Biofluorescent particle counters

Alternative technologies for microbial detection are available. First marketed around 2010, these systems can differentiate between inert and biologic particles. Known as biofluorescent (or spectrophotometric) particle counters, these devices provide real-time indications of probable airborne microbial contamination. The term ‘probable’ is used advisedly, as these instruments can be susceptible to false positives. 

In addition, there is no direct quantification from a detected event with actual microbial cells.
Nevertheless, biofluorescent particle counters can serve as effective early warning tools, signalling potential out-of-control environmental situations and enabling timely corrective action.

In terms of their operation, these devices analyse laser-induced fluorescence pulses emitted by airborne microbiological particles as they are drawn into the counter. Internal detectors are tuned to identify biologic entities, specifically the fluorophores tryptophan, reduced Nicotinamide Adenine Dinucleotide (NADH) and riboflavin (detected using a blue laser with 405-nm wavelength as its excitation source). These metabolites are commonly found fluorophores in bacterial cells ³. 

The instrument stimulates and then collects the total fluorescence signal from each probed aerosol particle captured, allowing the determination of the ratio of elastic scattered light at the laser wavelength to fluoresced light. Simultaneously, the device will allow particle sizing - much like a conventional particle counter - through the collection of the elastically scattered laser light and a flux measurement, by counting the particles that pass ⁴.

Advantages

These counters have several uses and connect with the Process Analytical Technology (PAT) paradigm. One use is the monitoring of aseptic processing environments, enabling filling runs to be stopped if biologic events are detected. Having a ‘real time’ capability eliminates the inherent delays of culture-based methods ⁵. The counters can provide improved risk-reduction as well as providing a rapid indication of the success (or otherwise) from root cause analysis and in putting in place remedial actions ⁶.

Moreover, the counters provide a higher estimation of contamination, helping to overcome the limitations of culture-practices. Since culturing the organisms is not necessary for detection, biofluorescent counters can detect Viable but Non-Culturable (VBNC) microorganisms.

Limitations

Biofluorescent particle counters do have limitations. One limitation is false positives, where the fluorophores present in artificial products can interfere with airborne assessments, leading to the incorrect detection of microorganisms when these materials are present as airborne particles in a cleanroom. Fluorophores can arise from clothing fibres, spray-droplets of disinfectants or as aerosolised debris made of fluorinated rubber product ^{7, 8}.

Manufacturers have sought to limit these false positives, with technologies advancing since their inception. The verification of biofluorescent counters can be improved using inkjet aerosol generators, where the use of two types of monodisperse test particles - inert particles and fluorescent particles - can be used to assess the potential for false positives and enable better tuning to eliminate the rate of false occurrences ⁹. 

Challenges: Colony Forming Units (CFUs)

Another issue with biofluorescent particle counters is how the data recorded for a suspected biologic event translates to actual microbial cells. The reported unit is commonly referred to as an Autofluorescence Unit (AFU), which is not dependent upon growth, as is the traditional method ¹⁰. Traditional methods are reliant on counting visible (by humans) or subvisible (through blue light laser excitation) colonies.

It is not an easy task to make a comparison and hence validating non-growth based alternative methods, compared to the growth-based compendial method, can be fraught with difficulty (some microbiologists advise against even attempting to make a comparison) ¹¹.

The reason for the discrepancy relates to the dependency on growth on agar plates so that CFUs can be counted. Since not all microbes will grow on agar and considering the VNBC issue above, standard methods for incubation do not support the growth for all microbes. For this reason, the measured values of biofluorescent systems are typically much higher than the values measured by conventional microbial tests ¹².

Experimental attempts to correlate AFUs to the traditional CFUs are not necessarily appropriate and there is no consistent correlation factor between the two units of measure ¹³. A negligible to low correlation between biofluorescent particles and CFU has been reported. In contrast, a strong correlation exists with conventional particle counter measurements for the total numbers of particles ¹⁴. 

Applications

With such limitations accepted, biofluorescent particle counting can be used in other applications beyond monitoring aseptic processing environments. Examples include assessing any transfer of contamination from airlocks into higher graded cleanrooms and with setting the maximum numbers of permitted personnel in changing rooms ¹⁵. The technology can also be used to assess how long cleanrooms take to recover after facility shutdowns or unplanned events like power cuts.

Conclusion

Biofluorescent particle counters have been commercially available to cleanroom users for over 15 years. Their cost remains high and this is undoubtedly a reason why small to medium pharmaceutical companies cannot afford them. Those companies that have selected them tend to use them for investigational purposes and for determining facility recovery times. On the issue of false positives, as with any environmental monitoring system, the strength is with gathering as much data as possible and trending it.