November 14, 2019 - Transferring Frozen Larvae into Ethanol Part 1

Today, I began helping Laura with transferring frozen Olympia oyster larvae from her experiment studying temperature and food effects on the parental generation and the subsequent effects that may have been experienced by the larvae.

Background of Experiment

Olympia oysters were placed in four different treatment groups that varied in food and temperature. When the oysters began larval release, these larvae were collected for further analysis.

The larvae samples that were collected were then measured, but the sequential way in which the larvae were measured and possibly high levels of ethanol used may have contributed to an unexpected trend seen in the data. So, the next step moving forward is to find an optimal percentage of ethanol to preserve the larvae in as well as to measure the larvae in a randomized fashion to confirm if the trend seen in the data is accurate or not.

Current Lab Work

General Tips and Tricks Gained

  1. Freezers: Always TRIPLE CHECK to make sure that the freezer is closed
    • Dry Ice: When working with dry ice, don’t hold the ice for too long or it may start to hurt
  2. Lab work:
    • Pipettes: When drawing liquid from the pipette tip, go down to the first “stop” point. When releasing the liquid, go down to the first “stop” point, and then release any remaining liquid by going down to the second “stop” point.
    • Gloves: When removing gloves, remove the first glove by grabbing the bottom of the glove (where the opening is) and pulling out so that the glove turns “inside-out”. To remove the other glove, slide index finger underneath the opening of the glove and pull glove out to turn it “inside-out”. This way, both gloves are removed safely without touching any materials that may be on the glove.
    • Sample tubes: To gather tubes from their respective bag, do not reach hand into the bag (since this may cause the tubes to be contaminated). Instead, pour out tubes from bag. Also, close the tube caps once tubes are gathered.
    • Samples: For the frozen larvae samples, try to keep them frozen as much as possible. If the sample is too frozen to collect larvae from, let it sit out for ~30 seconds, but do not leave it out for minutes.

Different Ethanol Concentrations Experiement

Before actually putting larvae into a set percentage of ethanol, we began testing different levels of ethanol to see which level would be optimal to preserve the larvae. Using 190 Proof Ethanol, we calculated the number of uL needed to have 1000 uL (1 mL) of solution for each of the following ethanol percentages:

Materials 70% 75% 80% 85% 90% 95%
Ethanol (95%) 737 uL 789 uL 842 uL 895 uL 947 uL 1000 uL
DI Water 273 uL 211 uL 158 uL 105 uL 53 uL 0 uL

We used two samples of larvae (7 and 50) to test the different ethanol concentrations. Sample 7 was surprisingly softer than what Laura was expecting, so having sample 50 tested as well might be a good way to accurately see how well the ethanol at different percentages preserve the larvae.

Next Steps

Laura has made a spreadsheet that has a randomized list of the sample numbers, so that the order in which the measurements are made are randomized as well.

  • Next week, Laura will look back at the different ethanol concentrations and find the optimal preservation concentration for the larvae.
  • Once the optimal concentration is found, a bigger volume of this concentration will be made to begin transferring all larvae samples into ethanol.
Written on November 14, 2019