4 23 2015 Heat/Mechanical Shock 24 Hour Time Point

Today I completed the 24 hour time point collection and the collection of the controls for the experiment. Tonight I ran out of liquid nitrogen before doing the control groups. I put them on ice and placed them in the -80 ASAP.

Heat Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml screw cap tube dropped into liquid nitrogen -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Mechanical Stress

• 24 hours post exposure 8 animals each pop staggered by 1 hour
  • N pop first, H pop next, S pop last
  • collected with ethanol flame sterilized forceps and scissors
    • tools kept in 100% EtOH
    • ran through EtOH flame
    • allow EtOH to burn off
    • Collect tissue
    • replace in 100% EtOH
    • wipe occasionally with paper towel and resterilize
  • ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
  • 2nd ctenidia sample in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
  • mantle sample in 1.5 ml tube dropped into liquid nitrogen - then to -80°C
  • remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
  • shells placed in labeled plastic bags so correspond to sample including oyster id

Controls

For controls sample 8 from each group, this should be done on day 2 as less things are going on. Protocol is the same. These oyster should have remained in holding tank-throughout and should simply be removed and sampled. There is no treatment. Using sterile techniqes.

• ctenidia sample (50-100 mg) homogenized in 1 ml RNAzol stored at -80°C
• 2nd ctenidia sample (any remaining) in 1.5 ml screw cap tube dropped into liquid nitrogen then to -80°C
• mantle sample (50-100 mg) in 1.5 ml tube dropped into liquid nitrogen then to -80°C
• remaining tissue placed in 15 ml conical with 75% EtOH at -20°C
• shells placed in labeled plastic bags so correspond to sample including oyster id