Improved methods for ATP analysis

ANALYTICAL

BIOCHEMISTRY

60, 102-114 (1974)

Improved SUE CHEER, National

Matine

Methods JOHN

for ATP Analysis

H. GENTILE,

AND

C. S. HEGRE

Water Quality Laboratory, Environmental Narragansett, Rhode Island 02882

Received May 7, 1973; accepted December

Protection Agency, 19, 1973

An investigation of the techniques required for the analysis of adenosine triphosphate (ATP) in marine systems led to refinements in methodology. Adaptation of the DuPont luciferin-luciferase system to the liquid scintillation spectrometer resulted in a sensitivity range of 1 X lOme-2 X lo-’ pg ATP per 10 &ters sample. Examination of sample collection techniques showed no significant difference between direct extraction of unfiltered samples and extraction of filtered samples. Liquid nitrogen and dry ice proved to be satisfactory for the preservation of filtered samples prior to extraction, whereas freezing at -20°C resulted in ATP losses as great as 39%. Investigation of the stability of frozen ATP standards revealed no losses in activity for months. The luciferin-luciferase proved to be stable for hours when kept chilled and retained 89% of its activity for at least 1 mo when frozen.

Adenosine triphosphate (ATP) appears to be an excellent measure of the concentration of living material. Numerous researchers are using ATP analysis to determine the biomass in such diverse systems as sewage sludge (1)) marine (2,3) and fresh water (4,5) systems, foods (6)) as well as in laboratory studies of bacteria (7,8), and algae (9,10), etc. All of these studies utilize the analysis of ATP by the ATP-dependent light yielding reaction of the firefly lantern luciferin-luciferase system (11) : luciferase

LHZ (luciferin)

AMP.LH,

+ ATP f

+ PP

M@+

LH2.AMP

+ 3$0, --t L.AMP’

(excited) + Hz0 -+ L*AMP

+ H20 + hv (light)

The amount of light emitted in this reaction is proportional to the concentration of ATP. A number of detection systems have been used by various investigators to quantify the light emitted in the ATP-dependent reaction; however there has been a paucity of comprehensive information published on the techniques required for routine ATP analysis. In the development of this assay for use in the establishment of marine water quality criteria, some unforseen problems were encountered. We have made a detailed examina102 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

IMPROVED

METHODS

FOR

ATP

ANALYSIS

103

tion of these problems as well as of procedures described in the literature. These investigations resulted in several modifications and improvements in the methodology. In this laboratory a liquid scintillation spectrometer (Packard TriGarb, Model 3380) was used as the light detector and some of the methods herein described were developed for this instrument, but may well be applicable to other detection syst.ems. We have adapted the highly purified DuPont luciferin-luciferase assay system to the liquid scintillation spectrometer, designed and built an automatic sample injector and evaluated sample collection and preservation techniques. In addition, we have accumulated data on the stability of ATP standards, enzyme-substrate, and ATP extracts. METHODS

AND

MATERIALS

In the process of ATP analysis the materials to be examined must sometimes be concentrated by filtration (or possibly centrifugation) prior to extraction with boiling Tris buffer. The buffer quickly denatures thermolabile proteins (ATPases) and simultaneously releases ATP into the buffer. An aliquot of this extract is directIy assayed with the luciferinluciferase system and its ATP concentration determined from a standard curve. The standard curve is prepared by comparing the amount of emitted light measured in counts per minute (cpm) for a series of known ATP concentrations. Water. All water used in the process of ATP analysis was derived from a Culligan ultra-high purity system and had a resistivity of 12-18 MQ cm (20°C). W7ater in this system is circulated through a train composed of ultraviolet radiation, activated charcoal, a mixed bed ion exchange resin, with a 0.45 ,um final filtration. This water was excellent for ATP studies as reagents prepared in it showed no ATP activity. Buffer Preparation. Tris buffer (Nutritional Biochemicals Corp. 3 x crystalline for enzyme research trishydroxymethyl-amino methane) was used both for extraction of samples and for preparing ATP standard solutions. A 0.02 M stock solution of Tris was prepared by dissolving five grams of the buffer crystals in 2 liters of deionized water. This solution was brought to pH 7.75 with concentrated HCl and was stored at 4°C. ATP ~ta~durds. Ten milligrams of ATP (12.3 mg of A grade disodium adenosine-5-triphosphate tetrahydrate, MW 623.2, Calbiochem) were dissolved in 1 liter of high purity water. This primary stock solution was very stable and could be kept frozen at -20°C for as long as 30 months. Fresh dilutions of such primary stocks showed little or no loss in activity (Table 1). Any differences in the activity of these standards could be accounted for by variations in the weighing and dilution of the ATP.

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TABLE 1 of Frozen Primary ATP Stock (10 mg/l)” 5 pg ATP/liter

Age of stock (years) 2.5 2.0 1.0 Fresh

Mean cpm 1.80 2.02 1.90 1.92

X x x x

106 106 106 106

10 pg ATP/liter CP 6 2 3 1

Mean cpm 3.34 3.76 3.69 3.98

x X X x

106 lo6 lo6 106

c, 4 4 5 2

0 5 and 10 &liter dilutions were prepared just prior to analysis. Data are in cpm for a 10 al injection of standard into 0.1 ml enzyme-substrate. b C, = Coefficient of variation = (standard deviation/mean) X 100%.

standard dilutions, in pH 7.75 Tris, were prepared from the primary stock and 2 ml aliquots were dispensed into two dram screw cap vials for storage at -20°C. The usual series of ATP standards contained 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10.0 pg of ATP/liter. Numerous vials of each of these concentrations were prepared annually. These diluted standards were also very stable and lost no activity in the course of a year (Fig. 1). Contrary to previous reports (1,12) repeatedly thawed standards remained stable for weeks at -20°C when kept on ice for the duration of the experiments. In a routine series of analyses spanning several days, a complete standard curve was not usually prepared. Two to four levels of ATP were selected to bracket the activity level of the samples. Unless the enzyme

ATP

CPM

FIG. 1. Activity in counts per minute ATP standard solutions.

(cpm) of fresh (X)

and year old (0)

IMPROVED

METHODS

FOR

ATP

105

ANALYSIS

showed greatly altered activity, reference was made to a previously prepared standard curve. Enzyme-Substrate. The DuPont luciferin-luciferase system (DuPont Luminescence Biometer Reagent Kit) was selected for its simplicity of preparation and use, as well as for its great stability. The reagent kit included 20 vials of a lyophilized, purified enzyme-substrate powder, 20 buffer-salt tablets (morpholinopropane sulfonic acid buffer and MgSO,) and 500 reaction cuvettes. Each buffer-salt tablet was dissolved in 3 ml of 18 Ms~ water. This solution was used to gently dissolve the contents of one vial of enzyme-substrate powder. Often two or three vials of enzyme-substrate were necessary for large experiments. Using an automatic pipetter, the prepared enzyme-substarte was dispensed in 0.1 ml aliquots into the reaction cuvettes provided with the DuPont kit. The enzyme solution was kept iced from this time on and was not used for ATP analysis for at least 30 min after dispensing. This time allowed the inherent luminescence of the preparation to attenuate. Undue shaking or thermal agitation interfered with the ATP assay. Thermally caused light emission was minimized by holding the enzymesubstrate on ice. As the temperature optimum for the reaction is 25~ (11) the enzyme has greater sensitivity at room temperature (2O-23°C) than when chilled at 0°C (Fig. 2). Since chilling the warmer preparation caused a reduction in the enzymatic activity and the assay was run in a refrigerated instrument, the problem of possible random loss of activity due to cooling was avoided by maintaining all enzyme-substrate, samples, and ATP standards on ice. Under these conditions, the enzyme-substrate preparation and ATP solutions were stable and lost no activity in the I o.o-

x

5.0-

CPM

FIG.

2.

Standard curves for

0°C

(0)

and

23°C

(X

) ambient

temperature,

enzyme.

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AND

HEGRE

course of an entire day. This increased convenience may compensate for the sensitivity loss suffered in using a refrigerated instrument. For instruments normally operated at room temperature, the reverse phcnomenon should be guarded against; that is, variable response as a function of variable warming times. Unused enzyme, previously dispensed into the assay reaction cuvettes, remained active after overnight storage at 4°C. Enzyme activity of 8090% was retained after several weeks of storage at -20°C in sealed containers such as scintillation vials (Fig. 3). This stored enzyme-substrate could have been used for future analysis provided that ATP standards were reanalyzed at that time. While there is some variation in the slope of ATP standard curves for different lots of enzyme substrate, the trend was toward a relatively consistent luminescent response (Table 2). Sample Preparation. The preparation of ATP samples from laboratory cultures of marine plankton was carried out either by directly pipetting samples into boiling Tris buffer, or by extracting a filter pad on which the organisms had been collected. In the case of a direct extraction of marine samples, at least a 25fold dilution of the sample by Tris was recommended (7) since NaCl apparently inhibits the luminescence reaction (11). In these studies 0.2 ml of culture were extracted in 5 ml of boiling Tris. Fdr less dense cultures 25 mm membrane filters (Millipore, 0.45 pm) or 24 mm glass fiber filters (Whatman GF/A) were used to collect a sufficient number of organisms for analysis. The data showed that filtration with either filter type or the direct pipetting of sample

FXG. 3. Activity of frozen (0) to the original enzymatic activity

and

(A).

refrigerated

(X

) luciferin-luciferase

compared

IMPROVED

Evaluation

METHODS

FOR

Mean cpm 4 4

0.25

0 5 10 ‘2 5 5 0 10.0

107

ANALYSIS

TABLE 2 of ATP Standard Activities (cpm) over a 12 Month Period

rg ATP/liter 6.1

ATP

8.38 x 1Oa 2.82 x 104

12

9.39

x

104

13 5 23 11

2.66 x 10” 7.56 X 105 1.73 x 106 3.82 x 106

Standard deviation

c,

0.95 x 103 0.53 x 104 2.0

x

11

19 21 15 16 16

104

0.40 x 105 1.20 x 105 0.29 x 106 0.66 x 106

17

a I\: refers to the number of experiments in which t.hat standard was analysed.

yielded about the same levels of ATP (Table 3). As a further check 14C labeled algae were collected on both filter types and no significant difference was observed in the recovered levels of radioactivity. HOWever, in sample collection by filt.ration, a small amount of the medium must cover the organisms on the filter pad. This was particularly important if it were necessary to rinse the tower and pad or when further sample was added. If the filter became dry and further sample or a rinse was passed through the pad, the cells apparently lysed and ATP recovery was drastically reduced (Table 4). As with laboratory cultures, field samples could also be collected by filtration. Whatever the sample source, it was necessary to stop all enzymatic (phosphatase) activity at the time of sampling in order to maximize ATP recovery. This was accomplished in the laboratory by immediate extraction in boiling Tris. If the filtered samples were frozen at -20°C for several days prior to extraction as little as 2&30% of the ATP remained. Slow freezing and the freezer temperature (-20°C) apparently allowed considerable ATPase activity with the resultant loss of ATP. TABLE 3 Comparison of ATP Recovered (rg/l sample) from Unfiltered (UF), Glass Fiber (GF) or 0.45 pm Millipore (MP) Filtered Samples Sample Cyclotella

nanaD

Cyclotella nana Rhodomonas Rhodamanas

Natural

balticu

baltica marine sample

(1Nomenclature change. Cyclotella psezldonana (Hustedt) Hasle.

UF

GF

MP

-

6.8 33.0 82.5 1.33

6.8 70.0 1.31

35.0 71.2 72.5 nana Hustedt

--

has been changed to Thalassiosira

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CHEER,

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AND

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TABLE 4 ATP Recovery from the Unicellular, Marine Alga Rhodomonas baltica. Direct Extraction Compared with Filtration and Washing Methods

rg ATP Treatment 1. 2. 3. 4.

Filter 2 ml of Filter 2 ml of Filter 2 ml of No filtration.

l/20 diln No wash of filter pad. l/20 diln Wash “wet” filter pad with 5 ml sterile seawater. l/20 diln Wash ‘(dry” filter pad with 5 ml sterile seawater. Direct extraction of 0.10 ml undiluted culture.

per liter culture 68.8 70.0 1.0 71.2

To eliminate ATPase caused losses from field samples where long holding periods were required from the time of collection until extraction, the filtered samples were rapidly frozen with liquid nitrogen or dry ice. ATP samples held in liquid nitrogen or on dry ice were stable for several hours and showed no loss in ATP when compared to samples extracted immediately. Field samples were collected on a 24 mm glass fiber filter in a 25 mm Swinnex filter holder (Millipore Corp.). The water sample was poured or pipetted into a 50 ml Luer-Lok disposable plastic syringe with the Swinnex attached to the Luer-Lok. Filtration by positive pressure required extreme compression to clear the filter pad of the last few ml of sample water. This pressure apparently damaged the more fragile organisms. Therefore, the plunger was left out of the first syringe and a second 50 ml syringe (with plunger in) was connected by tubing to the Swinnex outflow. The sample was placed in the first syringe (reservoir) and a negative pressure was generated by pulling the plunger out on the lower syringe. The use of negative pressure Swinnex filtration appeared to be a much gentler treatment when tested on a laboratory culture of the flagellate Rhodomonas baltica (Table 5). Immediately after the liquid phase cleared the filter pad in the Swinnex holder, the pad was transferred with a pair of plastic insulated forceps to an individual container of liquid nitrogen or placed in direct contact with dry ice. In the field, the liquid nitrogen was kept in vented one quart thermos bottles to prevent explosion of the expanding gas. Labeled Nalgene beakers (30 ml) were fitted into a covered Styrofoam box with labeled cup-shaped depressions. The beakers were filled with liquid nitrogen and a filtered sample was placed in each beaker immediately after collection. Liquid nitrogen from the thermos was added to the beakers as needed in order to keep the pads immersed until extraction. All liquid nitrogen and dry ice treated samples appeared to yield at least, as much ATP as the immediately extracted control samples regardless of

IMPROVED

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ATP

109

ANALYSIS

TABLE 5 ATP Recovery of Samples Collected by Vacuum (- 5 psi) or Swinnex Filtration and/or Treatment with Liquid Nitrogen (LN2) or Dry Ice. Controls (1OOojn Recovery) Were Vacuum Filtered and Immediately Extracted. Tabular Values are Percent of Control. Sample

Filtration

method

Immediate extraction

LN2 treated

Dry ice treated 107 -

Vacuum Swinnex Negative Swinnex

100 90

122 98

117

129

nana

Vacuum Swinnex

100 79

97 97

Natural marine sample

Vacuum Swinnex

100 101

105

Rhodowwnas baltica

Cyclotells

106 -

sample source or method of collection. Some experiments with liquid nitrogen and dry ice actually showed an improved recovery of ATP after this treatment (Table 5). To determine whether the liquid nitrogen had any effect on the analysis itself, Tris extracts of filters containing the particulates from boiled seawater were subjected to liquid nitrogen treatment. ATP standard was then added to this extract. Results of this experiment indicated that t,he liquid nitrogen did not appear to affect the enzymatic ATP assay. Therefore, liquid nitrogen and dry ice probably improved the recovery of ATP from the organisms by reducing possible ATP lossesbetween filtration and extraction. Such losses could have been reduced by rapid freezing, preventing membrane and cell wall leakage of ATP and inhibiting membrane bound ATPases. Extraction. Extraction of either a suspension of organisms directly or of organisms collected on a filter pad were carried out in boiling Tris buffer, pH 7.75. Five milliliters of the buffer were dispensedinto disposable, 20 ml scintillation vials. The buffer had to be maintained at 100 +- 5°C for effective extraction. A boiling water bath was originally used for this purpose. However, the disadvantages of working with boiling water and the inconvenience of maintaining an adequate water level in the course of an experiment prompted a change to a sand bath. This bath consisted of an ordinary electric fry pan filled with about one inch of washed, beach sand, heated at 200°C for 12 hr to remove volatile organic material. The sand bath temperature was maintained at 105-115°C to ensure a Tris temperature of close to 100°C after addition of sample. The Tris-containing vials were capped and preheated for about 5 min. Immediately after

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TABLE 6 Effect of Extraction Time on Recovery of ATP. A. ATP Extracted from 2 ml of Filtered C. nuna Culture. B. ATP Standards (0.5 and 1.0 fig/l) iu Boiling Tris A. C. nanu Min. in boiling Tris

cpm X 1W

2 4 6 8 10

9.23 10.08 8.93 9.17 8.34

B. ATP standards rgATP/liter extract 3.40 3.55 3.15 3.35 3.00

0.5 fig/liter cpm X lo4

1.0 pg/liter cpm X 105

7.89 8.12 8.32 7.60 7.78

2.26 2.30 2.40 2.55 2.38

a sample was filtered, it was immersed in the boiling buffer, the vial was recapped and the sample extracted for 5 min. Longer extraction times were not necessary (Table 6A). At the extraction temperatures, ATP appeared to be stable for at least 10 min (Table 6B) so that in the 5 min extraction period most of the ATP should have been extracted with no loss of ATP through thermal or enzymatic decomposition. In the case of samples which were preserved in liquid nitrogen, either the latter was allowed to boil off just prior to extraction, or the frozen filter pad was carefully removed with insulated forceps. The pad was then held over the vial of heated Tris until it thawed enough to be inserted into the vial without shattering. These samples were then capped and extracted. After 5 min, each sample was transferred to an ice bucket and the cap loosened immediately to prevent loss of sample due to vacuum formation in the cooled vials. After the vials cooled the caps were retightened. At this time the samples were ready for immediate assay or they could be stored indefinitely at -20°C. At the time of analysis, the freshly thawed or extracted samples were transferred to graduated, conical centrifuge tubes. If necessary, the volume was adjusted to 5 ml, with Tris buffer rinses of the vial and filter pad. The 5 ml sample was then mixed on a vortex mixer and kept on ice throughout this procedure and the subsequent assay. Analysis. The ATP-dependent luminescence was detected with a Model 3380 Packard Tri-Carb Liquid Scintillation Spectrometer. Tritium settings (quick set equals 50% gain, 50-1000 discriminator divisions) were found to be most satisfactory. Manual control of the instrument was used for sample loading, starting count, printing count and unloading. The instrument was set for a 30 set counting period, 960,000 preset count, and no background subtract was used. To facilitate the use of the DuPont reaction cuvettes, a holder for

IMPROVED

METHODS

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ATP

ANALYSIS

111

+-20mm-r: I-:. I APUNCHED RUBBER

6.0mm

Frc. 4. Modified

scintillation

SEPTUM

vial.

the cuvette was constructed from a nylon scintillation vial (Fig. 4). Openings were cut in both the cap and in the bottom of the scintillation vial. The cap was fitted with a polyethylene guide backed by a punched rubber septum to hold the cuvette firmly suspended within the scintillation vial. For rapid disposal, the next cuvette to be used was inserted from the top causing the used cuvette to drop out through the bottom of the scintillation vial. Background activity of enzyme-substrate was checked just prior to use. A background of O-100 cpm was considered accept’able. Cuvettes with a higher background were set aside on ice for possible future use if the background level dropped to the acceptable range. Samples or ATP standards (10 ~1) were injected into the enzymecontaining cuvette and immediately loaded and counted. The time elapsed between injection and count, initiation was 3.0 -+ 0.5 sec. To achieve the required precision, a rapid, consistent delivery and mixing of sample was necessary. Use of a hand-operated microliter syringe often gave erratic results; therefore, a pneumatic injector, with automatic refill capability, was designed and built for the ATP analysis (13). RESULTS

AND DISCUSSION

ATP standards were usually run in the anticipated range of the samples extracted. Occasionally a more detailed analysis of standards was carried

112

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out to examine the potential range of the system and the configuration of an expanded standard curve, Most standards were run in the range of 0.1-10.0 pg ATP/liter giving an activity range between 103-lo6 cpm. When necessary, standards were assayed which were as low as 0.01 pug/liter (23 X l(y! cpm) and as high as 20.0 pg/liter (27 X lo6 cpm). However, since sample volumes were flexible, the extracts could easily be prepared to fall within the desired ATP concentration range. Thus, only a portion of the entire standard curve need be run for a given experiment. In general, the entire standard curve (ATP concentration vs cpm) for the range of 0.1-10 pg consisted of two linear portions (Figs. l-3). There was usually a change in slope at 0.5-1.0 pg/l. For the duration of any one experiment, the values for the standards remained constant. Overall, the slopes changed somewhat from day to day with different vials and lots of enzyme or with new standards, but generally, the activities were relatively predictable (Table 2). Analysis of very different sample types (i.e., algae, microcrustaceans) was facilitated by the reliability of the results obtained when either the sample itself or the extract was varied. For example, samples of increasing volumes of the same cell density gave proportionately increasing values for ATP in their extracts. Good agreement was seen in the final ATP per unit culture volume calculated on the basis of these variable sample sizes (Table 7A). Furthermore, samples of variable volume containing the same total number of cells yielded the same concentration of ATP in the extracts, and the calculated values for ATP in the original undiluted culture correlated very well (Table 7B). Therefore, confidence may be placed in the ATP values found regardless of the original sample volume or density of organisms used, as long as those values fall within the range of the standard curve. For samples with very high ATP levels, the extract itself could be diluted before analysis (Table 7C). Again, the ATP levels found in the diluted extracts was proportional to the dilution factor and the final values of ATP per unit of original culture were consistent. Such constancy in the ATP values generated from various sampling and analytical manipulations allowed considerable flexibility in the types of samples routinely examined for ATP content. In the case of natural marine samples taken from near-shore surface waters, a sample volume of 204% ml was suitable, whereas, larger volumes would be needed for less productive areas or great depths, In some instances the natural samples were so rich that the extracts required dilution to facilitate the assay. In addition, the possible enzymatic interference due to high levels of organic or inorganic solids and adsorption of ATP to particulates in such extracts would dictate the assay of a

IMPROVED

METHODS

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ATP

113

ANALYSIS

TABLE 7 Effect of Sample Size Variation (A) Sample Density Variation (B) and Extract Dilution (C) on Final ATP Recovery from C. nuna A. ATP from various volumes of a l/10 diluted C. nana culture. Sample volume of l/10 dilution 1 ml 2 ml 5 ml 10 ml

pg ATP/I

extract-analyzed 0.093 0.15 0.36 0.94

3.72 3.00 2.88 3.76

B. ATP from samples of variable density containing extract. Sample volume 1 ml 5 ml 10 ml 50 ml 100 ml C. Dilution

pg ATP/I

pg ATP/l culturecalculated

extract

0.84 0.78 0.78 0.81 0.77

same number of total cells in the pg ATP/I

culture

3.36 3.12 3.12 3.24 3.08

of ~&act of 4 ml of C. nunu culture. Dilution None l/2 115 l/10

l/20 l/50

pg ATP/l 3.55 1.83 0.65 0.35 0.16 0.067

extract

pg ATP/l

culture

3.55 3.66 3.25 3.50 3.20 3.35

diluted extract (4). Such contaminated field samples showed a lO-20% increase in sample ATP concentration upon dilution of the extract. This might have arisen from the limitations of the scintillation counter itself, since the undiluted extracts were often at the extreme upper range of the instrument’s resolving ability (10-20 pg/liter) . In general, ATP analysis has proved to be a reliable assay technique which we have utilized for several purposes. For example, it has provided a quick, simple test for the contamination of “sterile” water systems. Also ATP analyses have been used for field investigations of the effects of thermal pollution on the microplanktonie biomass. A long term study of local natural waters using ATP as a biological parameter has been initiated. Using both laboratory and natural samples, studies have been

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carried out to determine the ATP content per cell (organism) and per unit biomass (pm3) for numerous marine algal and zooplankton species. In addition alterations in ATP content and its relationship to other physiologic parameters are currently being investigated in both stressed and unstressed populations. These and other studies were facilitated by our investigation of the processes required for ATP analysis. The improvements and modifications which we incorporated into our analysis system have considerably enhanced its reliability, sensitivity and simplicity. The sample preparation technique has been shown to be a critical point for a dependable analytical procedure, requiring care in the filtration, extraction and preservation phases. The utilization of the automated injector and the sensitive, reliable spectrometer contributed greatly to the practical application of this assay. Enzyme-substrate and ATP standard stabilities significantly increased the reliability of the resulting data and reduced the time and effort required. In short, the present state of the technical aspects of ATP analysis should encourage its adoption as a valuable biological parameter. ACKNOWLEDGMENTS We wish to thank assistance.

John

Cardin

and Dr. Peter

Rogerson

for their

technical

REFERENCES 1. PATTERSON,

J. W., BREZONIK,

P. L., AND PUTNAM,

H. 0. (1970) Environ.

Sci. Tech.

4, 569. 2. HOLM-HANSEN, O., AND BOOTH, C. R. (1966) Limnol. Oceanogr. 11, 510. 3. HOLM-HANSEN, 0. (1969) Limnol. Oceanogr. 14, 740. 4. LEE, C. F., HARRIS, R. F., WILLIAMS, J. D. H., ARMSTRONG, D. E., AND SYERS, J. K. (1971) Soil Sci. Sot. Amer. Proc. 35, 82. 5. LEE, D. F., HARRIS, R. F., WILLIAMS, J. D. H., ARMSTRONG, D. E., AND SYERS, J. K. (1971) Soil Sci. Sot. Amer. Proc. 35, 86. 6. SHARPE, A. N., WOODROW, M. N., AND JACKSON, A. K. (1970) J. Appl. Bacterial.

33, 758. 7. HAMILTON, R. D., AND HOLM-HANSEN, 0. (1967) Limnol. Oceanogr. 1.2, 319. 8. SCHMIDT, G. L., AND KAMEN, M. D. (1971) Arch. Microbial. 76, 51. 9. ST. JOHN, J. B. (1970) And. Biochem. 37, 469. 10. HOLM-HANSEN, 0. (1970) Plant Cell Physiol. 11, 11. STREHLER, B. L. (1966) in Methods of Biochemical

16, p. 99, Inter-science, New York. 12. Instruction Manual 760 Luminescence Biometer & Co., Inc., Wilmington, Delaware. 13. JOHNSON,

R. GENTILE,

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AND CHEER,

689. Analysis

(Glick, D., ed.), Vol.

(1970) E. I. DuPont

S. (1974)

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De Nemours 60, 115.