Asking the pikas politely…
Upon completion of sampling, 0.2μm filters containing collected aerosol particles are carefully removed from filter units and stored frozen at approximately −20°C in individual sterile Petri dishes. Samples are preserved on site until they can be transported in frozen batches to CSU
Sample processing and resuspension
Each filter is placed in a sterile 50mL polypropylene tube with 7-10mL of 0.1μm-filtered deionized water, depending on expected aerosol loading. Samples are re-suspended by rotating the tubes end-over-end for 20 minutes. Dilution series are prepared using the suspensions and 0.1μm-filtered deionized water, typically including 11-fold dilutions
Quantities: 7-10mL of 0.1μm-filtered deionized water per 50mL tube, 11-fold dilutionsDuration: 20 minutes for resuspensionConditions: Laboratory conditions
Equipment: 50mL polypropylene tubes, 0.1μm-filtered deionized water, rotation system
INS analysis
Each suspension and its dilutions are dispensed into blocks of 32 aliquots (50μL each) in single-use 96-well PCR trays. The trays are placed in the aluminum blocks of the INS and cooled at 0.33°C min⁻¹. Freezing is detected optically using a CCD camera with 1-second data resolution. HEPA-filtered N2 continuously purges the headspace to prevent condensation build-up
Quantities: 32 aliquots of 50μL each per sample, cooling rate of 0.33°C min⁻¹Duration: Temperature range from 0°C to approximately −27 to −30°CConditions: HEPA-filtered N2 atmosphere, continuous purging
Equipment: 96-well PCR trays, aluminum incubation blocks, cold plates, CCD camera, HEPA-filtered N2
Heat and peroxide treatments
Approximately one-third of samples undergo specific heat and peroxide treatments to identify INP composition. Heat treatment involves heating 2.5mL of sample suspension to 95°C for 20 minutes. Peroxide treatment is performed by adding 1mL of 30% H2O2 to 2mL aliquot to create a 10% solution, followed by heating to 95°C for 20 minutes under UVB illumination
Quantities: 2.5mL for heat treatment, 2mL sample + 1mL of 30% H2O2 for peroxide treatmentDuration: 20 minutes at 95°C for both treatmentsConditions: 95°C heating, UVB illumination for peroxide treatment
Equipment: heating system, 30% H2O2, UVB light source, catalase for neutralization