Resazurin with stressed oysters 06/24-06/25

Procedure- 

1. A resazurin stock solution was prepared by mixing 9.890 mL of salt water with .100 mL of resazurin.
2. 2 mL of this solution and 7.990 mL of salt water was used to make a 1:5 dilution, and 1 mL of this solution and 8.990 mL of sea water was used to make a 1:10 dilution.
3. Then 0.010 mL of DMSO was added to each solution to keep it constant across each solution.
4. After the stock solution, 1:5 dilution, and 1:10 dilution are prepared, then 2 mL of each solution are added to their respective row with 4 wells filled per row, and each row is a different concentration.
5. Small oysters are then added to 2 columns where one column is stressed oysters that have been in freshwater and the other are controlled oysters in salt water. 1 column is left as a control solution without any oysters, and one column had a random size assortment of stressed and non-stressed oysters. 
6. An initial intensity measurement is then taken with the spectrophotometer, and subsequent measurements are taken every 15 minutes for 4.5 hours.
7. Then, measurements where taken every hour for 23.5 hours.

Results-

Control Wells (Only Resazurin no Oysters)-

Different Sized Oysters-

Note: DZ0.02 is a stressed oyster

Controlled & Stressed Oysters-

SO- Stressed Oyster, CO- Control Oyster, DZ- Different Sized Oysters, C- Control Solution no Oysters

Numbers after this represent concentration

Observations-

Photo was taken at 4.5 hours, and column type from left to right is control, stressed oysters, control oysters, and different size oysters. Then rows from top to bottom are 1:10 dilution, 1:5 Dilution, and 0.1 M resazurin. There was a significant color change in the stressed oyster 1:10 dilution, and the 1:10 & 1:5 dilution of the different sized oysters.

This photo was taken after 23.5 hours, and has the same layout. Unfortunately, a lot of water evaporated overnight but there was still a noticeable change in color for all of the oysters compared to the control on the left column.

Discussion-

The color change happened much faster in the stressed oysters, this could be seen the in both pictures, and the graph from the spectrophotometer. It also was faster with the larger oysters, as there was a noticeable color change after only 4 hours. And the color change seemed to be the strongest in DZ0.2 as it had a very light pink color. However, a color change was eventually seen in all wells with oysters, and they all had a strong upward trend in intensity that was measured by the spectrophotometer. Furthermore, it doesn’t seem like there was a notable change in the intensity measured with different dilutions, however, changes in color were first seen with the lower dilutions.

Unfortunately, these were also the ones that evaporated the fastest which is something that needs to be taken into consideration in the future. Another thing to take into consideration in the future is the effect the oyster has on the measurement from the spectrophotometer, especially with the bigger oysters as it is less accurate because it interferes with the solution reading. This is also likely the cause of the weird jumps in the graphs of the different size oysters (along with evaporation).