Before testing our proposed density gradient centrifugation technique on INS suspensions, we tested the density gradient centrifugation technique using ultrapure water to establish if the technique introduces INS into the low-or high-density isolates. In these experiments, the high-density isolates overlap with the ultrapure water controls, indicating that the density gradient centrifugation technique was not introducing significant amounts of INS into the high-density isolates (Figure ). The freezing curves of the low-density isolates overlap with the ultrapure water controls at temperatures less than approximately -23 °C (Figure ). At warmer temperatures, the INS concentrations in the low-density isolates were significantly higher than in the ultrapure water controls in two of the three experiments. This suggests that some INSs were being introduced into the low-density isolates at some point in some experiments. INSs could be introduced from the lab air, the density gradient medium, materials used during density gradient centrifugation such as centrifuge tubes or pipette tips, and materials used in droplet freezing assays such as hydrophobic glass slides. Ultimately, the concentrations of INSs introduced are small and did not limit us from additional tests of the density gradient centrifugation technique. Information Classification: General