An Alternative Methodology for Preparation of Bacterial Samples for Stratospheric Balloon Flight: Comparison Between High Density Wet Pellet and Medium Density Glycerol Solution

Abstract

Introduction: Micro-aerosols containing bacteria can be found in extreme conditions of the terrestrial stratosphere. Lyophilization is a standard method for preparing bacterial samples for stratospheric balloon flights. Here we compare the viability of bacteria prepared according to different methods: high- density wet pellets and medium-density broth suspension with glycerol before and after a stratospheric balloon flight in order to establish an effective and accessible method alternative to lyophilization.

Methods: Subjects of our study were bacteria causing hospital-acquired infections. One set of samples was prepared as a wet pellet. The second set of samples was obtained by adding brain heart infusion broth with 15% glycerol solution to the wet pellets. Each set had an additional group with protective layer of aluminum foil. A viable count assay was performed by preparing series of dilutions and calculating Colony Forming Units (CFU/ml). Next, we compared the data between two sets before and after a single stratospheric flight.

Results: Majority of the bacterial strains (72,8%) showed a higher survival rate suspended in a glycerol solution. Carbapenem-resistant E. coli pellet samples had higher viability. Results for P. aeruginosa were inconclusive, because CFU numbers varied between different sets and groups. Therefore, no obvious trend in survival could be observed.

Conclusions: Although lyophilization of bacterial samples may still be the most popular method, it is not necessarily required to perform basic stratospheric balloon flight experiments. Both our methods used with this set of bacteria allow most of them to survive a stratospheric balloon flight, however the glycerol supplement seems to give better protection. It is possible that different species require different approach.

Appendix

The experimental mission was 2 h 29 min long and it was launched on 18th July 2020 at 10:30 CEST at Queen Jadwiga Astronomical Observatory in Poland, geographical coordinates: 49.7761 latitude, and 21.0901 longitude. 4 m$^3$ of hydrogen gas (Linde) was inflated into the 1600 g latex balloon. Scientific payload weighting 2 kg (with two cameras onboard and one tracker and parachute), was attached to the balloon. The balloon flight occurred according to the planned time, because the flight predictions were correct. That means that the balloon landing was planned to be located in a safe area within the borders of Poland. Flight predictions have been made using predict.habhub.org software integrated with Google Maps, available on internet. The flight track is visualized on Fig. 1 and Fig. 2.

Fig. 1.

Maps revealing the flight prediction and the flight tracking using SPOT GEN3 satellite tracking system during the STRATOS mission in 18th July 2020.

Fig. 2.

STRATOS mission flight profile. The flight was 2.5 h long, with about 1 h exposition to high levels of UVA, UVB, UVC light. Balloon burst occurred in the stratosphere at about 31 km altitude. The graph on the right side visualizes temperature fluctuations at different altitudes. Curve was obtained as average from 4 temperature sensors located on the payload capsule.

The balloon ascending time was 1 h 43 min, while descending was only 46 min. Average ascend velocity was 4,825 m/s. The balloon burst at 31 km altitude. Tracking system was provided by SPOT GEN3 satellite GPS messenger. This device was used for flight tracking using 100% satellite technology.