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Monitoring of airborne bio burden is
part of the classic repertoire of pharmaceutical microbiology. Currently, rapid methods are finding their way into
the aseptic production environment as
diagnostic tools. This article describes a
method for counting airborne bacteria in
real time together with its basic principles and technical design. It also discusses
fields of application, such as its limitations
in relation to conventional methodology.
Counting Airborne Bacteria in Real Time
Autofluorescence systems and their fields of application
Classic methods and pharmaceutical regulations
The applicable standards are the driving force behind microbiological monitoring within the pharmaceutical industry.
The FDA „Guidance for Industry, Sterile Drug Products“ and
the corresponding EU „Guide to Good Manufacturing Practice“ dominate everyday production life. These documents
only take account of airborne bacteria that are able to metabolise as any bacteria that is present should be collected
in appropriate culture media and reproduced by incubation.
After completing the reproduction phase, bacteria colonies
are counted and designated as so-called colony-forming units
(CFU). The EU/FDA has stringent requirements for aseptic
production areas: less than one CFU per cubic metre of clean
room air is specified for grade A areas (EU) and critical areas
(FDA) respectively. It stands to reason that a microbiological
assessment should be available as quickly as possible with
such narrow acceptance criteria. Unfortunately, the conventional method of determining CFUs only provides reliable
counting results after a few days. Moreover, sampling by means of sedimentation plates or active airborne bacteria samplers requires only a short break in production. Damaged or
inactive bacteria are just as hard to recognise as microbiological systems that do not feel stimulated to reproduce by the
culture medium used. The detection of bacteria that is damaged by the physical collection process (e.g. impaction) is also
extremely misleading. Overall, these are reasons enough for
microbiologists to want methods that detect any microbiological air pollution without a delay. Conventional methods are
now being given precisely this addition with the availability of
new laser light sources.
The preliminary stage - flow cytometry
Numerous biological systems can be easily stimulated energetically with short-wave light and react to lower frequency
light with re-emission. This fluorescence effect was used in
the recent past for microbiological analyses. In so-called flow
cytometers, fluorescence dyes are linked to microorganisms
by means of metabolic processes. This process generally takes
place in an aqueous solution. The organism/dye combination
finds its way into the interaction zone of one or more lasers.
Whilst passive, non-metabolic particles only emit diffused light
flexibly (emitted light wavelength corresponds to a coupled
light wavelength), metabolic organisms are disclosed by means of an additional fluorescent signal. For this reason, flow
cytometry represents a completely viable analysis technique
to determine the bacterial count. Due to its principle, liquid
samples are only analysed with a previous sample preparation.
Linking of the fluorescence dye also takes a certain amount
of time. As a result, a typical laboratory method is available in
discontinuous operation. An extension of the principle to do
without the use of dyes is required to be able to analyse clean
room air - in real time.
Autofluorescence method
This method is based on the idea of stimulating the characteristic molecules of a microorganism towards independent
fluorescence. Detailed spectroscopic analyses show which key
molecules can be stimulated with preferably a single wavelength.
In the analysis technique presented, NADH (a coenzyme involved in cellular metabolic reactions) and Riboflavin (Vitamin
B2; important for metabolic processes) are stimulated as representative types of molecules in vegetative bacteria. Dekosa
pantanoic acid (DPA – a fatty acid) also emits fluorescence and
can be found in bacteria spores. The fluorescence emission
curves of all three molecules described overlap in an interesting way in near UV and in the violet spectral range.Therefore,
it makes sense to use high quality diode lasers with a 405 nm
wavelength (violet light) to stimulate fluorescence.
Technical design
Figure 1 shows a diagram of the optical configuration of such
a device. In principle, references to the conventional diffusedlight laser particle counters (OPC) can be found in the construction design.The gas sample is drawn through the measuring
Lufteingang
Düse Eingang
Photo
Multiplier
Multi - Reflektor
Detektor
405 nm
Laser
Düse Ausgang
Luftauslass
Figure 1: Optical configuration of an autofluorescence bacteria
counter
Sensitivity and a comparison with conventional methods
Initially, an autofluorescence airborne bacteria counter detects conventional airborne bacteria with an active metabolism (classic CFUs) and airborne bacteria in their spore state
(potential CFUs). Moreover, damaged and even destroyed
airborne bacteria that conventional culture media methods
never register are also detected. It is immediately apparent to
any microbiologist that the counting results of an autofluorescence device must be better than those of a conventional
culture media method. The factor of continuous monitoring,
with resolutions within the second area, also leads to significantly improved counting statistics. Autofluorescence airborne counters „see“ significantly more occurrences than
processes that are based on reproduction with subsequent
CFU counting. Furthermore, they have an incomparably better temporal resolution with improved counting statistics. A
typical example of this is shown in figure 3 of the analysis.The
vital question remains on how these rapid methods should be
integrated into everyday operations.
100000
10000
Normalized Counts
device and formed into the optimum interaction geometry
by means of nozzle systems. The main difference from the
conventional OPC is the use of a fairly short-wave 405 nm
laser and the integration of a second detection channel. A secondary electron multiplier (photo multiplier) registers weak
autofluorescence signals from the biological elements in the
air. Simultaneous detection of flexible and inflexible diffusion
processes enables biological systems to be recognised and
classified by size. The optical resolution ensures that spores
and bacteria are displayed. The detection of 405 nm diffused
light in a forward direction supplies conventional particle
data whose size channels conform to ISO 5 clean rooms as
auxiliary information. The 5.0 um particle channel in particular can emphatically support the interpretation of biological
counting results.The overall technical design of such a system
(figure 2) is not substantially different to that of a conventional particle counter. All practical insights relating to the
best possible sampling (isokinetic sampling probe; BevALine®
tubing, reducing the tube lengths etc.) can be adopted for the
new method of measuring.
E. coli aerosolized
Red bars: IMD
Blue bars: Andersen sampler
r2=0.936
1000
100
10
1
10
20
30
40
50
Run #
Figure 3:Test results from the collection of airborne bacteria from an
E. coli aerosol. Measuring results of an autofluorescence counter vs.
counting results of an Anderson sampler.
Fields of application
First of all, it is clear that the counting results from the new
method are considerably superior to those of conventional
CFU counting. Therefore, a 1:1 exchange of method is not an
option - and also not entirely sensible. These classic methods
are deeply entrenched in pharmaceutical regulations and
should initially remain the basis of communication with the
supervisory authorities. The autofluorescence device has now
replaced the classic methodology as a powerful diagnosis and
qualification tool. The rapid method is called upon when problems arise in conventional microbiology. Larger measuring
volumes with greater statistical significance do not just reveal
individual problems but also actually enable stable trending
in clean room areas with a small number of counting occurrences. The rapid method helps microbiologists to investigate
problems or even identify at an early stage that the microbiological integrity of an aseptic production is starting to become
unfavourable.
Summary and outlook
Pharmaceutical processes can be continuously monitored
and individual occurrences and level displacements can be assessed using autofluorescence bacteria counters. Therefore,
disproportional actions can be avoided or significantly weakened in the future after the counting of the individual CFUs.
User reports also show that autofluorescence bacteria counters play a vital role in the initial qualification of production
plants. Applications within the area of continuous GMP monitoring and product release (key word: parametric release) are
in the pipeline and will be discussed in forthcoming technical
papers.
Author: Herr Jörg Dressler, PMT Partikel-Messtechnik GmbH