The application of bioluminescence
technology to monitor clean room hygiene
By Gerard Ruth
Bioluminescence
based hygiene technology is a proven and accepted standard to monitor efficacy
of cleaning and sanitizing procedures in the food and beverage industries.? The technology rapidly measures residual
adenosine triphosphate (ATP). ATP is present in the cells of all organisms
which makes it an ideal biomarker of microbial and organic contaminants.? ATP technology has recently been adapted for
use in a clean room environment to monitor critical contact surfaces, water,
samples, and personal hygiene.
Clean rooms play
an essential role in the manufacture and assembly of microelectronics,
medical instrumentation, pharmaceuticals, and spacecraft components. They are
also critical to nanotechnology research.?
More recently, clean rooms have been utilized in the aseptic
manufacturing of foods1, beverages and nutraceuticals. The modern
clean room is constructed and controlled under stringent
quality-controlled conditions of air quality, humidity, temperature, and
circulation. Biocontamination in clean rooms is minimized through air
filtration and positive differential air pressure measures that lower the
density of aerosolized and airborne particulates within clean rooms.? This in turn, lowers the environmental
contaminants from inorganic, organic and biological residues.?
Contamination control
certification is based on the maximum number of particles greater
than 0.5 ¦Ìm per cubic foot of air2. The air within class
10 clean (ISO 4) rooms is maintained at fewer than 10 particulates
per cubic foot, compared to class 10K (ISO 7) clean rooms which allow 10,000
particles per cubic foot. Hygienic monitoring of clean rooms is a significant
challenge due to the high standards that need to be achieved, the potential to
foster conditions that allow microorganisms to survive and contaminate
products, and the ¡°clean ability¡± of custom equipment used in clean room
manufacturing.? Bacteria can survive
under extreme stress3 conditions in clean rooms, even if cells are
injured and/ or viable but non-culturable.
ATP
bioluminescence provides an objective, broader in scope, rapid, and more sensitive
method to measure residual levels of organic soil and microbial contamination
in a clean room. This non specificity is an advantage as organic product
residues may provide a nutritious medium for microbial growth and act as
barriers to the direct action of both sanitizers and disinfectants. ?The ATP
procedure requires a single service swab device with pre-measured luminescence
reagents and a pre-moistened swab tip. It is recommended that these swabs be shelf stable and double bagged
by the ATP swab manufacturer, which allows the outer bag to be stored and removed
in the garment changing area. The swab itself should be pre-moistened with a
biofilm breaking agent as biofilms, if present on a surface are difficult to
remove as extra cellular material produced in biofilms literally ¡®cement¡¯ the
cells to the surface.? An area of
10 cm by 10 cm is swabbed, re-engaged in the swab device, and then inserted
into a luminometer, and a RLU (Relative Light Unit) value is displayed in
seconds. The light is generated when ATP is hydrolyzed in a reaction that
utilizes a luciferin substrate and luciferase enzyme (Fig. 1).? Photons of light are directly proportional to
the amount ATP present in extra cellular and cellular bacterial cells. The
greater the amount of light produced, the greater the bioburden.?
Fig. 1.
RLU levels do not correlate linearly to
conventional plate count methods.?
However, ATP bioluminescence provides an instant assessment of the
hygienic status of the clean room.?
Ideally, ATP systems should be flexible to allow swabs to be sampled and
collected for later readings.? Methodology
has recently been developed to rapidly detect low levels of ATP using enhanced4
luminescence chemistry and detection systems.?
Systems that once detected 10 femtomoles (10 x 10-15 moles) of
ATP can now detect 0.01 femtomoles.? This
establishes new benchmarks for the level of cleaning that can be achieved on clean
room surfaces.? As an example, a
reduction5 of RLUs as a percentage is a more efficient method for
determining the best cleaning and sanitizing procedure (Fig. 2).
Fig.
2.
The highest acceptable standard is ¡°0¡±
Post RLU and 100% RLU reduction.? This
can be realistically achieved through careful consideration to clean room
design and diligent efforts to optimize clean room SSOPs (Sanitation Standard
Operating Procedures).? When the
reduction of RLUs becomes a challenge in remediation, this can be used to
initiate a complete disassembly and removal of equipment from the clean room
for more thorough cleaning. A unique ATP threshold can be established by
environmental surface type.? If the limit
is exceeded on a swab sampling point the area should be cleaned and re-tested
until an acceptable ATP level is achieved.?
Once an SSOP is optimized, a clean room¡¯s adherence to its written SSOP
will demonstrate knowledge of a commitment to good sanitation and maintaining a
safe clean room production process.? The
SSOP must also document the monitoring and verification procedures used,
including the frequency and recordkeeping processes associated with monitoring
and corrective action procedures.
Advanced
luminescence detection systems can now identify microorganisms in liquids such
as process water, rinse water, emulsions, and high-purity water samples at low
levels (< 10 cfu/ml) using enhanced luminescence systems and membrane
filtration.? Air samplers have also been
integrated with ATP based environmental monitoring systems.? The air is first filtered to remove particles greater than 0.5 microns. Filters or filtered
collection sites can be swabbed directly to measure total ATP, or can be
treated with pyrase, an ATP eliminating enzyme reagent, and followed by a
detergent that frees up ATP for testing from viable microorganisms. Either way,
the amount of ATP detected provides a rapid indirect indicator of the bioburden
quality present in the air sample.?
An advantage to ATP
detection over air sampling agar methods and surface agar contact plates is
that results are obtained instantly. Conventional methods require incubation
times of 24 to 72 hours before colony-forming units can be counted.
Significantly more time is spent labeling and handling the plates while
collecting samples. ?In contrast, RLU
readings that fail the luminometer threshold can be re-cleaned and re-sanitized
immediately if undesirable results are given. The RLU data is stored with
customized software programs, and when integrated with conventional
microbiological results, a macro picture of the overall success and lapses of
hygiene standards is recorded.
Methicillin resistant Staphylococcus aureus, Clostridium perfringens, and E.coli and other harmful bacteria live
in and on the human body, especially around the face and hands, so personal
hygiene must be reinforced to avoid cross contamination of clothing and the
clean room environment.? Personal
hygiene, especially before employees don garments, must be controlled in
sanitary changing areas each time employees enter or re-enter the clean room to
prevent the transfer of harmful microorganisms. ATP bioluminescence systems may
be used to assess efficacy of hand washing compliance.? Higher RLU pass/ fail thresholds are
required, as ATP is associated with squamous epithelial cells.? Testing for ATP also serves a useful training
tool as it educates clean room employees in real time on the origins and
dangers of cross-contamination.
ATP measurement systems, while not
replacing traditional microbiological techniques, actually complement them by
providing an early warning capability, to measure and document environmental
surface cleanliness on a continuous basis, and to evaluate the effectiveness of
remediation programs. The flexibility to adapt enhanced sensitivity ATP systems
for surface, air and liquid monitoring assures greater accuracy in hygienic
monitoring of clean rooms. In every sense ATP measurements provides a true
measure of 'hygiene' and 'cleanliness' by detecting both microorganisms, and
organic product residues present on surfaces. In real time, ATP tests
demonstrate due diligence and adherence to quality standards in cleaning and
sanitizing, thereby providing documentary evidence, of corrective actions
taken. Good sanitation should be an ongoing strategic objective as improved
hygiene and cleaning standards directly result in fewer micro-organisms, and
ultimately, a safer clean room environment to produce product.?
Gerard Ruth, Charm Sciences
in Lawrence, Ma., can be reached at 978-687-9200 or gerardr@charm.com; www.charm.com
References:
. ISO 14644-1, Part 1: Classification of Air
Cleanliness; Institute of Environmental
Sciences and Technology; www.iest.org/iso/iso1.htm
4. Comparison
of Visual Inspection, an Allergen-Specific Method (ELISA) and Nonspecific
Methods (Sensitive ATP and Total Protein) to Detect the Presence of Allergenic
Food Residues on Food-Contact Surfaces; F. Al-Taher1 and L.S. Jackson, Food and
Drug Administration, NCFST, Summit-Argo, IL; Illinois Institute of Technology,
National Center for Food Safety & Technology (NCFST), Summit-Argo.
International Association of Food Protection, Annual Meeting, 2007. http://www.charm.com/images/stories/pdf/atp/iafp_2007_poster.pdf
5. Using Bioluminescence Technology to
Monitor Kennel Sanitizing Procedures, Jesse McPherson, and John Savarino, Johnson &
Johnson, Pharmaceutical Research & Development, LLC, TechTalk, Vol 12/ No.
6 December 2007
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