Technical Bulletin

AccuCount™ Technology
The ATP+AMP Assay

Introduction techniques (such as the ATP test) have challenged

that reality, leading to a new understanding that such
nd
Building on the revolutionary capabilities of 2 techniques tend to underestimate the total population
Generation ATP measurement foundation, the new by orders of magnitude.
AMP Enzyme reagent is a proprietary formulation
In spite of the recent developments that are capable
modified from ATP Enzyme that enables the
of quantifying metabolically active cells (e.g. ATP), or
detection of both Adenosine Triphosphate (ATP) and
the total (alive + dead) bioburden level in the sample
Adenosine Monophosphate (AMP) and available
(e.g. qPCR), there remains a need to be able to
exclusively in AccuCount test kits. This additional
distinguish separately those cells that are
capability empowers users with new insight into
metabolically active and those that are alive yet
microbial populations, including the ability to detect
metabolically dormant. This is where the ATP+AMP
metabolically inactive (dormant) cells as well as to
assay provides a niche. Dormant cells present a
determine the overall metabolic status (stress level,
challenge because they can become active when the
or the proportion of active to dormant cells) or
time and conditions are right, leading to a perceived
general vigor of the microbiological community. Even
„explosive growth‟ that occurs without warning. This
with this new feature, AccuCount technology is not
can have disastrous consequences for the reasons
meant as a direct replacement for traditional
noted above. In general, microbiological populations
methods, but instead should be considered as
exist in one of two varieties as shown in Figure 1.
complimentary to traditional test methods by creating
a more detailed description of the sample‟s
microbiological profile.

Why Measure ATP+AMP?

In the majority of industrial situations, anti-microbial
initiatives (e.g. chemical biocides) are under-applied
in an effort to reduce operating costs or minimize
environmental impact. As a consequence, microbial
contamination is an ever-present challenge facing
operators and if unnoticed or uncontrolled, it can lead
to damaging outcomes such as human health
hazards, equipment deterioration, and increased cost
of process operation.
In general, it is difficult to justify the application of
anti-microbial treatments in the absence of positive
detection. Process control initiatives have
traditionally been based on culture-based techniques,
which by design select for specific sub-sets of the
total population and only detect those
microorganisms that are culturable or viable. Recent
developments in molecular biology monitoring
Figure 1: ATP & AMP Balance in Populations
In summary, the ATP+AMP assay when combined
with the ATP assay provides a new level of insight
into the total microbiological risk for a given process.
It is better able to detect the presence of both active
and passive contamination, which enables the
justification of anti-microbial control where none has
been applied previously, or simply an increase on
treatment initiatives where they are currently
inadequate. Routine measurement of both ATP and
ATP+AMP is the most proactive tool on the market
which when combined with anti-microbial treatment

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Technical Bulletin – AccuCount ATP+AMP Technology
initiatives affords a new level of risk reduction when it
comes to microorganisms.
How is AMP Enzyme Applied?
AMP Enzyme has been optimized and validated for a
all applications and sample types found in the oilfield
sector. AMP Enzyme is integrated as a separate
assay following the normal measurement of ATP
using any of the mentioned kits. In brief, the
measurement process involves adding 100µL of
prepared, diluted extract to 100µL of AMP Enzyme,
waiting for a 1 minute incubation period, and then
measuring RLU in a luminometer. The result from the
AMP Enzyme assay is representative of the total
concentration of ATP+AMP in the sample; the
concentration of AMP is calculated by subtraction of
the ATP concentration as measured with ATP
Enzyme.
The measurement of ATP+AMP should always be
accompanied by the measurement of ATP via ATP
Enzyme so that the following information can be
ascertained for the sample:
 [AMP+ATP]  Total microbial population size.
 [ATP]  Total active microbial population.
 [AMP]  Total dormant microbial population.
 AMPi  AMP Index; calculated as the ratio of
AMP to ATP. Provides an indication of the
relative dormancy of the total population.
Note that different systems and organism types will
have their own characteristic responses to a
biological control program, and AMP Enzyme and
ATP Enzyme will uncover the population
characteristics and reveal the proper control
measures.
Will This Save Time & Money?
Measuring for AMP concentration will allow for
greater sensitivity than ATP alone, as it also allows
for quantification of dormant cells within a system.
Dormant cells can be seen as a „passive‟ risk of
growth or regrowth that may cause biological fouling
if the conditions within the system changes in favor of
the organisms. Furthermore, peaks or persistent high
quantities of dormant and highly stressed cells can
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indicate ineffective treatment for biofilm removal.
AMP testing will allow the operator to quickly assess
the risks beyond active cells and help guide what
actions need to be taken to keep the system within
operating specifications. This add-on uses all of the
same sample processing steps included in its
standard ATP test kits with the addition of a short 1-
minute assay. This additional measurement will give
operators even more useful knowledge about the
system and enhanced control.
Overall, AMP Enzyme can help save money by
having more efficient and proactive anti-microbial
control programs that may reduce biological fouling
or improve process efficiency giving increased yield
of high-quality product.
Who should use AMP Enzyme?
Most any industry concerned with microbial growth
control can benefit from AMP Enzyme, but the risk
associated with metabolically-dormant cells is
especially important in oilfield applications.
What does AMP Enzyme do?
AMP Enzyme combines the existing ATP test kit with
AMP cycling technology, allowing for the accurate
measurement of both ATP and AMP in a sample.
Through a short 1-minute incubation period, AMP is
quantitatively converted into ATP which is then
measured via the same light output reaction as
achieved when using ATP Enzyme. AMP is a natural
product of the reaction between luciferase and ATP.
This cyclical reaction allows for a negligible signal
loss of the originally present ATP in the sample
during the conversion period of AMP to ATP. The
result is a quantitative combined measurement of
ATP and AMP, and when compared to the previously
determined ATP concentration, the AMP
concentration can be calculated on its own.
The ratio of AMP to ATP (“AMP index”, abbreviated
to “AMPi”) is another valuable parameter as an
overall indicator of total microbial population stress
and dormancy. The reaction is as shown below in
Figure 2:
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Technical Bulletin – AccuCount ATP+AMP Technology

ATP Enzyme: 8

Log RLU
4

0
0.01 0.1 1 10 100
AMP Enzyme:
Concentration (ng/mL)

Figure 3: ATP Calibrator/AMP Calibrator Linearity in

AMP Enzyme

Table 1: ATP Calibrator/AMP Calibrator Linearity in

AMP Enzyme
2
Standard Measured RLU % CV R
AMP Cal 100 2,459,640 7%
AMP Cal 1 26,582 2% 1.00
AMP Cal 0.01 300 1%
Figure 2: ATP & AMP Measurement Processes
ATP Cal 100 2,523,116 2%
ATP Cal 1 25,216 8% 1.00
ATP Cal 0.01 264 6%
The AMP index is calculated simply by dividing the
AMP concentration by the ATP concentration, as
shown below: Range of Measurement & Repeatability
[ ] In addition to validating repeatability and linear
[ ] response when used with ATP and AMP standards,
hundreds of samples have been tested using AMP
Enzyme through the course of its development and
How Do I Know it Works? validation. Example results of typical variability and
AMP Enzyme has been validated through extensive results can be found in Table 2.
testing for various types of samples. AMP Enzyme
has been shown to give reliable and consistent Table 2: Triplicate Sample Repeatability
results across several replicate measurements. Sample pg AMPi %CV*
Linearity ATP/mL
Metal Working Fluid 20,843 0.03 6%
For example, AMP Enzyme was validated for linear Latex Polymer 63 16.97 18%
response with both ATP and AMP standards (Figure Sour Water – Oil field 319 0.03 24%
2 and Table 1). Stock Lab Culture 1,877 1.15 5%

Overall, AMP Enzyme has the same range of

measurement for AMP + ATP as does standard ATP
Enzyme for ATP concentration.

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Technical Bulletin – AccuCount ATP+AMP Technology

Activity in the field, such occurrences are exceedingly rare

and therefore are not of concern. The other potential
AMP Enzyme uses the same ATP standard solution,
failure point is in the conversion of AMP to ATP. This
ATP Calibrator 1, as ATP Enzyme. With the addition
has also been minimized for all but the most extreme
of a new AMP standard solution AMP Calibrator 1, a
cases in the present test kit, but the test kit alone
1 ng AMP/mL solution is used to confirm the potency
does not take into account the change within AMP
of the conversion of AMP to ATP reaction. By
Enzyme itself over time. This is accounted for
comparing the ratio of ATP Calibrator 1 to AMP
through the LAAF. With this, the operator can be
Calibrator 1 signals, the user can be confident that
confident that both reactions are occurring as
the AMP in the sample is being quantitatively
designed. An example of validation against
converted to ATP. Confirmation of this conversion is
interferences can be found below in Table 3.
achieved through determination of the Luminometer
AMP Activity Factor (LAAF). Calculating the LAAF is
a simple comparison between the RLU of ATP Table 3: Check for Inhibition in AMP Conversion
Calibrator 1 to the RLU of AMP Calibrator 1, Sample [AMP] [AMP + [AMP] % vs.
performed as shown below: Spike] Increase Control
Control 1 22,014 62,105 40,091 97%
Control 2 23,686 65,844 42,158 107%
Sour Water 1 50 41,467 41,417
Sour Water 2 45 39,399 39,354

AMP Enzyme Stability & Accuracy Over Time

Normally, LAAF should be >0.5. When the LAAF is
To date, AMP Enzyme has shown excellent long term
less than 0.3, the AMP Enzyme should be replaced
stability as a frozen liquid, with at least four months of
with a fresh bottle. The low value indicates that it
useful life after frozen storage. There have been
cannot be guaranteed that a complete AMP to ATP
significant improvements in stability at warmer
conversion is occurring. Note that as demonstrated
temperatures through freeze drying. These stability
in Figure 4, linearity of AMP and ATP measurement
trials are still ongoing, but the most up to date results
is held over time.
are very promising as shown in Tables 2 and 3.

Table 4: Stability of Non-Freeze Dried AMP Enzyme

Temp. Duration % RLU LAAF
Remaining Remaining
-20°C 24 Weeks 65% 1.2
4°C 14 Days 100%* 0.9
22°C 4 Days 100%* 0.8
35+°C 1 Day 0% 0.0

Figure 4: Linearity with AMP Enzyme Age

Table 5: Stability of Freeze Dried AMP Enzyme
Temp. Duration %UC1 RLU LAAF
Interferences (°C) Remaining Remaining
4°C 9 Weeks 100%* 1.2
Since AMP Enzyme has two reactions occurring at 22°C 9 Weeks 100%* 1.2
the same time, there are two potential failure points in 30-40°C 5 Weeks 100%* 1.2
the reagent system. The first failure point is the same *Measured activity resulted in >100% activity remaining.
as in ATP Enzyme – inhibition of the ATP-powered
light reaction in complex sample matrices. Through
years of development, validation, and successful use

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Technical Bulletin – AccuCount ATP+AMP Technology
Extract Stability
Because of the unpredictability of the presence of
enzymes in prepared extracts that may create AMP
from ATP or ADP or enzymes that may deteriorate
AMP concentration, it is recommended that prepared
extracts be tested for AMP concentration within 4
hours of preparation.
What do I need to run the test?
You will need no additional equipment to perform a
AMP Enzyme test that is not already included in
AccuCount test kits. All processing steps and
materials are the same as for the ATP test with the
addition of two additional standard calibrations (using
the ATP Calibrator and the AMP Calibrator) and a
sample measurement.
How do I Interpret AMP Results?
In general, interpreting results from AMP Enzyme will
fall into three major categories:
1. A high ATP concentration and low AMP index
indicates a high portion of the population is
healthy, active cells and in applications where the
goal of biological control is to limit growth, the
treatment program is not effective and corrective
action complete with intensive monitoring is
recommended.
2. A medium ATP concentration and low to medium
AMP index indicates that a population is slightly
stressed and/or dormant. Treatment program is
questionable, and routine monitoring is
recommended.
3. A low ATP concentration and medium to high
AMP index indicates that a population is within
control, and that the treatment program appears
to be effective. Semi-routine monitoring is
recommended.
For application specific ATP values, refer to the ATP
test kit instruction that is being used at the time of
testing.
In addition to the above interpretation, the following
3-pronged approach can be considered for
application of ATP and AMP testing on industrial
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processes. Ideally, these 3 processes should be
enacted in the order they appear.
1. Site Surveys and Audits – measurements of
ATP and AMP concentration as well as
calculation of AMPi can be weighted and
combined into an overall „risk factor‟ that takes
into account active cells, dormant cells, and
the overall level of activity within the population
to rapidly identify areas requiring attention and
their priority. These factors can be established
by comparing each parameter to an absolute
magnitude (e.g. [ATP] < 100 pg/mL; AMPi <
1.0). For more details, refer to the appendix.
2. Antimicrobial Program Design – by measuring
the ATP concentration and AMPi of the starting
population to better select biocide dosages.
While not yet determined, it stands to reason
that criteria could be developed for biocide
types and dosages that are likely to be
successful based on a ratio of dosage to ATP
concentration with some factor associated with
AMPi. Eventually, a calculation could be
derived as follows: Biocide Dosage (mg/L) =
(Biocide type factor) * [ATP] * AMPi. Such
potential has been recently observed (Keasler
et al., ISMOS-3, 2011) as well as over the
years through multiple experiments and
observations – in general, a greater amount of
total microorganisms require more biocide to
suppress.
3. Antimicrobial Program Effectiveness
Monitoring – whether on the lab bench or in the
field, it appears that looking at ATP
concentration reductions and AMPi increases
are the surest way to determine if a particular
antimicrobial program is effective. A >95%
decrease (for ATP) and >5-fold increase (for
AMPi) serve as early estimates for good
performance but additional work is needed to
confirm these targets.
Of course, the 3-pronged approach above would
be enhanced via the use of complementary
chemical and microbiological data. The appendix
shows a chart that illustrates an example of how
to interpret AMP Enzyme data together with ATP
data.
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Technical Bulletin – AccuCount ATP+AMP Technology

What about ADP? (genus/species) organisms. Results from qPCR

typically take two to five days to be obtained, and
AMP Enzyme will not measure Adenosine it has a tendency to overestimate the viable
Diphosphate (ADP); the measurement of ADP is not population of a system as it does not differentiate
necessary in industrial water applications as its living and dead cells. For more information on
relative proportion of total energy (ATP+ADP+AMP) Nalco Champion‟s qPCR testing capabilities,
is very low. contact Energy Services technical support.
Culture tests and qPCR are both still valuable
Do I still need ATP? determining the organisms responsible for
contamination and should be used as a second-line
Yes! Measuring AMP is not a direct replacement for of defense following ATP and AMP testing. For a
measuring ATP. Measuring ATP is still required to relative comparison of what each method detects,
determine both the AMP concentration and AMP refer to Figure 5.
index of the sample, as AMP Enzyme measures both
ATP and AMP at the same time. The ATP-only value
rendered by use of ATP Enzyme is integral for this
purpose. ATP Enzyme can be used to measure ATP
independently of AMP Enzyme. But AMP Enzyme
cannot be used to measure the AMP index without
ATP Enzyme. The best information will always result
from the use of both ATP Enzyme and AMP Enzyme
as a pair.

What’s Different About AMP?

AMP Enzyme, when combined with ATP Enzyme,
provides a short, five-minute field ready method that
can detect active or dormant/stressed organisms and
provide an indication of the overall health. However,
measuring AMP and ATP do not indicate specific Figure 5: Microbial Method Comparison

types of organisms. Such requirements can be better

met through traditional culture-based testing or newer
Industry Acceptance
molecular microbiology methods such as qPCR.
Refer to the AccuCount technology page on myOFC
 Culturing organisms has a long tradition of being
for a suite of technical papers that have been
the go-to method for determining microbiological
developed to demonstrate the utility of the AMP test
load in a system; however it can only detect
method.
culturable organisms. This leaves out viable but
not culturable cells, dormant cells, and injured
cells. Culture-based tests also take a Interpretation Guidance
considerable amount of time to obtain results
from since incubation times can range anywhere Refer to the table on the next page for interpretation
two days for heterotrophic plate counts to thirty of the risk score when using ATP and ATP+AMP
days for sulfate reducing bacteria. assays in the context of routine moitoring and kill
 qPCR requires a lab outfitted with expensive studies.
equipment and samples must be taken to it.
qPCR can detect active and dormant cells, and
can be tuned for general (bacteria) or specific

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Technical Bulletin – AccuCount ATP+AMP Technology

Routine Monitoring

Risk Score Calculation User Explanation

<100 100 to 1000 >1000 <100 100 to 1000 >1000
AMPi ATP AMPi ATP Moderate quantity of active
0 1 3 Low quantity of active microorganisms. High quantity of active microorganisms.
microorganisms.

Moderate quantity of active

Low quantity of active microorganisms. High quantity of active microorganisms.
Low proportion of microorganisms. Low proportion of
Low proportion of dormant Low proportion of dormant
<1 0 0 1 3 <1 dormant dormant microorganisms. Moderate
microorganisms. Low priority for microorganisms. High priority for
microorganisms. priority for treatment and routine
treatment and routine monitoring. treatment and routine monitoring.
monitoring.

Moderate quantity of active

Low quantity of active microorganisms. High quantity of active microorganisms.
Moderate proportion of microorganisms. Moderate proportion
Moderate proportion of dormant Moderate proportion of dormant
1 to 10 1 1 2 4 1 to 10 dormant of dormant microorganisms. Moderate
microorganisms. Moderate priority for microorganisms. High priority for
microorganisms. priority for treatment and routine
treatment and routine monitoring. treatment and routine monitoring.
monitoring.

Low quantity of active microorganisms. Moderate quantity of active High quantity of active microorganisms.
High proportion of
High proportion of dormant microorganisms. High proportion of High proportion of dormant
>10 2 2 3 5 >10 dormant
microorganisms. Moderate priority for dormant microorganisms. High priority microorganisms. High priority for
microorganisms.
treatment and routine monitoring. for treatment and routine monitoring. treatment and routine monitoring.

Priority Description: GREEN Low priority for treatment and routine monitoring.
YELLOW Moderate priority for treatment and routine monitoring.
RED High priority for treatment and routine monitoring.
Kill Study

Risk Score Calculation User Explanation

<90% 90 to 95% >95% <90% 90 to 95% >95%
↑AMPi ↓ATP AMPi ATP Low reduction in active microorganism Moderate reduction in active High reduction in active microorganism
3 1 0
population. microorganism population. population.

Low reduction in active microorganism

Moderate reduction in active High reduction in active microorganism
Low relative increase in population. Low relative increase in
microorganism population. Low relative population. Low relative increase in
<1 0 3 1 0 <1 microorganism microorganism dormancy. Treatment is
increase in microorganism dormancy. microorganism dormancy. Treatment is
dormancy. not effective; change biocide or
Treatment is moderately effective. effective.
increase dosage.

Low reduction in active microorganism Moderate reduction in active

Moderate relative High reduction in active microorganism
population. Moderate relative increase microorganism population. Moderate
increase in population. Moderate relative increase
1 to 5 1 4 2 1 1 to 5 in microorganism dormancy. Treatment relative increase in microorganism
microorganism in microorganism dormancy. Treatment
is not effective; change biocide or dormancy. Treatment is moderately
dormancy. is moderately effective.
increase dosage. effective.

Low reduction in active microorganism Moderate reduction in active

High reduction in active microorganism
High relative increase in population. High relative increase in microorganism population. High relative
population. High relative increase in
>5 2 5 3 2 >5 microorganism microorganism dormancy. Treatment is increase in microorganism dormancy.
microorganism dormancy. Treatment is
dormancy. not effective; change biocide or Treatment is not effective; change
moderately effective.
increase dosage. biocide or increase dosage.

Priority Description: GREEN Treatment is effective.

YELLOW Treatment is moderately effective.
RED Treatment is not effective; change biocide or increase dosage.

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