Twin-plate Ice Nucleation Assay (TINA) with infrared detection for high-throughput droplet freezing experiments with biological ice nuclei in laboratory and field samples

Abstract

Abstract. For efficient analysis and characterization of biological ice nuclei under immersion freezing conditions, we developed the Twin-plate Ice Nucleation Assay (TINA) for high-throughput droplet freezing experiments, in which the temperature profile and freezing of each droplet is tracked by an infrared detector. In the fully automated setup, a couple of independently cooled aluminum blocks carrying two 96-well plates and two 384-well plates, respectively, are available to study ice nucleation and freezing events simultaneously in hundreds of microliter-range droplets (0.1–40 µL). A cooling system with two refrigerant circulation loops is used for high-precision temperature control (uncertainty <0.2 K), enabling measurements over a wide range of temperatures (∼ 272–233 K) at variable cooling rates (up to 10 K min−1). The TINA instrument was tested and characterized in experiments with bacterial and fungal ice nuclei (IN) from Pseudomonas syringae (Snomax®) and Mortierella alpina, exhibiting freezing curves in good agreement with literature data. Moreover, TINA was applied to investigate the influence of chemical processing on the activity of biological IN, in particular the effects of oxidation and nitration reactions. Upon exposure of Snomax® to O3 and NO2, the cumulative number of IN active at 270–266 K decreased by more than 1 order of magnitude. Furthermore, TINA was used to study aqueous extracts of atmospheric aerosols, simultaneously investigating a multitude of samples that were pre-treated in different ways to distinguish different kinds of IN. For example, heat treatment and filtration indicated that most biological IN were larger than 5 µm. The results confirm that TINA is suitable for high-throughput experiments and efficient analysis of biological IN in laboratory and field samples.

Macromolecular fungal ice nuclei in <i>Fusarium</i>: effects of physical and chemical processing

“…Fusarium acuminatum 3-68 showed the highest ice nucleation activity and F. langsethiae the lowest per gram of mycelium. The results are comparable to other IN-active microorganisms like Sarocladium implicatum (10 8 g −1 , Pummer et al, 2015), Mortierella alpina (10 9 g −1 , Fröhlich-Nowoisky et al, 2015; 10 10 g −1 , Kunert et al, 2018), and the bacterial IN-active substance Snomax ® containing Pseudomonas syringae (10 12 g −1 , Budke and Koop, 2015;Kunert et al, 2018). The size of the Fusarium IN was investigated by filtration experiments.…”

“…For Twin-plate Ice Nucleation Assay (TINA) (Kunert et al, 2018) experiments (see Sect. 2.3) the fungal mycelium was scraped off the agar plate and transferred into a 15 mL tube (Greiner Bio One, Kremsmünster, Austria).…”

“…The fresh weight of the mycelium was determined gravimetrically. Pure water was prepared as described in Kunert et al (2018). Aliquots of 10 mL pure water were added before vortexing three times at 2700 rpm for 30 s (Vortex-Genie 2, Scientific Industries, Inc., Bohemia, NY, USA) and centrifugation at 4500 g for 10 min (Heraeus Megafuge 40, Thermo Scientific, Braunschweig, Germany).…”

“…Ice nuclei of selected Fusarium species, which were long known for ice nucleation activity (F. acuminatum, F. avenaceum), as well as all the newly identified species, were further analyzed in immersion freezing mode using the highthroughput Twin-plate Ice Nucleation Assay (TINA) (Kunert et al, 2018). The aqueous extracts were serially diluted 10fold with pure water by a liquid handling station (epMotion ep5073, Eppendorf, Hamburg, Germany) to a dilution at which droplets remained liquid in the investigated temperature interval.…”

“…The temperature was measured with an accuracy of 0.2 K (Kunert et al, 2018). The obtained fraction of frozen droplets (f ice ) and the counting error were used to calculate the cumulative number of IN (N m ) with the associated error using the Vali formula and the Gaussian error propagation (Kunert et al, 2018;Vali, 1971). For each experiment, the cumulative number of IN was averaged over all dilutions.…”