Bioluminescence Technology
| Back
to Technical Main Page>
Rapid Detection
of Micro-Organisms using ATP Bioluminescence
Manufacturers across many industries are required
or recommended to test each batch of product for microbial
contamination. While agar plate testing has long been
a useful and reliable method of testing for such microbial
contamination, it does have drawbacks. In particular,
it can take up to five days to grow a culture on an
agar plate until a colony is formed which is large
enough for the eye to detect.
Testing cycles that last many days have a significant
impact on production timetables and efficiencies.
The implications of these delays have led to a search
for improved microbiological testing methods that
are simpler and faster; todays rapid
methods.
The rapid testing technologies
currently available or being developed are numerous
and diverse ranging from 'simple' analysis using
biochemistry and testing methods requiring sophisticated
analytical instruments to electrochemistry and immunoassay.
The first group includes several rapid testing methods
which use light in one form or another to measure
the presence of micro-organisms, such as dye reduction,
photometry, flow cytometry and bioluminescence.
Over the past ten years or so, a great deal of emphasis
has been placed on the use of bioluminescence technology
in the detection of micro-organisms. The generation
of light by a biological process (hence bioluminescence)
is most beautifully demonstrated by the American firefly
Photinus pyralis. The mechanism by which fireflies
produce a flash of light was first analyzed and identified
by William McElroy in 1947. McElroy found that central
to the light emission process was a specific enzyme
reaction catalyzing the consumption of adenosine triphosphate
(ATP). In microbes, ATP can only be detected when
living cells are present. It has since been established
that the amount of light emitted from this reaction
is directly proportional to the amount of ATP present.
Since all life forms contain ATP, applications in
microbiology are based on capturing the micro-organisms,
releasing the ATP from within the cell, and measuring
the amount of bioluminescence generated. A high reading
of relative light units (RLUs) indicates that a sample
contains a high number of micro-organisms, provided
the background ATP level is low. Unlike traditional
testing methods, results from a bioluminescent reaction
can be obtained quickly. Light is produced within
seconds and can be measured with a luminometer.
In summary, the mechanism of reaction is described
below:
Light generated by the reaction is measured in a
luminometer of which there are several types (Stanley,
1992). The differences between the machines generally
derive from the type of light detector used, e.g.
photomultiplier tube, photodiode, or avalanche photodiode
(Brown, 1989) and the degree of automation (single
or multiple assays). The types of ATP detecting bioluminescence
reagents are also varied, in kinetics and performance
as well as quality. Reagent performance and sensitivity
has improved greatly over the last 5 years (Schram
1991, Simpson et. al, 1990) and it is now possible
to detect attomolar concentrations of ATP derived
from micro-organisms. Theoretically, the ATP detected
is equivalent to less than 10 organisms with the assay
itself taking seconds to give a result.
Applications
ATP bioluminescence is widely used in industries
such as the pharmaceutical and personal care product
industries as well as dairy and food production plants
for a variety of applications including raw material,
in-process and finished product testing. Many food
manufacturers also use ATP bioluminescence based
assays for hygiene monitoring. The application of
ATP detection using bioluminescence to monitor personal
care products, toiletries and cosmetics was evident
in research laboratories during the 1980s. Towards
the end of that decade, research based procedures
reported the detection of 1 cfu/g after a 24 hour
enrichment phase (Nielsen and Van Dellen, 1989).
At very low bioburden levels, the procedure requires
an enrichment stage to allow for the amplication
of microbial ATP that can be distinguished from the
background.
Since then, procedures have been optimized and the
technique adopted by major manufacturers as a rapid
test for the detection of contaminated product. In
addition to screening for low level contamination
using an enrichment step, ATP bioluminescence can
also be applied as a direct test, without enrichment,
as a disaster check where high levels of contamination
are suspected. Similarly, trend analysis may be performed
by monitoring for changes or spikes in typically
low level RLU readings.
The procedure is simple to implement provided the
correct screening and validation procedures are followed.
A brief summary of the most relevant parameters which
may affect bioluminescence testing is given below
together with data illustrating recent technical
advances which have helped to improve the reliability
and general applicability of the technique.
The
General Procedure
The procedure for the detection of low level contamination
using an enrichment step requires three simple steps:
- Disperse sample in broth (1% - 10% w/v)
- Incubate at 32 degrees C (18 to 28 hours depending
upon kit and screening criteria)
- Assay aliquot (50 - 100 microliters) by bioluminescence
A number of considerations must be taken into account
when validating the system including:
Sample effects
- Light or enzyme inhibition
- Background ATP
Growth Media
- ATP Level
- Influence on extraction
- Preservative neutralization
Extractant
- Efficiency of ATP extraction
Sensitivity of reagent
- Level of ATP easily detected
- Precision of test
Sample Effects
Not surprisingly, the effect of the sample itself
on the bioluminescence assay is sometimes unpredictable.
Occasionally, products will contain detergents and
salts that will impact the bioluminescence reaction
(Simpson and Hammond, 1991). Some sample ingredients
may be derived from natural sources and can contain
high levels of non-microbial ATP. High background
ATP levels can give false positive results. Typically,
however, these ingredients are present in the finished
product at levels that do not interfere with the
testing. A quick sample effects test is recommended
to qualify the sample before performing a full validation.
Of hundreds of products examined, rarely has there
been a product that cannot be tested for microbial
contamination in some way using ATP Bioluminescence.
Growth
Media
The broth used to incubate the sample must be capable
of supporting growth of viable organisms, be able
to neutralize any preservative systems, contain relatively
low levels of ATP and have good batch to batch consistency.
Letheen broth is an example of a general growth
medium that meets these criteria. Table One below
illustrates ATP content of a variety of broths including
Letheen.
| Type of Broth |
ATP content (pM) |
Letheen Broth
Nutrient Broth
Tryptic Soy Broth
Sabouraud Liquid Medium
MTGE Broth
Sabouraud 2% Dextrose Broth
Fluid Thioglycollate
Antibiotic Broth
Eugon LT100 |
26
453
2774
420
469
239
5055
1392
700 |
Table 1; Results of testing for ATP content of a
range of growth media
Consistency in ATP content from batch to batch is
also important in the selection of an appropriate
broth for enrichment. Table 2 shows the results of
an assessment of ten batches of Letheen broth for
ATP content.
| Batch of Letheen Broth |
ATP content (pM) |
1
2
3
4
5
6
7
8
9
10
Mean ATP concentration |
18
38
63
26
42
47
42
59
19
32
38.6 pM
Standard deviation 7.3pM |
Table 2; Results showing ATP concentration in 10
batches of Letheen broth
This is demonstrated for SLM (Figure 1) at two concentrations
of the enzyme. ATP depletion at higher apyrase activity
(0.25 U/ml) was very rapid, while the lower enzyme
activity (0.05 U/ml) gave a more gradual rate of depletion.
Significantly, apparent ATP concentration leveled
off at a similar amount for both activities of apyrase,
demonstrating the presence of a luminogenic substrate
other than ATP. This residual bioluminescence is typically
even higher with other types of growth media. Fractionation
of Tryptone Soya Broth (TSB) using HPLC demonstrates
the presence of at least three luminogenic substances
other than ATP. The fractions were separately treated
with 0.24 U.ml-1 apyrase (Table 2). Fractions 18,
42 (expected fraction for ATP) and 51 all decreased
in bioluminescence with time, whereas fraction 34
remained bioluminescent after 26 hours of treatment.
Treatment with an ATPase enzyme or a kinase removes
only part of the signal. The phenomenon demonstrates
that the problem in reducing the bioluminescence blank
signals stems from luminogenic substrates for firefly
luciferase, which either are not substrates for apyrase
or are physically protected from degradation.
Using a proprietary technique, the level of media
derived ATP post apyrase treatment can be further
reduced by a factor of 10 to 100 fold depending on
the starting material (Table 3). The most significant
advantage of such an ATP depleted medium is that the
number of organisms required to give a light signal
at a significant level above background is reduced.
With any ATP reduction procedure care must also be
taken to validate the nutritional property of the
treated media. In the above experiment growth properties
of Lactobacillus acidophilus, Candida albicans and
Penicillium expansum were checked and found to be
unaffected by the treatment.
Extractant
The detection of micro-organisms using luciferin-luciferase
reagents requires an extractant to lyse the cells
and release ATP into solution. There are stringent
and partially conflicting criteria for the extractant:
It must be sufficiently aggressive to attack a broad
range of microbes with very different morphologies
- It must not affect the activity of the luciferase
enzyme
- It should exhibit some chaotropic action in order
to inactivate intracellular enzymes which would
use the free ATP to begin repairing the bacterial
structure (Lundin, 1989: Stanley, 1986)
- The efficiency of an extractant is normally derived
by comparing the amount of ATP released from a given
number of organisms with the amount of ATP released
using a reference method employing 2%-10% trichloroacetic
acid (TCA) as the lytic agent (Lundin, 1992).
Table 4 lists the organism types which were used
in a study to determine the efficiency of a broad
spectrum extractant developed in this laboratory.
The mean extraction efficiency for the study was calculated
to be 98% (SD = 30%) with a mean ATP level of 1.4
x 10 -18 moles per bacterial cell.
This extractant also has the advantage that it is
not as chaotropic as TCA and does not affect ATP after
extraction. It is, however, powerful enough to fully
extract very quickly. Figure 2 shows extraction of
ATP from an E.coli sample over 10 seconds.
Reagent Sensitivity
Simpson et al, (1990) demonstrated the high sensitivity
of bioluminescence reagents. Although important, sensitivity
is not the only performance criterion to consider.
Consistency of light signal and reagent stability
are also of great practical interest. This is, of
course, the responsibility of reagent manufacturers
and, apart from understanding the basic requirements
when performing bioluminescence reactions and exercising
good microbiology laboratory practice, there is little
the user can do to ensure that reagent performance
is adequate. End-users must insist manufacturers have
carried out correct Q.C. procedures (e.g. as part
of the Quality Assurance program) and have validated
production procedures so that reagent consistency
is maintained.
Summary
Many rapid microbiological methods are unsuitable
when applied to cosmetics, toiletries and other personal
care products because of interference from product
constituents, inadequate sensitivity, difficulties
in detecting certain types of micro-organism, complexity
of procedures and/or instrumentation and cost. Bioluminescence
can overcome many of these considerations to give
a true advantage over other techniques.
The database of bioluminescence information increases
daily at Celsis, and this technique is being adopted
in an ever increasing range of research interests
and industrial laboratories. ATP bioluminescence is
beginning to show real financial benefits by reducing
inventory costs and consumable spending. More than
ever, the manufacturing industry has to consider not
only the necessity of reducing costs but also the
absolute requirement to produce goods of the desired
quality under the watchful eye of the regulators and
consumers alike. One example of the dilemma facing
manufacturers is the drive toward excluding many synthetic
preservatives from final product formulation with
the possible penalty of reducing shelf life. The logistics
required to produce goods fit for use puts even more
pressure on the often rate limiting step of testing
using conventional microbiology. This situation provides
an example where the 24hr ATP bioluminescence test
is being evaluated and will prove to be of considerable
benefit.
In this laboratory, ATP bioluminescence remains a
technology that is used for many applications requiring
rapid tests. Future rapid methods are now being developed
which combine the unique sensitivity and speed of
bioluminescence with other emerging techniques especially
in the areas of molecular biology, immunology and
enzymology. Rapid micro-organism identification tests,
toxin test and various residue tests will emerge which
will aid the long awaited, but hopefully welcome,
change to traditional microbiology.
References
- Brown R G W, (1989). ATP Photon Counting using
Avalanche Photodiodes. In Rapid Methods in Microbiology.
Eds. Stanley P E, McCarthy B J and Smither R. Oxford
Blackwells Scientific Publications.
- Lundin A, (1989). ATP assays in routine microbiology:
from visions to realities in the 1980s. In: ATP
Luminescence. In Rapid Methods in Microbiology.
Eds. Stanley P E, McCarthy B J and Smither R. Oxford
Blackwells Scientific Publications.
- Lundin A, (1992). Method for extraction of intracellular
components. International Patent: PCT/GB92/00056.
- McElroy W D, (1947). The energy source for bioluminescence
in an isolated system. Proceedings of the National
Academy of Sciences USA, 33: 342-345.
- Schram E, (1991). Evolution of Bioluminescent
ATP assays. In: Bioluminescence and Chemiluminescence:
Current Status. Eds. Stanley P E, Krika L J.
- Simpson W J, Hammond J R M (1991) The effect of
detergents on firefly luciferase reactions. J. Bioluminescence
and Chemiluminescence, 6: 97-106.
- Stanley P E (1986) Extraction of Adenosine Triphosphate
from Microbial and Somatic Cells: In: Bioluminescence
and Chemiluminescence part B: Methods in Enzymology.
Eds. Colowick S P and Kaplan N 0 London. Academic
Press.
- Stanley P E, (1992) A Survey of more than 90 Commercially
Available Luminometers and Imaging Devices for Low-Light
Measurements of Chemiluminescence and Bioluminescence,
Including instruments for Manual, Automatic and
Specialized Operation, for HPLC, LC, GLC and Microtitre
Plates. Part 1: Descriptions. J. Bioluminescence
and Chemiluminescence, 7: 77-108.
