David Coil edited Materials_and_Methods_Sample_collection__.md  over 8 years ago

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A set of bacterial plates were created for each aspect of the study: growth in microgravity on the ISS (space plates), or growth on earth (ground plates). The plates were created using clear agar to facilitate optical density (OD) measurements. 1.5 g of Gelzan™ CM agar (Sigma-Aldrich) was added to 1 liter of lysogeny broth (LB). Each well of a flat-bottomed 96-well plate (Costar) was plated with 200 μl of agar. The plates were flamed to remove bubbles and incubated for 48-72 hours at room temperature (~20 °C) to ensure sterility before adding bacteria. Fresh overnights of each bacterial isolate were diluted to .01 OD600 and made into 8% glycerol stocks. For plating, 10 μl of each thawed stock dilution was added to 2 wells per 96 well plate. 6 replicate plates were made. The bacteria were placed into different locations on each plate in order to account for drying at the edges or any other positional effects on the plates. The plates were then sealed with adhesive polypropylene film (VWR #60941-072), into which a grid of micron-diameter holes were cut with a laser to allow for airflow. The ground plates were stored at -80 °C at UC Davis, and the space plates were mailed on dry ice to the National Aeronautics and Space Administration (NASA) Johnson Space Center in Houston, TX before transfer (at -80 °C) to Cape Canaveral, FL for launch.  This payload was flown on the CRS-3 launch of the Space Exploration Technologies (SpaceX) Dragon spacecraft, on a Falcon 9 v1.1 rocket which successfully launched April 18, 2014. After six days, the space plates were removed from the MELFI (Minus Eighty Lab Freezer For ISS) and partially thawed. However, technical problems arose and the space plates were placed back into the MELFI until December 8, 2014. At that time, all three plates were thawed and the OD600 of each well (3x3 grid) was measured at time 0 (60 minutes after removal from the freezer) and then every 24 hours for 4 days. Measurements were performed in a Molecular Devices SpectraMax M5e plate reader which was modified for integration onto the ISS. On these same days, equivalent measurements of the ground plates were taken in a Molecular Devices SpectraMax M5e plate reader at UC Davis. The exception to this was the initial partial thawing, which was not replicated with the ground plates since the amount of thaw was not reported by the astronauts.  After the experiment, the ground plates were placed back at -80 °C and the space plates were placed back into the MELFI. In February 2015, the space plates were transferred to a -95 °C freezer on board a Dragon spacecraft. The vehicle splashed down in the Pacific Ocean on Feb 10, 2015. The space plates were then mailed to UC Davis on dry ice and were transferred to -80 °C when received. Once the plates arrived, we thawed all six plates and performed a high-density measurement in a Tecan M200 plate reader. OD600 readings were taken in a 5x5 grid covering the entire well, these 25 measurements were then averaged within each well.