108:1072-1086, 1971. MONTAGUE, M. D., and DAWES, E. A.: The Survival of Peptococcus prevotii in Relation to the Adenylate Energy Charge. J. of Gen. Microbiol. 80:291-299, 1974. WALKER-SIMMONS, M. and ATKINSON, D. E.: Functional Capacities and the Adenylate Energy Charge in Escherichia coli Under Conditions of Nutritional Stress. J. of Bacteriol. 130:676-683, 1977. BENY, M., and DOLIVO, M.: Separation of Firefly Luciferase Using an Anion Exchanger. FEBS Letters 70:167-170, 1976. ADAM, H.: Adenosine-5'-diphosphate and Adenosine-5'-monophosphate, in Bergmeyer, H. V. (ed): Methods of Enzymatic Analysis, New York: Academic Press, Inc., 1963, pp 573-580.
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
The biochemical function, adenylate energy charge (AEC), correlates with the viable count of S. mutans. AEC may be used to estimate the percent viable fraction of bacteria in dental plaque samples. An interactive computer program designed to process the AEC data is described. J Dent Res 68(D):2192-2197, November 1979
Introduction.
(AEC) was used. The AEC is the ratio of the usable phosphate bond energy to the total adenine nucleotides, ATP, ADP (adenosine diphosphate), and AMP (adenosine monophosphate), and is calculated by the expression: AEC=(ATP+1/2ADP) / (ATP+ADP+AMP). The AEC is a more stable and reproducible value than the measurement of ATP alone, is relatively independent of the phase of bacterial growth, 2-1 5 and has a numerical limit below which an individual cell cannot be recovered as a viable unit.1 3 The derivation of AEC and its use to describe the regulation of intracellular metabolism has been reported by Atkinson,1 2 but its potential use to quantitate living cells has been largely ignored. This paper describes
A detailed knowledge of the microbial composition of dental plaque is of prime importance in understanding the pathogenesis of dental caries. The microflora is complex1 2,3 and, although particular components of the flora may be identified and quantitated,3,4 the procedures entailed are so tedious that only limited numbers of samples may be processed. If meaningful data are to be obtained from epidemiologic studies the methods for AEC determination of of the relationship of suspected odontopaths pure cultures of bacteria and dental plaque, to dental caries, it will be necessary to pro- the use of AEC to estimate the viable cess numbers of samples in excess of those fraction of bacterial cells in dental plaque, which can be handled by conventional and the computer program which I have developed to handle the data obtained. microbiological methods. Recently, a method has been developed Materials and methods. which circumvents the need to culture Plaque samples were obtained from large numbers of plaque samples on bacteriological media.5 It has been demonstrated three female Rhesus monkeys (MTlacaca that viable cell mass may be estimated mulatta). The procedure for sample collecfrom extractable adenosine triphosphate tion and extraction has been previously (ATP),5-10 and our laboratory has applied described.5 this technique to estimate the viable cell Streptococcus mutans 6715-14 (Brathall mass in samples of dental plaque.5 However, serotype g) and Escherichia coli K12, used because the specific ATP content in pure in this study, were from a stock culture cultures of bacteria depends on the phase collection routinely maintained in the of growth,1 1 estimation of viable bacterial laboratory. Starvation cultures of S. mutans and units in dental plaque by extractable ATP may be inaccurate. To overcome this E. coli were prepared from a 500 ml batch problem, the adenylate energy charge culture of organisms grown overnight in Brain Heart Infusion Broth* with a
*This paper was the winner in the Predoctoral Candidate category of the 1979 Hatton Awards
*Difco Laboratories, Detroit, MI Competition. 2192 Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
VoL 58, Special Issue D
AEC & VIABLE CELL MASS IN PLAQUE
N2:C02 (95%:5%) atmosphere at 370C. The cells were aseptically harvested at 40C using a Sorvall** RC-5 centrifuge at 1 6,300xg for 20 min., the supernatant liquid discarded, and the cell sediment resuspended to approximately 50 ml in 0.01 M phosphate buffered saline, pH 7.0. The washed cell suspension was transferred to a sterile flask and incubated aerobically in a rotary shaker+ at 370C and 100 rpm. A direct particle count of a 1: 100 dilution of the cell suspension was obtained with the aid of a Petroff-Hausserl counting chamber under phase contrast microscopy. Aliquots (3 ml) of the washed cell suspension were removed at various time intervals for viable cell and adenine nucleotide analysis. A portion of sample for the adenine nucleotide analysis was frozen immediately in a dry ice-ethanol bath and stored at -700C until extracted as described below. For the viable cell determinations, serial tenfold dilutions were made from 1 ml of the remaining sample, and 0.1 ml aliquots of the appropriate dilution were inoculated onto five replicate plates of Mitis Salivarius Agar* without tellurite (S. mutans) or Eosin Methylene Blue Agar* (E. coli). Viable counts were obtained after the plates were incubated in a N2 :CO2 (95%:5%) atmosphere at 370C for 72 hours. Extraction of the adenine nucleotides was carried out in 0.02 M trishydroxymethylaminomethane* -HC 1 (Tris-HC 1) buffer, pH 7.75, as previously described,5 except that an ethylene glycol bath at 1 100C was used to heat the extraction solution. Internal standards of ATP,+* ADPi* and AMP were included in the extraction mixtures for the AEC calculation of a pure culture. The luciferase used to measure ATP in the plaque determinations was prepared as previously described.5 The ATP quantitation in the pure culture experiments utilized luciferase purified by gel filtration and ion exchange chromatography, according to the method of Beny and Dolivo.16 **E. I. DuPont DeNemours and Co., Inc., Newton, CT +New Brunswick Scientific Co., New Brunswick, NJ tC. A. Hausser and Son, Philadelphia, PA *Sigma Chemical Co., St. Louis, MO *Difco Laboratories, Detroit, MI
2193
Purification removes adenine nucleotides present in the crude enzyme preparation, thus the inherent background of the reaction is decreased. In addition, these chromatographic procedures remove nucleotide diphosphokinase and adenylate kinase which can synthesize ATP from other nucleotides present in the sample extract. Thus, purification of the luciferase markedly increases the sensitivity of the analysis. In order to quantitate the ATP present in the sample extracts, the light emitted from the reaction of ATP with firefly luciferin-luciferase was analyzed using an Aminco Chem-Glo photometer * equipped with an integrator timer.+* Specifically, a 6x50 mm disposable culture tube containing 25 ,ul of 0.6 M glycylglycine§ with 0.24 M MgSO4 pH 7.4, 25 ,l of 0.06 M KH2PO4 with 0.024 M MgSO4 pH 7.4, 10 ,l of 0.3 mg/ ml luciferin, and 400 ,l of sample or a dilution of ATP standard was mixed on a vortex agitator and placed in the sample chamber of the photometer. Luciferase enzyme (100 pl) was injected through a light-tight septum into the sample tube using a 5 ml gas-tight syringe¶ housed in a repeating dispenser.T A switch attached to the dispenser activated the integrator-timer, a 5 sec. portion of the light emission peak was integrated, and the data were recorded manually. The ADP and AMP present in the sample were measured using the luciferase reaction following enzymatic conversion to ATP by pyruvate kinase* and myokinase' as described by Adam.17 For the synthesis of ATP from ADP, 0.4 ml of a sample containing at least 2.5 picomoles of adenine nucleotides was added to the following: 0.55 ml of 0.1 M Tris-HCl, pH 7.75, containing 2.4 mM MgSO4, 0.09 M KCI, and 0.09 mM ethylenediaminetetraacetic acid' (EDTA), 33 pl 0.02 M phosphoenolpyruvate' and 10 units of pyruvate kinase in a volume of 20 ,l. The final volume of the mixture was 1 ml. The mixture was incubated at 240C for 15 min., frozen
#American
Instrument Co., Silver Spring, MD § Calbiochem, La Jolla, CA ¶ Hamilton Co., Reno, NV
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
21 94
JDent Res November 1979
KEMP
in a dry ice-ethanol bath, and kept frozen until analyzed (within two hrs.). ATP was obtained from AMP in the same manner but with the addition of 6 units of myokinase* in a volume of 10 ,ul to the mixture. Dilutions of ATP used for the standard curve were also incubated to compensate for losses or gains in concentration during the synthesis. Results from preliminary experiments showed that the synthesis was quantitative and that the net recovery as ATP for ADP and AMP was 99.3% and 96.2%, respectively. The ATP content of the standard solution was assayed as previously described. 6 ADP and AMP standard solutions were quantitated with pyruvate kinase, myokinase, and lactic dehydrogenase* by the method of Adam.1 7 A computer program was written in Basic language to assist in the analysis of data collected from the experiments described above. The program is designed to work on a Digital§ PDP-8, PDP-11, or PDP-1 0 system, with parameters such as the number of standards, samples, replicates, the dilution factor, blank values for standards, and sample values being entered into the program through a direct interactive process. Raw data collected from the adenine nucleotide analysis were entered in numerical form with the use of data statements. The data collected from the standard curve were subjected to a linear regression analysis and the slope, intercept, and correlation coefficient of the best-fit line were computed and included in the printout (Table 1). The slope of the line was used to convert the sample data into picomoles of ATP which was then normalized to pmol ATP/ml. The AEC of the sample was then calculated and the data were listed in tabular form to indicate the sample number, pmol ATP/ml, pmol ADP/ml, pmol AMP/ml and the AEC value (e. g., Table 2). 1
Results and discussion. The linearity and sensitivity of the luciferase assay for ATP using purified enzyme is illustrated in Figure 1. Using the methods described above, it is possible to quantitate 10 14moI of ATP (10 femtomoles). This degree of sensitivity is essential for the accurate analysis of the low levels of ATP found in dental plaque samples from in-
'Sigma Chemical Co., St. Louis, MO § Digital
Equipment Corp., Maynard, MA
TABLE 1 SAMPLE PRINTOUT OF THE COMPUTER PROGRAM USED TO CALCULATE AEC VALUES
AEC Enter data values for standards and samples on lines 940-1000. INTERACTIVE SECTION OF PROGRAM* how many standards did you run ?5 how many replicates are there ?3 how many samples did you run ?3 what is the dilution factor ?66.667 what are the blank values ?0,80,85t the blank for today is 82.5
LINEAR REGRESSION RESULTS FOR THE STANDARD CURVE std.# b 1 2 3 4 5
pmol ATP 0
0.55 1.1 5.5 11 55
avg.
light units-blk. 0
11.5 43.5 192.5 360.5 1765.5
slope = 3.12435E-2 pmol ATP/light unit 7-intercept = 6.44196 correlation coefficient = 0.999938 RESULTS FROM DATA ANALYSIS OF SAMPLES what are the blank sample values ?78,80,81 the sample blank for today is 79.6667
sample#pmol ATP pmol ADP pmol AMP AEC
is 2s SEE TABLE 2 FOR NUMERICAL RESULTS 3s
*The first five lines of this section require direct input from the operator. tThis illustrates the ability to correct for missing sample values as discussed in Results and Discussion.
dividual teeth and pure cultures of organisms containing small numbers of viable cells. The analysis of the ATP pool has been used to estimate the viable fraction of pure cultures of microorganisms and dental plaque samples.5 Figure 2 shows the relationship between the ATP pool of E. coli K 12 and viable count during a period of
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
2195
AEC & VIABLE CELL MASS INPLAQUE
Vol. 58, Special Issue D
TABLE 2
ADENINE NUCLEOTIDE CONCENTRATIONS AND AEC VALUES FOR DENTAL PLAQUE SAMPLES OBTAINED FROM MONKEYS Sample # 1 2 3
pmol ATP/ml
pmol ADP/ml
pmol AMP/ml
AEC
252.0 254.1 81.2
1693.4 1918.3 1635.1
377.0 168.7 0
0.473 0.518 0.523
prolonged starvation. Within the initial 6.5 hrs. of starvation the concentration of ATP drops sharply as the viable count rises slightly. There appears to be only a minimal relationship between the ATP pool and viable count until the cell suspension has reached a steady state (30.75 hrs of starvation). After this time, a definite correlation between viable count and the concentration of the ATP pool is observed. Although the ATP level falls close to baseline values in the period between 73.0 and 170.0 hrs., these values are well above the limit of ATP detection with purified luciferase. E. coli was chosen for this experiment because the organisms' simple nutritional requirements allow growth on the products of the dead cells in the suspension. This is evident in the periodic oscillation in the number of viable cells and amount of extractable ATP. The organisms in dental plaque are probably comparable to the portion of Figure 2 in which the viable count of E. coli and the quantity of extractable ATP are directly related to each other; however, this might not always be the case. A plaque sample could be exposed to some localized nutrient source, markedly affecting the ATP pool while only slightly altering the AEC value. Thus, interpretations using AEC should be much less equivocal than those based on raw ATP measurements. An important corollary of the ideas originally proposed by Atkinson,12 and TABLE 3 VIABLE COUNT AND A.E.C. VALUES FOR S. MUTANS 6715-14 DURING PROLONGED STARVATION
Elapsed Time (hr.)
0"i 4.25 22.5
Viable Count x10-9 8.30 6.74 0.74
A E C
0.084 0.046 0.014
documented by data obtained from procaryotic and eucaryotic cells,1 3 was a minimum AEC value (0.45) that a cell must maintain in order to remain viable. We have obtained values for dental plaque samples corresponding to a mass of totally viable organisms from individual animals and different specific sites (Table 2). The ATP concentration is substantially lower in all cases than the ADP concentration, indicating that the cells are in a resting state without any readily available energy source. This is in marked contrast to the high ATP pool found in the E. coli suspension during the initial phase of starvation. Table 2 also illustrates that the AEC value is independent of the concentration of ATP, as evidenced by the fact that the ATP level in sample 3 was the lowest of the samples assayed. However, the AEC for sample 3 was similar to the other AEC values. The relationship of AEC and viable count was studied using a washed cell suspension of S. mutans 6715-14. The cells underwent starvation conditions identical to those used with E. coli K12, and AEC was calculated at three points during a period of 22.5 hours (Table 3). After 22.5 hrs. 8.9% of the zero time viable count and 16.3% of the zero time AEC value was recovered, the AEC decreasing concomitantly with decreasing viable count. The AEC values at zero time are substantially below those expected for a totally viable population. When the viable and direct counts of the suspension of cells were compared at the start of the experiment, it was found that only 50 percent of the direct count was recovered as colonyforming units, resulting in a substantial reduction in the AEC by the nonviable fraction of the population. This effect is compounded by the fact that one-third of the population enumerated in the direct count were pairs or chains of organisms, each of which would produce a colony-
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
2196
JDentRes November 1979
KEMP
-6_~ 9-
S
0
-3°
2 s s s _ _*
C) v v
O
5 PICOMOLES ATP
_
*
*
D~~~
120 OF STARVATION (Nis.
80
DURATION
Fig. 1. - Standard curve for the analysis of ATP with the luciferin-luciferase reaction using purified luciferase. All points represent the average of three individual determinations.
Fig. 2. - Viable counts (closed circles) and extractable ATP (closed diamonds) of a washed cell suspension of E. coli K12 during prolonged starvation.
forming unit if only one of the cells were viable. Automatic data processing methods are essential for the numerical calculations which are necessary in this work. All of the ATP standards were assayed in triplicate at each concentration, and the other nucleotide phosphates were converted to ATP by enzyme-catalyzed reactions before subsequent analysis with luciferase. Internal standards are used during the incremental phosphorylations to account for any possible loss. The combination of data obtained for ATP standards, in addition to the adenine nucleotide concentrations of the samples (with and without internal standards), greatly increases the amount of numerical computations involved in the AEC calculation. One valuable aspect of the computer program described is its ability to sense an incomplete set of replicates and make adjustments for the calculation of mean values. In addition to the use of this program in the calculation of AEC, it is clear that, with minor modifications, the program would be applicable to any analysis in which the standards are linearly related to the type of reading, as is the case with the majority of the analyses which are commonly used in the laboratory.
tion. The computation of AEC is applicable to the analysis of dental plaque, and the samples that were analyzed had AEC values consistent with a totally viable cell mass. AEC has been related to, and the function may be used as, a direct measure of the fraction of viable cells in a population. Processing large numbers of samples requires the use of automatic data analysis methods, without which the necessary numerical calculations would limit the number of samples which could be analyzed. AEC analysis, combined with automatic data processing methods, should permit the reliable analysis of the viable cell mass for large numbers of dental plaque samples, and is applicable to epidemiologic studies and other studies where large numbers of samples are required. Another obvious application is in screening the effectiveness and persistence of various anti-plaque agents. The level of sensitivity obtainable with the purified luciferase is such that AEC calculations can be made from very small plaque samples, such as those obtained from rodents.
Conclusion. Adenylate energy charge is a more suitable function for the analysis of viable cells than extractable ATP because it is independent of the physiologic state of the constituent organisms in the popula-
Acknowledgments. This work was sponsored and supervised by Dr. Stanley A. Robrish of the National Caries Program. I also wish to acknowledge the assistance of Drs. J. P. Carlos, W. H. Bowen, D. B. Mirth, and M. F. Cole of the Caries Program, also Dr. K. R. Wagner of the Neurological and Communicative Disorders and Stroke Institute.
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
VoL 58,
Special Issue D
AEC & VIABLE CELL MASS IN PLAQUE
REFERENCES 1.
9.
GIBBONS, R. J.; SOCRANSKY, S. S.; ARAUJO, W. C.; and VAN HOUTE, J.: Studies of the Predominant Cultivable Microbiota of Dental Plaque. Arch. Oral Biol.
2.
3.
4.
5.
6.
7.
8.
9:365-370, 1964. LOESCHE, W. J.; HOCKETT, R. N.; and SYED, S. A.: Predominant Cultivable Flora of Tooth Surface Plaque Removed from Institutionalized Subjects. Arch. Oral Biol. 17:1311-1325, 1972. BOWDEN, G. H.; HARDIE, J. M.; and SLACK, G. L.: Microbial Variations in Approximal Dental Plaque. Caries Res. 9:253-277, 1975. GORDON, N. F.; STUTMAN, M.; and LOESCHE, W. J.: Improved Isolation of Anaerobic Bacteria from the Gingival Crevice Area of Man. Appl. Microbiol. 21:10461050, 1971. ROBRISH, S. A.; KEMP, C. W.; and BOWEN, W. H.: Use of Extractable Adenosine Triphosphate to Estimate the Viable Cell Mass in Dental Plaque Samples Obtained from Monkeys. Appl. Environ. Microbiol. 35: 743-749, 1978. FORSBERG, C. W., and LAM, K.: Use of Adenosine 5'-triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents. Appl. Environ. Microbiol. 33:528-
537, 1977. LEE, C. C.; HARRIS, R. F.; WILLIAMS, J. D. H.; ARMSTRONG, D. E.; and SYERS, J. K.: Adenosine Triphosphate in Lake Sediments. I. Determinations. Soil Sci. Proc. 35:82-86, 1971. LEE, C. C.; HARRIS, R. F.; WILLIAMS, J. D. H.; SYERS, J. K.; and ARMSTRONG, D. E.: Adenosine Triphosphate in Lake Sediments. II. Origin and Significance. Soil Sci. Proc. 35:86-91, 1971.
10.
11.
12.
13.
14.
15.
16.
17.
2197
LEVIN, G. V.; CHEN, C. S.; and DAVIS, G.: Development of the Firefly Bioluminescent Assay for the Rapid Quantitative Detection of Microbial Contamination in Water. Aerospace Medical Research Laboratory Document no. TR6771. Defense Documentation Center, Alexandria, Va., 1967. PATTERSON, J. W.; BREZONIK, P. L.; and PUTNAM, H. D.: Measurement and Significance of Adenosine Triphosphate in Activated Sludge. Environ. Sci. Technol. 4:569-575, 1970. FORREST, W. W.: Adenosine Triphosphate Pool During the Growth Cycle in Streptococcus faecalis. J. Bacteriol. 90:1013-1018, 1965. ATKINSON, D. E.: The Energy Charge of the Adenylate Pool as a Regulatory Parameter. Interaction with Feedback Modifiers. Biochemistry 7:40304034, 1968. CHAPMAN, A. G.; FALL, A.; and ATKINSON, D. E.: Adenylate Energy Charge in Escherichia coli During Growth and Starvation. J. of Bacteriol. 108:1072-1086, 1971. MONTAGUE, M. D., and DAWES, E. A.: The Survival of Peptococcus prevotii in Relation to the Adenylate Energy Charge. J. of Gen. Microbiol. 80:291-299, 1974. WALKER-SIMMONS, M. and ATKINSON, D. E.: Functional Capacities and the Adenylate Energy Charge in Escherichia coli Under Conditions of Nutritional Stress. J. of Bacteriol. 130:676-683, 1977. BENY, M., and DOLIVO, M.: Separation of Firefly Luciferase Using an Anion Exchanger. FEBS Letters 70:167-170, 1976. ADAM, H.: Adenosine-5'-diphosphate and Adenosine-5'-monophosphate, in Bergmeyer, H. V. (ed): Methods of Enzymatic Analysis, New York: Academic Press, Inc., 1963, pp 573-580.
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 18, 2015 For personal use only. No other uses without permission.
