## 2 Materials and Methods
### Cleanroom and Equipment  
For this experimental study, a training room at Testo Industrial Services in Kirchzarten, Germany, was used. The room is designed with a Class C/ISO 8 finish with pharma grade floors, walls, doors, and ceilings. A variable HVAC system with two H13 filters, mounted at the room inlet prior to swirl diffusers, allows ACRs from \(<\)10 to 70 h\({}^{-1}\). This enabled the test execution with the chosen ACRs. The test room was cleaned and disinfected prior to test execution. See Figure 2 for room layout details. The BioTrak(r) BFPC was set up with an airflow rate of 28.31/min and an aggregation period of 1 min. This measuring system combined two different detection units: One ISO-compliant particle counter and the MCP counting unit below. A schematic drawing of the system is given in [28]. The laser excites particles with a wavelength of 405 nm. To differentiate viable particles from other fluorescent substances the system takes three parameters for analysis: the normal scattered light at the same wavelength, a re-emission at a higher wavelength between 430-500 nm, and another between 500-650 nm. By this, the system can distinguish biological from non-biological particles and also viable, potentially bacterial, MCPs from other biologic particles, such as pollen [29]. The Merck Millipore RCS(r) High Flow Touch collects air with a flow rate of approx. 100 L/min, and uses centrifugal force for an impaction of MCPs on agar test-strips [30]. The high sampling volume is an advantage of this system. Depending on the used garment the test duration and, consequently, the sampling volume, the set-up was as follows:  
**ISG/IgG**: 7 min/strip--total 683 L (four strips, 28 min + 2 min changeover time)  
Figure 2: Room layout. For a comparison of the BioTrak® Biofloorescent Particle Counter (BFPC), (TSI Incorporated, Shoreview, MN, USA) with the classic Microbial Air Sampler (MAS), two different measuring systems were used: Merck Millipore RCS® Merck Millipore, Darmstadt, Germany (1)) and ImpactAir® Iso-Con Slit-to-Agar, Bridgend, UK (2)). All instruments were placed inside a process unit in the middle of the room. **NCG**: 7 min/strip--total 683 L (four strips, 28 min + 2 min changeover time) plus 5 min with last strip (488 L)  
The ImpactAir(r) Iso-Con Slit-to-Agar MAS uses 90 mm tryptic soy agar (TSA) petri dishes. Inside the sampling device, the agar-filled plate slowly rotates below a very narrow inlet slit [31]. The air flow was setup with 15 L/min. The run time and the resulting sampling volume was 7 min/plate, equaling 105 L for l8G/l9G experiments, and 8 min/plate (120 L) for NCG. For the MAS systems, Merck Millipore 90 mm/30 mL TSA plates and test strips for the RCS(r) air sampler were used [32; 33]. The collected plates and strips were incubated at 32 \({}^{\circ}\)C. A daily CFU count was done, starting at day two and finishing after day four, as no further CFUs were observed. In industrial processes, rooms of this size are typically run by two operators. Consequently, all experiments were performed by two persons to generate realistic cleanroom process operations. As three persons were available, one was exchanged after each test to allow breaks. Hand disinfection during the experiments was done with 2-propanol at the table in the back of the process unit. A small process unit, consisting of a filled water tank, pump, connecting pipes, and instrumentation, was placed in the middle of the room to simulate a production process (Figure 3). The circulation pump was operated continuously during the three days of the test execution, which also created realistic conditions with regard to equipment heat generation and motor fan-induced air turbulence. The air supply rate, to measure and control the anticipated ACR, was determined by a Testo 420 volume flow hood. All test equipment was calibrated before the start of the tests. All tests were recorded by a full HD-Camera (Canon EOS 5D Mark IV, Tokyo, Japan) for later analysis of the counter results in relation to the operator's behavior.