United States
 Environmental Protection
 Agency
Municipal Environmental Research,
Laboratory
Cincinnati OH 45268
Research and Development
 EPA-600/S2-81-014  Apr. 1981
Project  Summary
Firefly  Luciferase ATP  Assay
Development for  Monitoring
Bacterial  Concentrations in
Water  Supplies
Grace L Picciolo, EmmettW. Chappell, Jody W. Deming, Richard R.Thomas,
D. A. Nibley, and Harold Okrend
  This research program was initiated
to develop a rapid, automatable system
for measuring total viable microorga-
nisms in potable drinking water supplies
using the firefly luciferase adenosine
triphosphate (ATP) assay.
  The ATP assay was adapted to an'
automatable flow system that, in less
than 2 minutes, provided assays with
sensitivity comparable with establish-
ed methodology (105 bacteria/mL).
Quality controls for required reagents
were established. To achieve the sensi-
tivity necessary for bacterial measure-
ments in water, the sample must be
concentrated before assay. Filtration
systems were evaluated for ability to
concentrate bacteria from large volume
samples rapidly, efficiently, and with-
out damage to the organisms. Results
indicated most filtration systems test-
ed had a limited capability to meet
project criteria. Promising results
(200- to 600-fold  concentration and
up to 88% recovery of bacteria) were
obtained using hollow-fiber concentra-
tion systems modified to incorporate
repeated backwash steps.
  This Project Summary was devel-
oped by EPA's Municipal Environmen-
tal Research Laboratory, Cincinnati,
OH, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction

  Current methods for examining bac-
teriological quality of potable water
supplies depend on time-consuming
culture procedures. Efficient control of
contamination breakthroughs into fin-
ished waters or detection of water
quality deterioration in distribution
networks is delayed until results from
the coliform test and standard plate
counts become available. A method for
rapid measurement of bacterial concen-
tration in potable water would greatly
enhance water quality maintenance
and control. Criteria for an automated
assay include sensitivity comparable to
established methodology, minimum
mechanical manipulations, real-time
analysis, and quality controlsfor required
reagents.
  Intracellular adenosine triphosphate
(ATP) extracted from microorganisms
can be detected and quantified with the
use of the firefly luciferase ATP assay.
This assay is a rapid,  "wet chemical"
reaction that is easily automated. Rela-
tive ATP concentrations could be used
to monitor changes in the levels of
microbial populations as well as detect
contamination breakthroughs into fin-
ished water supplies or water quality
deterioration in network distribution
systems in "real time." If the majority of
microbes in the water supply are assumed
to be bacteria, ATP levels could  be

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further used to estimate the total number
of bacteria present. Experimental data
indicate that the deviation of ATP con-
tent of bacteria, accounting for  differ-
ences  between species and variation
during the growth cycle, does not exceed
one order of magnitude.
  The sensitivity limit of the ATP assay
with pure cultures of bacterial strains is
about 10s bacteria/mL Assuming 500
bacteria/mL as an acceptable  upper
limit for the standard plate count popu-
lation in drinking water, contaminating
levels of bacteria must be concentrated
by a factor of at least 200 to permit
detection by an ATP assay. For example,
a 20-L sample volume containing 500
bacteria/mL would have to be reduced
to 100 mL with complete retention  of
bacteria to provide a concentration
equal to the sensitivity limit of the assay.
To ensure operation above the minimum
sensitivity limit, bacterial concentration
greater than 105 cells/mL would be
desirable.
  This study focused on development of
an automated ATP assay and a suitable
technique for concentrating low levels
of bacteria from large sample volumes.
The concentration technique had to: (1)
be  compatible with automation,  (2)
provide a high concentration factor, (3)
have a rapid concentration rate, and (4)
yield complete recovery of intact, undam-
aged bacterial cells in a reduced volume
that could be diverted  to an automated
ATP assay system.
Automated A TP Assay
  The established method for bacterial
ATP assay generally includes adding
ATP extractant by pipet to the bacterial
sample in a polypropylene test tube,
diluting by pipet, mixing, distributing the
enzyme mixture into individual cuvettes,
and finally, injecting 0.1 mL extracted,
diluted sample into a luciferase-contain-
ing cuvette positioned in front of a
photomultiplier tube.
  The firefly luciferase ATP assay was
adapted to an automated flow system
that greatly reduced the  mechanical
manipulations required (Figure 1). Buch-
ler peristaltic pumps move both sample
and reagents through interconnected
tubes. Relative flow rates shown in
Figure 1 provide the optimal concentra-
tion of nitric acid extractant (0.1  N), with
minimal sample dilution (50%),  and 0.2
mL of  luciferase enzyme solution.  The
luciferase enzyme is pulsed into the
flow system only as the final processed
sample reaches the glass coil positioned
next to the photomultiplier tube. A
Chem  Glow photometer* (American
Instrument Company) equipped with a
coiled  flow cell was used to measure
maximum light output. Total light pro-
duction was measured by coupling an
Aminco Integrator-Timer to the photo-
meter. Sample ATP concentrations
were compared with ATP standards(0.1
fjg ATP/mL); the latter were prepared by
diluting purified ATP (Sigma Chemical
Company) in sterile, deionized water.
  ATP  concentrations were converted
to bacterial numbers using the conver-
sion factor of 2.5 x 10~10 /ug ATP/cell,
which represents the average ATP
content of 19 bacterial  species. For
application  of the flow ATP assay to
potable water analysis, the ATP conver-
sion factor should be determined using
bacteria isolated from actual water
samples.
  Figure 2 shows typical ATP concentra-
tion curves determined for Escherichia
coli in saline and in tap water using the
flow ATP assay.
  Nitric acid (0.6 N) extraction was most
suitable for bacterial ATP and was
optimized for use in the  flow  system.
Use of acid extraction requires  that the
                            final sample be buffered to pH 7.75 to
                            optimize the luciferin-luciferase enzyme
                            reaction.
                              Luciferin-luciferase extracted from
                            dessicated firefly lanterns was obtained
                            in purified  form from E. I.  Dupont de
                            Nemours and Company, Inc., or was
                            prepared in the laboratory. Use of highly
                            purified  luciferase  requires adding
                            synthetic luciferin and results in 100-
                            fold increase in test sensitivity.
                              Purified luciferase is rehydrated in
                            0.25 M TRIS buffer containing 0.01  M
                            MgS04 and 0.001 M  Cleland's reagent
                            (dithiothreitol). When  Cleland's reagent
                            is included, stability of rehydrated
                            luciferase increases from  4  hours at
                            10°C to 8 hours at 10°C. At least 60% of
                            functional  activity can be preserved at
                            10°C in the dark for 48 hours by adding
                            0.001 M  EDTA. Since luciferase is an
                            enzyme, precaution must  be  taken to
                            avoid denatuation due to overheating or
                            exposure to marked temperature fluctu-
                            ations. For ATP assay, the  enzyme
                            preparation should be brought to 20°C.
                            To minimize  inherent light from the
                            luciferase  mixture, rehydrated enzyme
                            should be  incubated at room tempera-
                            ture for 30 minutes before assay.Re-
                            maining inherent light should be less
            Relative
           Flow Rates
           J.20 ml/min
                                                                  60 sec
                                                                Residence
                                   Drain
                                                           Flow Head

                                                               Glass Coil
                                                         Photomultiplier
                                                              Tube
                                                       Chem Glow
                                                       Photometer
                  0.25 M TRIS-pH 8.2
                    0.01 M MgS04
               0.001 M Cleland's Reagent
                                                       Recorder
                                      To Drain
Figure 1.
Schematic of automated firefly lucifera.se flow system for detecting
bacterial A TP including nitric acid extraction and subsequent dilution.
"Mention of trade names or commercial products
does not constitute endorsement or recommenda-
tion for use.

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 Is
 u.
 •Q
 r
                                                       Legend:
                                                       O in Saline
                                                       • in Tap Water
                      1
Figure 2.
                     6789
                        Log Bacteria/'mL by Plate Count

            Concentration curve ofE. coli in saline and in tap water; bacteria/mL by
            A TP flow vs. plate count.
than  10%  after 30 minutes, and an
 ppropriate control cuvette will indicate
the inherent light correction needed.
  ATP standards were prepared with
the use of purified chemical ATP (Sigma
Chemical Co.) in 0.001 M EDTA and
0.01 MMgSCv The purified ATP is used
for daily quality control and for preparing
ATP standard curves for determining
sample ATP concentrations.
  Loss of sample bacterial ATP due to
hydrolysis by nitric acid extractant was
minimized  by 50% dilution  after 60-
second extraction, followed immediately
by reaction with the luciferase enzyme
system.
                                        for subsequent assay using the ATP
                                        flow system. Recovery in a small volume
                                        of water rather than on a membrane
                                        surface would permit measurement of
                                        the bacterial population by standard
                                        plate count, Coulter count, or other
                                        method to confirm ATP assay results.
                                          Additionally, the concentration pro-
                                        cedure must be able to process large
                                        volume samples (at least 10 L) in a time
                                        period compatible with the desired
                                        sampling frequency without damaging
                                        the cells or significantly altering the cell
                                        ATP content.
                                          Potential concentrator! techniques
                                        were tested for concentration of known
     densities of E. co//from sterile, deionized
     water. After a minimum of three test
     runs, if a  concentration factor of 10
     could not be achieved with 50% recovery
     of bacteria, further work with the tech-
     nique was abandoned. The percentage
     of bacteria recovered in the final concen-
     trate was determined by enumerating
     the bacteria before and after concentra-
     tion. Standard plate counts, ATP assays.
     Coulter counts, luminol  iron porphyrin
     assays, or a combination of these methods
     was used to determine cell concentration.
        Four basic concentration techniques
     were tested; for some techniques, more
     than one type of unit was evaluated. The
     techniques tested were: centrifugation;
     direct in-line filtration, flat-surface
     membrane filtration, and hollow fiber
     filtration.

     Centrifugation
        Continuous  flow centrifugation pro-
     vided large sample volume processing
     with about 75% bacterial recovery, but
     concentration factors were very low.
     Mechanical manipulations involved in
     collecting the bacterial concentrates
     were not suitable for automation, so
     work on this procedure was discontinued.

     Direct In-Line Filtration
        Sample under pressure was passed
     through a 0.45 /urn cellulose acetate
     filter (Swinnex filter) unit to concentrate
     bacteria. After sample filtration, attempts
     to backwash with small volumes of
     deionized water to resuspend cells for
     the ATP assay  were unsuccessful.
     Adequate seals to prevent leakage could
     not be made using Nucleopore polycar-
     bonate filters.
       With 10-fold concentration of bacter-
     ial cells, attempts to assay ATP using
     direct extraction  of ATP  after filtration
     resulted in an average of only 18%
     recovery (Table 1).
Bacterial Concentration
Procedures
  To detect contaminating levels of
microorganisms in potable water using
the firefly luciferase ATP assay, the
organisms must first be concentrated to
achieve at least the minimum sensitivity
cell concentration of 105 cells/mL. For
samples containing  500/mL, 200-fold
or greater concentration must be achieved.
If concentrated by membrane filtration,
the cells must be recovered intact and
undamaged in a small volume of water
                                        Table 1.    Test Results Using Mill/pore In-Line Filter Concentration with In-Line
                                                   Extraction
                                                       In-Line Extraction
                                            Sample       fag A TP/mL)
External Extraction
   fag ATP/mL)
 % Recovery with
In-Line Extraction
1
2
3
4
5
3.3 x 1Q-*
3.5 x W*
2.5 x W4
8. 1 x 10~*
1.0x10-*
1.9 x 10~3
1.85 x 10'3
1.8 x 10~3
1.08 x 10'3
3.3 x W*
17
19
14
7
30

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Flat-Surface Membrane
Filtration

  Sample concentration by membrane
filtration systems that use flat surfaces
and high sample flow velocities parallel
to the membrane filter surface was
explored. The sample is concentrated by
volume loss through the filter, but
particles are retained in a decreased
sample volume (the retentate) (Figure
3). Sample flow parallel to the membrane
surface reduces particle buildup on the
membrane surface. Three systems
utilizing this principle were tested;
these were Uni-pore Radial and Stirred
Flow Cells (Biorad Laboratories), Sartor-
ius Ultrafiltration System (Sartorius
Corporation), and the Pel I icon Cassette
Molecular Filter (Millipore Corporation).
  All flat-surface systems yielded poor
results when tested with bacterial
suspensions, and recoveries were less
than 50%. A backwash step was added
in an attempt to improve bacterial
recoveries from such systems. Back-
washing alone proved to be insufficient
to recover the bacteria, and the volume
of backwash required often negated any
concentration effect. Adding Triton X-
100 (a nonionic detergent) or 0.1%
Rhozyme (proteases and glucosidases
mixture isolated from Aspergillus oryzae)
to samples as a filtration  aid did not
consistently improve bacterial recovery
with any of the flat-plate systems, and
concentration factors greater than 10
could not be achieved (Table 2).
Hollow-Fiber Membrane
Filtration

  Hollow-fiber membrane filters also
incorporate sample flow parallel to the
membrane surface, but the membrane
configuration is tubular and has pores of
controlled dimensions (Figure 4). The
sample solution  is pumped through  a
bundle of the hollow fibers, and particles
larger than the pore size of the particular
hollow-fiber  filter in use are retained.
Particle concentration occurs when low
molecular weight solutes and water
pass through the membranes into the
filtrate. The sample volume is recircu-
lated to achieve further volume reduction
and consequent sample concentration.
Backwashing was also necessary with
the hollow-fiber membrane filter sys-
tems. Two hollow-fiber member filter
systems, the Bio-Fiber 80 Mini-plant
(Biorad Laboratories) and the Diaflow
Hollow Fiber Concentrator (Amicon
Corporation), were tested.
  The Bio-Fiber 80 Mini-plant yielded
up to 83% bacterial recovery at concen-
tration factors of 15 to 250 with the use
of repeated backwashing and reconcen-
tration steps. However, the unit was
removed from the market and is no
longer available. The Amicon Diaflow
Hollow Fiber unit, with a surface area of
1,000 cm2 and 100,000-molecular
weight (m.w.) cutoff, yielded nearly
100% bacterial recovery at 10-fold
concentration without backwashing
(Table 3). When the concentration factor
was raised to 100 fold, recoveries
dropped to only 32%. A dual cartridge
unit with 50,000-m.w. cutoff cartridges,
operated to provide 100-fold concentra-
tion without backwashing, gave incon-
sistent recoveries that ranged from 43%
to 100%. After modification to permit
backwashing, a 10,000-cm2 cartridge
yielded recoveries of 53% (single back-
wash) to 88% (three backwashes).
  Hollow-fiber concentrators modified
for backwash capability proved to be the
only concentration systems tested that
allowed greater than 200-fold concen-
tration of bacteria with adequate recov-
ery of the organisms (88%). The hollow
fibers of the Amicon Diaflow  unit are
made of noncellulosic  polymers that
should be durable for a period of months.
For extended use, cleanup steps may be
necessary to prevent the buildup of
bacteria within the fibers. Although a
30-minute  rinsing procedure using
sterile deionized water was effective
between samples during testing, use of
0.1  NaOH  followed by sterile deionized
water rinse may be necessary during
long-term  use.
  Manpower constraints forced Goddard
Space Flight Center to  discontinue
projects not directly related to the space
mission. They were, therefore, unable
to evaluate and develop a satisfactory
concentration system to go with the
flow system for the ATP assay. Although
preliminary results appeared promising,
extended  testing of the  hollow-fiber
concentrator (backwash modified) using
                          Retentate
                 Buchler
                Peristaltic
                  Pump
                                              Sartor/us
                                            Ultrafiltration
                                              System
                                                                    Filtrate
                                                        Backwash
   Sample
Reservoir and
 Concentrate
  Collection

Figure 3.    Schematic of Ultrafiltration System using tangential flow and backwash.
             System is diagrammed in concentrating mode; valves are rotated 90°
             for backwashing provided by pressurized tap source of sterile,
             deionized water.
                                        Table 2.    Test Results Using
                                                   Sartorius Ultrafiltration
                                                   System with Tangential
                                                   Flow and Backwash

                                        Concentration    %
                                           Factor*    Recovery Filtration Aid
2
4
10
10
10
5


5

79
63
21
90
92
50


80

_ - _ _

	
With TX
With Rhozyme
Cellulose
Acetate Filter,
TX
Polycarbonate
Filter, TX
                                        *Each factor represents a separate
                                         test.

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drinking water samples is needed before
final recommendation can  be made. If
performance of the concentrator proved
satisfactory with potable water samples,
additional testing of the system, includ-
ing the  automated  flow  ATP assay
would be necessary to establish overall
system performance,  sensitivity, repro-
ducibility, and reliability.
  The full  report was submitted in
fulfillment of Interagency  Agreement
No. EPA-IAG-D6-0982 by NASA/God-
dard Space Flight Center under the
sponsorship of the U.S. Environmental
Protection Agency
Concentration Mode:
    Sample
J_

T , .1.

J '
    Backwash and
   Collection Mode:
                                                       Filtrate
Concentrate .
1 — ' 1 — '
1


A


                                                        Water
                                                     Backwash

Figure 4.    Schematic of Amicon hollow fiber cartridge modified for backwash
            capability.
Table 3.    Test Results with the Use of Amicon Hollow Fiber Cartridge with
           Backwash
                         Maximum
    Cartridge Size        Concentration         Test
   Filter Area (cm)
Factor
Procedure
^Standard deviations calculated on the basis of test runs.
< Recovery + o*
1,000
10.000








60 Backwash
600 Backwash
Backwash,
Refilter,
Backwash
Backwash,
Refilter.
Backwash,
Refilter,
Backwash
95 ± 5
53 ±21

83 ± 14



88 ± 12



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Grace L Picciolo, Emmett W. Chappell, Jody w. Darning, RichardR. Thomas, and
  D. A. Nibley were with the NASA/GoddardSpace Flight Center at the time this
  research was performed; Harold Okrend was with Howard University. All
  inquiries should be directed to the EPA Project Office.
Donald Reasoner is the EPA Project Officer (see below).
The complete report, entitled "Firefly Luciferase ATP Assay Development for
  Monitoring  Bacterial Concentrations  in   Water Supplies,"  (Order  No.
  PB 81-163 271; Cost: $8.00, subject to change) will be available only from:
        National Technical Information Service
        5285 Port Royal Road
        Springfield. VA 22161
        Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
        Municipal Environmental Research Laboratory
        U.S. Environmental Protection Agency
        Cincinnati, OH 45268
                                                                                     > US GOVERNMENT PRINTING OFFICE: 1961-757-012/7075

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