Limits of Applicability of the Firefly
Luminescence ATP Assay for
the Detection of Bacteria
in Clinical Specimens
R E X B. C O N N , M.D.,
PATRICIA C H A R A C H E ,
AND E M M E T T W. C H A P P E L L E ,
M.D.,
M.S.
Department of Laboratory Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205,
and NASAIGoddard Space Flight Center, Greenbelt, Maryland 20771
ABSTRACT
Conn, Rex B., Charache, Patricia, and Chappelle, Emmett W.: Limits of
applicability of the firefly luminescence ATP assay for the detection of
bacteria in clinical specimens. Am J Clin Pathol 6 3 : 4 9 3 - 5 0 1 , 1975. ATP
measurement can be used as an indicator of biological mass, and the
extreme sensitivity of the firefly ATP assay has led to its use in bacterial
detection systems. Clinical specimens present problems not encountered
with cultured isolates of known bacterial species. T h e lower limit of
sensitivity for detecting bacteria using the firefly assay is 100,000 bacteria
per ml. Non-bacterial ATP, which is probably present in all clinical
specimens, produces false-positive results unless it is completely destroyed,
and this destruction must be carried out under conditions that do not affect
bacterial ATP. A cause of false-negative results is the presence in all urine
specimens of unidentified materials that inhibit the luminescent enzymic
reaction. These considerations indicate that application of the firefly ATP
assay in bacterial detection systems for clinical specimens is feasible only if a
preparatory step separates bacteria from interfering materials and from
non-bacterial sources of ATP, and concentrates microorganisms to measurable levels. These limitations sharply curtail the applicability in diagnostic
microbiology of this exotic chemical reaction. (Key words: Luciferase;
Bacteriuria; Adenosine triphosphate; Bacterial detection.)
AVAILABILITY of specific and effective
antimicrobial agents has enhanced the
need for more rapid methods in diagnostic microbiology, preferably methods that
Received November 11, 1974; accepted for publication December 9, 1974.
This work was carried out at T h e Johns Hopkins
Hospital and was supported by Contract #GSFC
71-3(5) NASA/Goddard Space Flight Center.
Address reprint requests to Dr. Conn, Department
of Laboratory Medicine, The Johns Hopkins Hospital, 601 N. Broadway, Baltimore, Maryland 21205.
493
do not require growth of microorganisms.
Standard cultural technics, because of the
growth requirement and the necessity for
intermediate decisions during the identification process, introduce obligatory delays in laboratory processing. This problem can be approached by separating the
overall procedure into its component
steps: (1) detection of microorganisms, (2)
quantitation, (3) identification, (4) antibiotic susceptibility testing.
494
CONN, CHARACHE, AND CHAPPELLE
Chemical methods for detecting bacteria present two major problems: (1)
most constituents and metabolic products
of bacteria are common to the host and
therefore are found in clinical specimens;
(2) because of the small total mass of
bacteria in clinical specimens, few
methods offer sensitivity sufficient to detect their presence. A chemical method
that has been studied extensively is based
upon measurement of adenosine triphosphate (ATP) using the firefly luciferase
reaction. 8,11 Although ATP is ubiquitous
in living organisms, the extreme sensitivity of the firefly assay makes this measurement attractive for use in bacterial
detection systems. All procedures require
separation or destruction of non-bacterial
ATP, and two approaches have been
used: those which require initial separation of bacteria from other constituents in
the specimen, and those utilizing direct
chemical measurement. Filtration or centrifugation can aid in the separation of
bacteria from interfering materials prior
to measurement of ATP, and with these
technics bacteria may be concentrated to
increase sensitivity by processing larger
volumes of sample. Direct extraction procedures, without filtration or centrifugation, offer the advantages of simplicity
and the possibility of automation. The
major difficulty encountered with direct
extraction procedures has been interference by non-bacterial sources of ATP;
they share with all methods the problem
of limitations set by inherent sensitivity.
Chappelle and Levin 2 first reported use
of the firefly luminescence ATP assay for
detecting microorganisms, although previously other investigators had utilized it
to measure bacterial ATP. D'Eustachio
and Levin 6 measured ATP levels in several species of bacteria and found a
relatively constant level throughout all
phases of growth. D'Eustachio and
Johnson 4 extended these observations to
a d d i t i o n a l species of bacteria and
A.J.C.P. —Vol. 63
examined the effects of various extraction
procedures on ATP recovery. These authors later compared bacterial ATP measurement with standard cultural technics
and discussed use of the ATP method for
detecting bacteria in various situations. 5
T h e development of instruments for
measuring light emitted in the luciferase
reaction has understandably influenced
the application of ATP measurement in
various areas. The intensity of the initial
light flash resulting from the chemical
reaction may be measured with a recording photometer, and several instruments
for this purpose are available (discussed
below). Another approach is to measure
the decay of the flash using a liquid scintillation spectrometer. 3 Picciolo, Kelbaugh,
Chappelle, and Fleig10 have extended the
instrumental approach to include automation of the preparatory steps required to
utilize ATP measurement for detecting
the presence of bacteria in clinical specimens. These investigators developed a
direct perchloric acid extraction method
for measuring bacterial populations in
urine specimens and constructed a fully
automated instrument for carrying out all
steps in this procedure. 7 Royalty-free
nonexclusive licenses for commercial use
of this instrument are being offered by its
developers. 9
The intriguing prospect of utilizing
chemical measurements for direct detection of bacteria, and perhaps the rather
exotic approach of using firefly tails in a
chemiluminescent reaction, may have induced a proclivity to overlook specific
limits of applicability of the luciferase
reaction in bacterial detection systems.
The purpose of the study has been to
identify and quantitate these limitations.
Materials and Methods
All direct extraction methods tested
utilized the same sequential steps for
detecting bacterial ATP: addition of a
April 1975
FIREFLY ATP ASSAY FOR DETECTING BACTERIA
lysing agent to destroy mammalian cells;
treatment with apyrase (an ATPase isolated from potatoes) to hydrolyze ATP
released from the mammalian cells or
already present in solution; destruction of
apyrase activity; rupture of bacterial cells;
extraction of released ATP and measurement of the latter.
Reagents for the luciferase reaction
were obtained as the Luminescence
Biometer Reagent Kit from E. I. DuPont
DeNemours and Company, Wilmington,
Delaware. The manufacturer's directions
were followed in preparing reagents and
using the kit. Other reagents were prepared using sterile, deionized, distilled
water which was monitored by determining the "blank" ATP level. Other materials used included Tris buffer (trishydroxymethylamino methane), Triton X100 (octylphenoxypolyethoxyethanol) and
potato apyrase from Sigma Chemical
Company, St. Louis, Missouri, and ATP
for p r e p a r i n g standards from Calbiochem, La Jolla, California. Reagentgrade perchloric or nitric acid, diluted
appropriately, was used to rupture bacterial cells. All other chemicals used were
reagent grade.
Except where noted, the following procedure was used:
(1) Urine, 1 ml., was mixed with 0.1 ml.
1% Triton X-100 and 0.1 ml. of
apyrase (50 mg. per ml. in 0.03 M
CaCl2) and allowed to stand at room
temperature for 10 minutes.
(2) The treated sample was then placed
in a boiling water bath for 10 minutes
to destroy the apyrase activity.
(3) Bacterial cells were ruptured by adding 0.1 ml. of 1 N perchloric acid to
the sample and allowing it to stand for
5 minutes.
(4) The perchloric acid was neutralized
by the addition of 0.1 ml. of 1 N
KOH. The final pH of the sample was
brought to 7.4 by addition of 0.1 ml.
of 1.5 M Tris buffer, pH 7.4.
495
(5) 0.1 ml. of the sample was then used in
the luciferase-luciferin reaction.
When all components of the luciferase
reaction without added ATP are placed in
the instrument cuvette, a low light emission is observed due to residual ATP
bound to luciferase molecules and possibly to protein contaminants. Injection of
distilled water into the reaction mixture
will cause a slight and variable increase in
this endogenous luminescence, possibly
due to displacement of additional ATP
from binding sites. To achieve maximum
sensitivity and to maintain precision at low
ATP levels it is important that endogenous luminescence be low and, more important, constant during the period when
instrument readings are taken. Freshly
prepared enzyme solutions may give significant light readings in the absence of
a d d e d A T P , but this e n d o g e n o u s
luminescence decays to a low, constant
level if the solution is permitted to stand
at room temperature for 60 minutes.
T w o i n s t r u m e n t s were used, the
Luminescence Biometer, manufactured
by E. I. DuPont DeNemours and Company, and the Chem-Glow Photometer,
manufactured by American Instrument
Company, Division of Travenol Laboratories, I n c o r p o r a t e d . Both instruments were designed for measurement of
luminescent reactions. In the Biometer an
analog signal from the photomultiplier
tube is amplified and utilized to charge a
"memory" capacitor to a voltage proportional to the peak amplitude of the light
measured. The peak voltage is then converted into a digital display by measuring
the time r e q u i r e d for a calibrated
linearly-rising signal to become equal in
amplitude to the signal stored in the
memory capacitor. This design reduces
instrument response time to virtually zero
and provides the convenience of a digital
display. The instrument also provides
automatic range change over 5 decades to
496
CONN, CHARACHE, AND CHAPPELLE
A.J.C.P. —Vol. 63
Table 1. Effect of Extraction Mixture on ATP Measurement
% Recovery
ATP
(Concentration
/i.M per ml.)
Extraction M xture
ATP
Tube 1
Tube 2
Tube 3
Tube 4
Control
Triton
Ca
Apyrase
PCA
10"4
10"6
0
0
100
92
73
64
0
100
80
75
63
0
+
0
0
+
+
0
0
0
0
+
+
0
+
+
+
+
+
+
+
+
accommodate a wide range of light intensities. T h e Chem Glow consists of a
reaction chamber-photomultiplier assembly which is used with the Aminco
10-222 Microphotometer. Either peak
height or build-up of bioluminescence
may be measured, the readout being by
meter, strip chart recorder, or oscilloscope. Four decades of range change are
provided; however, range selection is
manual, and if the range selected is
inappropriate for the sample being measured, the entire reaction must be repeated after changing the range.
Results
Sensitivity, Linearity of Response and Precision
of Measurement of Standard ATP Solutions
The limits of sensitivity and reproducibility of the luciferase ATP assay determine the absolute limits of these attributes
in any bacterial ATP detection system.
These absolute limits for aqueous solutions containing known quantities of ATP
were confirmed in our laboratory as an
initial step in the evaluation. Two buffer
systems were tested: Tris buffer (0.01 M,
pH 7.4) and the MOPS buffer (morpholinopropane sulfonic acid, 0.01 M, pH
7.4), which is included in the reagent kit
manufactured by DuPont. Both buffer
solutions contained 0.01 M MgS0 4 .
Identical standard lines were obtained
+
+
+
with the Tris buffer and the MOPS buffer, indicating that the two are equally
suitable for the luciferase ATP assay. The
Biometer and Chem Glow were linear
over the same range, and neither instrument was clearly superior to the other in
regard to the lowest ATP concentration
that could be measured. With aqueous
ATP solutions, the lower practical limit
for measurement was approximately
5 x 103 fg. per ml. (fg. = femtogram
= 10~15 Cm.). At this concentration,
the instrument reading was approximately 2.5 times the blank reading,
and reproducibility was poor. Concentrations of one-tenth this gave readings identical to the blank. Acceptable
precision was obtained over a concentration range from 104 to 5 X 107 fg. per
ml. At higher concentrations instrument
response became non-linear.
Effects of Extraction Mixtures
on A TP Measurement
In all types of direct extraction methods
(i.e., those in which the bacteria are not
isolated prior to extraction), the extractants, although diluted, are carried
through into the cuvette in which the
luciferase reaction takes place. Luciferase,
like most enzymes, is affected by a variety
of inhibitors. Use of extracting agents that
subsequently inhibit the luciferase reaction results in a decrease in sensitivity, but
April 1975
497
FIREFLY ATP ASSAY FOR DETECTING BACTERIA
Bacterial
ATP Equivalent
(fg/ml) (cells/ml)
FIG. 1. Residual ATP, also expressed as equivalent numbers of
bacteria, after treatment of leukocyte suspensions with Triton X-100,
apyrase and perchloric acid. ATP
content of bacteria is assumed to be
2 x 10"' femtograms (fg.) per cell.
2x10'
10",8
2xlOc
10'
2x10°
I0 C
2x10
10
2x10°
10"
10°
\0H
\Qr
I0 C
10'
3
Leukocytes (cells/mm )
process also contain leukocytes. Many
clinical specimens contain erythrocytes,
platelets, and epithelial and mesothelial
cells as well; however, polymorphonuclear
leukocytes are the constant companion of
bacterial inflammation. One of the criteria
for a satisfactory bacterial ATP assay is
that all non-bacterial ATP must be removed prior to lysing bacterial cells and
measuring bacterial ATP. There are
many potentially useful agents for lysing
mammalian cells, destroying ATP, and
then lysing bacterial cells, and the evaluation of all possible combinations is unmanageable. T h e problem was a p proached by selecting those agents that
were considered most effective and that
had been selected as optimal for use in an
automated instrument. 10
Leukocytes were obtained from the
buffy coat of whole blood drawn in
heparin and centrifuged. Cells were
washed with 0.9% NaCl, resuspended,
and diluted in saline solution. The suspensions were then treated with the same
Effects of Residual
procedures that would be used for assayLeukocytic ATP
ing bacterial ATP: Triton X-100, apyrase
Virtually all clinical specimens that con- and perchloric acid. The final ATP valtain bacteria resulting from an infectious ues, therefore, represent leukocytic ATP
if the concentration of extractant is constant these inhibitory effects may remain
constant and thus not impair precision. It
is, of course, essential that acidic extraction mixtures be adjusted to the pH
optimum for Iuciferase prior to carrying
out the reaction.
Triton X-100 was used for lysing nonbacterial cells in urine samples. A variety
of agents may be used for rupturing
bacteria and extracting ATP; perchloric
acid was found highly effective. Table 1
indicates that both Triton X and perchloric acid, as used in the technic described, either are inhibitory to the
Iuciferase reaction or destroy some of the
ATP present, and that these effects are
additive. While the inhibitory effects of
various extractants on the Iuciferase reaction do not prohibit their use in direct
bacterial ATP assays, such effects must be
considered in assessing and optimizing
the sensitivity of the system.
498
CONN, CHARACHE, AND CHAPPELLE
Table 2. Effect of Urine on ATP Recovery*
% Recovery
(ATP Concentration
jtiM per ml.)
Urine
io- 4
10~5
IO"7
Normal, first morning
Normal, midday
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
57
60
75
53
60
68
55
86
28
37
57
66
53
53
70
66
70
48
60
4
38
33
41
99
88
98
85
* Recoveries were calculated from duplicate measurements. Patients
were taking the following medications:
Patient 1, diazepam (Valium), diphenylhydantoin (Dilantin)
Patient 2. isoniazid. thioridazine, allopurinol, hydroxyzine, trimethobenzamide, acetaminophen, procarbazine
Patient 3, isoniazid. acetaminophen
Patient 4, penicillin, propoxyphene
Patient 5, cephalothin
Patient 6. methyldopa (Aldomet)
Patient 7. chlorothiazide, propoxyphene, dicloxacillin
that was not destroyed by this rather
drastic treatment. Results are summarized
in Figure 1. It would be desirable to
measure residual ATP at a lower leukocyte concentration than used; however,
this would result in ATP concentrations
below the limits of detection of the
luciferase method. Although it may be an
artifact due to methodologic imprecision,
the fact that the regression line does not
pass through the origin (or would not if
the scales on the ordinates were comparable) suggests that the number of leukocytes present is not the full determinant
of residual ATP. Absorption of released
ATP onto leukocytic debris, where it may
be unaffected by apyrase, is a possible
explanation, as is binding to proteins
released during cell lysis. Microscopic
examination of the centrifuged sediment
after cell lysis revealed no intact cells,
although it was not determined whether
subcellular particles, such as mitochondria, remained intact.
These results indicate that an essential
criterion for any method to detect bacterial
AJ.C.P.—Vol.
63
ATP in clinical specimens is that the
method must destroy all non-bacterial ATP
prior to disruption of bacterial cells.
Numerous approaches to accomplish this
objective were attempted, but none was
completely satisfactory. Figure 1 indicates
that the presence of non-bacterial ATP
contributed by mammalian cells in numbers commonly found in infected urine
would result in unacceptable loss of accuracy and production of false-positive results in any bacterial ATP assay system.
Effects of Normal and Abnormal Urine
on ATP Recoveries
One of the most useful applications of
the luciferase ATP reaction for detecting
bacteria would be to identify immediately
those urine specimens containing significant numbers of bacteria. However, urine
contains numerous unknown substances
inhibitory to enzymic reactions, and inhibition of the luciferase reaction by urine
would decrease the sensitivity of the bacterial ATP assay, making it more difficult
to detect clinically important numbers of
bacteria. Variation in concentration of
urinary luciferase inhibitors from specimen to specimen would compromise the
quantitative aspect of the assay as well.
Urine specimens were prepared by
filtration through 0.45-micron Millipore
filters to remove cells and bacteria. The
filtrates were treated by the standard
Triton X-100, apyrase, perchloric acid
method. This resulted in destruction of all
measurable soluble ATP. Known amounts
of ATP were then added to aliquots of each
specimen, and the luciferase assay was
carried out. Instrument readings obtained
on the ATP urine solutions were then
compared with readings obtained on
aqueous solutions of ATP having the same
concentrations. All assays were carried out
in duplicate. Results are summarized in
Table 2. Recoveries ranged from 4 to 99%,
with mean recoveries for all three concen-
April 1975
FIREFLY ATP ASSAY FOR DETECTING BACTERIA
499
Table 3. Comparison of Bacterial Counts Using Culture Technic
and ATP Assay on 91 Urine Specimens*
Colony
Count
C FU
per ml.
NG**
102
103
104
105
106
107
>10 7
TOTAL
Number of Specimens in Each Category
Grouped by Log (ATP Assay) Minus Log (Colony Count)
<-5
1
0
0
1
-4
-5
4
0
0
0
0
4
2
0
0
0
2
+2
+3
+4
+5
>+5
Total
0
0
1
1
1
0
2
0
1
1
6
1
2
0
0
11
1
0
0
8
3
0
3
0
0
19
0
5
11
12
11
3
19
33
11
18
12
6
3
5
3
91
-3
-2
-1
0
+1
2
0
0
0
0
0
2
2
0
0
0
0
0
0
2
0
0
0
1
0
0
1
2
11
0
0
0
1
0
3
2
17
1
* Conversion factor: 2 X 10" fg. = 1 bacterial cell. C.F.U. per ml. = bacterial colony forming units per ml. of urine.
** NG = no growth
trations of ATP used being approximately
60%. This extent of inhibition would
adversely affect the sensitivity of the assay.
More serious, however, is the extreme
variation in amount of inhibition, which
inevitably produces loss of accuracy. These
results indicate that direct determination
of bacterial ATP in urine specimens cannot
be carried out, at least with the technics
tested, unless the inhibitory effects of each
urine specimen on the luciferase reaction
are evaluated and used in calculating the
amount of bacterial ATP present.
In an attempt to circumvent the problems related to variable inhibition of the
luciferase reaction by different urine specimens, use of an internal ATP standard
for each specimen was instituted. This
technic involves addition of a known
quantity of ATP to an aliquot of each
urine specimen tested in order to calculate
the percentage inhibition (recovery) produced by that particular urine specimen. This technic would be invalid if the
degree of inhibition varied with the total
ATP concentration in the specimen. To
evaluate this possibility, several concentrations of ATP were prepared in urine specimens and in aqueous solution, and the
luciferase reaction was carried out. As
expected, inhibition varied from urine
specimen to urine specimen. In some experiments, a linear relation between ATP
concentration and instrument readings was
maintained, indicating proportional inhibition at all ATP concentrations. In other
experiments, strict linearity did not occur
(Table 2).
Comparison of ATP Assay and
Cultural Results
Experiments carried out at Goddard
Space Flight Center concurrently with
those described above resulted in modification of the procedure to include an
incubation of the sample-apyrase-TritonX mixture (using a 0.5 M malic acid-0.005
M Na arsenate buffer) at pH 3.75 to
dissociate ATP from binding sites on protein and cellular debris, thus making it
available for hydrolysis by apyrase. At
this pH apyrase is marginally active, but
activity was sufficient for complete destruction of ATP in aqueous solutions. Nitric
acid, 1.5 M, was substituted for perchloric
acid as well.
Urine samples were obtained from the
Bacteriology Laboratory of the Johns
Hopkins Hospital, and thus were from
patients suspected of having urinary tract
infections. Of those available, cloudy urine
500
CONN, CHARACHE, AND CHAPPELLE
A.J.C.P. —Vol. 63
samples were selected. Culture plates were clinical correlation. T h e results obtained
streaked using calibrated loops of two indicate that with present instrumentation
sizes, 0.01 ml. and 0.001 ml., and colony and reagents inherent limitations are sufcounts were made the following day. At ficiently great that Gram stain and culture
the same time urine specimens were cul- remain superior to bacterial ATP assays
tured, aliquots of each were used to quanti- for detecting bacteria in clinical specimens.
tate bacterial ATP. A T P measurements The findings suggest also that data prewere converted to bacterial counts of the viously reported by Picciolo, Kelbaugh,
basis of a factor of 2.0 X 10 _1 fg. per Chappelle, and Fleig,10 indicating a high
bacterial cell.
degree of reliability in the use of ATP
Results are presented in Table 3. In measurement for detecting significant
only 24 of 91 samples tested did the ATP bacteriuria (100,000 organisms per ml.)
assay and colony count agree within ± 1 may have been the result of leukocytes
order of magnitude, and 11 of these 24 present in infected urine and failure to
samples were found to be negative by destroy leukocytic or other non-bacterial
both technics. In 13 of the 91 specimens, ATP rather than actual measurement of
the A T P assay indicated fewer bacteria bacterial ATP.
present than were found by plate count.
In spite of its extreme sensitivity, the
In 61 specimens, the ATP assay indicated luciferase ATP reaction appears to have
more bacteria present than were detected limited potential for direct detection of
by plate count. Particularly significant bacteria in clinical specimens. Its use would
are the 19 samples that were sterile on be restricted to body fluids that are norplate count yet gave results by the ATP mally sterile, since it provides a gross
assay indicating >10 5 bacteria per ml. We measurement of the total number of bacattribute the latter findings to failure to teria present. Use of the assay for detecting
destroy non-bacterial A T P completely. Re- bacteria in blood or cerebrospinal fluid has
sults that indicate a lower bacterial popula- the potential for supplying misleading dition by ATP assay than actually found agnostic information, since the lower limit
by cultural technics could be attributed of sensitivity of the method is such that
to the presence of luciferase inhibitors only massive bacterial infections could be
detected. In positive blood cultures the
in the urine specimens.
number of organisms present rarely exceeds
102 per ml., far below the limit of
Discussion
detection of the luciferase method. A
A rapid, direct chemical method for negative report might delay vigorous applidetecting bacteria in clinical specimens cation of more standard technics and
would be a useful laboratory tool, and the prompt initiation of therapy, while failure
objective of these experiments was to to destroy all non-bacterial ATP would prodetermine the feasibility of this application duce spurious positive results that could
of the luciferase A T P assay and to define lead to inappropriate and possibly dangerthe limits of applicability. The numbers of ous administration of antibiotic agents.
bacteria estimated by Gram stain examinaIf average values for bacterial ATP
tion and by quantitative urine culture are are used in converting results of chemical
known to correlate with the presence or measurements to number of organisms
absence of infection in man. For this rea- present, an additional source of error is
son, grossly divergent estimates of bac- the variation in ATP content among bacterial counts calculated from the luciferase terial species. For example, Picciolo, Kelassay must be assumed to be without baugh, Chappelle, and Fleig10 found ATP
April 1975
FIREFLY ATP ASSAY FOR DETECTING BACTERIA
contents per cell to range from 0.5 x 10 _1
fg. per cell for Pseudomonas to 8.2 x 10 _1
fg. per cell for S. epidermidis. Other studies
have indicated similar variability in ATP
contents of bacteria. 6 The lower limit of
sensitivity for measuring ATP in aqueous
solutions was found to be 104 fg. per ml.
Even when effects of dilution during the
preparatory process, inhibition by urinary
constituents, and inhibition by the extractants are not considered, calculation of the
lower limits of bacterial numbers that can
be detected using the firefly luminescence
reaction substantiates other evidence that
direct extraction procedures cannot be
applied to clinical specimens. For Pseudomonas the limit is 200,000 organisms per
ml.; f o r £ . coli, 83,000 organisms per ml.;
for 5. epidermidis, 12,000 organisms per ml.
The luciferase ATP assay may have uses
in medical microbiology other than
the detection of bacteria in clinical specimens. Ames 1 has shown that ATP measurement can be used to quantitate bacterial
growth in antimicrobial susceptibility tests,
and no doubt it could be used to detect
growth in the speciation process as well.
In these applications the potential advantages in shortening the time required to
detect and quantitate bacterial proliferation must be weighed against the merits of
alternative approaches as well as the relatively high cost of using the firefly luminescence reaction for this purpose.
In summary, the use of ATP measurement for detecting and quantitating bacterial populations should be limited to
conditions in which all non-bacterial ATPcontaining cells can be completely excluded. Unless the assays are carried out on
pure strains, or the species of the organism is known, there may be as much as
16-fold error in calculation of bacterial
cells on the basis of average ATP content.
The medium in which the bacteria are
501
suspended should not be inhibitory in the
luciferase ATP reaction, or, if such an inhibitory effect is present, it should be circumvented by utilization of an internal
ATP standard for each specimen to arrive
at a calculated correction of measured
ATP concentration. Last, the limitations
imposed by the sensitivity of the luciferase
reaction must be recognized, and specimens must be concentrated to bring the
total number of organisms within the
measurable range.
Miss Elaine Budd, MT(ASCP) and Miss Amalia
Franco provided technical assistance.
References
1. Ames JS: T h e adenosine triphosphate assay
for determination of bacteriuria and antimicrobial sensitivity: 1-151. Unpublished
thesis (M.S. degree), University of Wisconsin,
Medical Microbiology Department, 1970
2. Chappelle EW, Levin GV: Rapid microbiological
detection. Navy Contractor Report 178-8097,
1964
3. Cheer S, Gentile J H , Hegre CS: Improved
methods for ATP analysis. Anal Biochem 60:
102-114, 1974
4. D'Eustachio AJ, Johnson DR: Adenosine triphosphate content of bacteria. Fed Proc 27:761,
1968
5. D'Eustachio AJ, Johnson DR, Levin GV: Rapid
assay of bacterial populations. Bacteriol Proc
68:13, 1968
6. D'Eustachio AJ, Levin GV: Levels of adenosine
triphosphate during bacterial growth. Bacteriol
Proc 67: p. 121-122, 1967
7. Kelbaugh BN, Picciolo GL, Chappelle EW, et al:
Automatic Instrument for Chemical Processing
to Detect Microorganisms in Biological Samples
by Measuring Light Reactions. U.S. Patent
3,756,920, September 4, 1973
8. McElroy WD, Seliger HH, White EH: Mechanisms of bioluminescence, chemiluminescence
and enzyme function in the oxidation of
firefly luciferin. Photochem Photobiol 10:
153-170, 1969
9. NASA Tech Brief. 71-10051, Goddard Space
Flight Center
10. Picciolo GL, Kelbaugh BN, Chappelle EW, et al:
An automated luciferase assay of bacteria in
urine. NASA GSFC Document X-641-71163, 1971
11. Strehler BL, Trotter JR: Firefly luminescence
in the study of energy transfer mechanisms.
I. Substrate and enzyme determination. Arch
Biochem Biophys 4 0 : 2 8 - 4 1 , 1952