1. PM Number Monitoring

1. Use an airborne laser particle counter (see Table of Materials) to determine the total PM number. Collect the PM by the air sampling port on the top of the airborne laser particle counter.
   NOTE: This particle counter is an automation equipment and it can work independently when the program including sampling time, interval, collection times, etc. has been set on its touch screen.
   1. Equip the instrument with a sensor to monitor temperature and relative humidity. Measure and record temperature and relative humidity data simultaneously every 5 min.
   2. In total, measure and record 6 different particle size classes (0.3-0.5 μm, 0.5-0.7 μm, 0.7-2.5 μm, 2.5-5 μm, 5-10 μm and > 10 μm) simultaneously every 5 min. Measure 4 other particle size classes (0.3-0.5 μm, 0.5-1 μm, 1-3 μm and ≥ 3 μm).
   3. Collect the air samples though the sampling hole on the top of the sampler and use the test modules inside the sampler to measure the particle size of each PM. Then the data is stored automatically. All the processes above can be performed automatically after the relative parameters, including sampling time and counting range, are set though the mini touch displayer of the airborne laser particle counter.
      NOTE: This particle counter is an automation equipment and it has test modules that count the number of PM of different particle size classes.
2. To assess experimental standard error, ensure that different particle size classes are counted with at least 15 replications. Ensure that readings are stored in the internal memory of the instrument and subsequently analyzed.
   1. Ensure that analysis of the primary data includes PM number concentration, the ANOVA test and the percentage of PM in each size class. For example, as shown in Figure 1A, the PM number concentrations in December (237.6 particles/cm³) were significantly higher than those in October (average: 110.2 particles/cm³) (ANOVA; P-value < 0.05). Figure 1B shows that the number of particles smaller than 3 μm accounted for more than 99% of the total number.
   2. Use the laser particle counter to monitor the PM number concentrations and store the data automatically. Use a USB flash disk to export data in the computer and perform the ANOVA test.

2. PM Collection by Cyclonic Aerosol Samplers

1. Perform the PM sampling in an open area without any nearby major pollution sources at ∼2 m above the ground.
   1. Record the position of the sampling site. For example, the campus of Beijing Institute of Technology is the following: 39°57’51.0’’ N; 116°19’38.5’’ E.
2. Use a cyclonic aerosol sampler to collect air samples. Use a sampler flow of 323 L/min, and a collection time of 6 h.
   NOTE: The sampler flow rate is fixed and cannot be changed. The collection time is controlled by manual time-keeping as there is only a Start and Stop button on this sampler.
   1. Use sterile water to wash the inside of the cyclonic aerosol sampler 3 times before collection, and use the automatic cleaning function 3 times by continuously pressing the collect button 3 times.
   2. Press the collect button to start sampling and press the pump button to stop sampling. Put the sampler on a shelf or floor with no one holding it in place.
   3. Preserve all samples in the dark at -20 °C until the subsequent analyses.

3. PM Collection by Filters

1. Perform the PM sampling in an open area without any nearby major pollution sources at ∼2 m above the ground.
   1. Record the position of the sampling site. For example, the campus of Beijing Institute of Technology is as follows: 39°57’51.0’’ N; 116°19’38.5’’ E.
2. Collect the PM samples on 20.32 × 25.4 cm² filters (see Table of Materials) by using a high-volume air sampler (see Table of Materials) at a flow rate of 1,000 L/min. Set the flow rate and collection time by relative auto-programming.
   1. Preserve all sampled filters in the dark at -20 °C until the subsequent analyses.

4. Biological Composition Analysis

1. For biological composition analysis, use each liquid sample from the cyclonic aerosol sampler or filter sample for DNA extraction by a multisource DNA extraction Kit (see Table of Materials) according to the manufacturer's protocols.
   1. For samples from the high-volume air sampler with filters, use 1/8 of each filter sample for DNA extraction. Put the filter into a 50 mL tube with the sample facing inward and the back facing towards the tube wall. Add 10 beads in the central sites towards the side of the filter containing the sample.
   2. Vortex the tube for 15 min at room temperature. Pipette the liquid in the tube into a clean 50 mL centrifuge tube to continue the DNA extraction process.
2. Extract the DNA from the sample by the processes as follows.
   1. Centrifuge the bacteria sample at 2,000 x g for 5 min, remove the supernatant, and suspend bacteria in 2 mL of sterile PBS buffer.
   2. Collect the suspension of bacteria in a 2 mL centrifuge tube and then centrifuge at 2,000 x g for 5 min to discard the supernatant solution.
   3. Add 350 μL of sterile PBS buffer containing the suspended bacteria to the solution.
   4. Add 0.8 mL of RNase A.
   5. Add 150 μL of Buffer CL and 8 μL of proteinase K, and mix immediately by vortex shaking for 1 min. After brief centrifugation at 1,000 x g for 30 s (no residues on the wall), put the centrifugal tube in 56 °C water for 10 min.
   6. Add 350 μL of Buffer PD, and mix for 30 s and then centrifuge for 10 min at 12,000 x g.
   7. Place the DNA preparation tube in a 2 mL centrifuge tube, and transfer the mixture made in step 4.2.6 to the preparation tube. Then centrifuge for 1 min at 12,000 x g.
   8. Discard the filtrate, put back the contents of the preparation tube into the original 2 mL centrifuge tube, and add 50 μL of Buffer W1. Centrifuge the mixture for 1 min at 12,000 x g.
   9. Discard the filtrate, put back the contents of the preparation tube into the original 2 mL centrifuge tube, and add 700 μL of Buffer W2. Centrifuge the mixture for 1 min at 12,000 x g. Repeat this step to wash again with 700 μL of Buffer W2.
   10. Discard the waste liquid, and put back the contents of the preparation tube into the original 2 mL centrifuge tube. Centrifuge the mixture for 1 min at 12,000 x g.
   11. Put the contents of the DNA preparation tube into another clean 1.5 mL centrifuge tube, and add 100 μL of eluent or deionized water to the middle of the membrane in the preparation tube (deionized water or eluent was heated to 65 °C). Allow the mixture to stand at room temperature for 1 min, and Centrifuge the mixture for 1 min at 12,000 x g.
3. Perform quantitative real-time polymerase chain reaction (Q-RT-PCR) to quantify the relative abundances of bacteria and fungi on the sampling filters.
   1. Use primers as follows: for bacterial 16S rDNA, 515F (5’- GTG CCA GCM GCC GCG GTA A-3’) and 806R (5’- GGA CTA CHV GGG TWT CTA AT-3’); and for the fungal ITS, ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS1 (5’-GCT GCG TTC TTC ATC GAT GC-3’).
   2. Run the Q-RT-PCR assays on a Real-Time PCR System (see Table of Materials). RT-PCR condition: predegeneration, 95 °C, 10 min; degeneration, 95 °C, 15 s; anneal and extension, 60 °C, 1 min; 40 cycles from degeneration to anneal and extension.
4. For bacterial and fungal community structure analysis, amplify the V1–V3 region of the bacterial 16S rDNA and the ITS region of the fungal rRNA operon by PCR.
   1. Use the primers as follows: for bacteria, V1-9F (5′-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG ACG AGT TTG ATC MTG GCT CAG-3′) and V3-541R (5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode-ACW TTA CCG CGG CTG CTG G-3′); and for fungi, ITS-3F (5’-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG CAC ATC GAT GAA GAA CGC AGC-3’) and ITS-4R (5’-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode-GCT CCT CCG CTT ATT GAT ATG C-3’).
   2. Ensure that the 20 μL mixture of PCRs included 2 μL of 2.5 mM dNTPs, 4 μL of 5× FastPfu Buffer, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA (see Table of Materials).
   3. Use the PCR program as follows: 94 °C for 5 min; followed by 10 cycles of 94 °C for 30 s, 55-60 °C for 45 s, and 72 °C for 90 s; 20 cycles of 94 °C for 30 s, 55 °C for 45 s and 72 °C for 90 s; and a final extension at 72 °C for 5 min.
5. Perform DNA purification, DNA quantification and pyrosequencing as described in a previous study ²¹.

5. Bioaerosol Sampling and Cultivation

1. Use an international standard Andersen six-stage sampler (see Table of Materials) to sample culturable airborne bacteria and fungi at a flow rate of 28.3 L/min. Use a sampling time of 35 min. Put the culture plate in each stage of the sampler. Sufficiently disinfect the sampler with 75% ethyl alcohol after each sampling.
   NOTE: The sampler flow rate is fixed and the collection time is controlled by manual time-keeping.
   1. Sample the six stages with the Andersen six-stage sampler, which is defined by the aerodynamic diameters of the airborne particles, including stage VI (0.65–1.1 μm), stage V (1.1–2.1 μm), stage IV (2.1–3.3 μm), stage III (3.3–4.7 μm), stage II (4.7–7.0 μm) and stage I (≥7.0 μm).
   2. Collect the bacterial particles and deposit onto the culture plate in each stage containing Soybean-Casein Digest Agar (see Table of Materials). There is a container with many air intakes on the top in each stage of the sampler.
   3. Culture the airborne bacteria collection plates at 37 °C for 24-48 h; then, count the colony numbers of bacteria on the sample dish for each stage.
2. To prevent the particles collected by the Andersen six-stage sampler from overlapping, correct the number of colonies at each sampler level by the following formula:
   
   where Pr: corrected number of colonies at each level,
   N: number of sampling holes of the sampler at all levels (400), and
   r: the actual count of colonies.
3. Calculate the unit of colonies per m³ of air as follows:
   
   where C: concentration (CFU/m³),
   N₁-N₆: the corrected number of colonies at each level,
   T: sampling time (min), and
   F: sampling flow rate (28.3 L/min).
4. Use methods from Steps 5.1 – 5.3 to study the bioaerosol in livestock farm, including four types of piggeries.
   NOTE: The livestock farm is located in Changchun, China. The central position 2 m above the ground in each piggery was selected as sampling location.) After culture of bacteria, treat all colonies in the plates as described in Steps 6.1 – 6.2.

6. Identification of Culturable Bacteria

1. After 48 h or 72 h of culturing, place the bacteria into a 2 mL centrifuge tube. Extract the DNA of these bacteria or fungi by using a multisource DNA extraction Kit (see Table of Materials). The DNA extraction process is described in section 4.2.
2. Use 200 μL DNA extraction sample for 16S rDNA sequencing. Use the same processes as described in Steps 4.2, 4.3 and 4.4 on biological composition analysis.