PRJ002AiptasiaMM Experimental start notes and T0 data
Objective: Experimental Setup of microbial Knockdown project.
Broader Context
This project will monitor the success of microbial knockdown under two seperate Antibiotic solutions being treated fro 7 days, and will also monitor the duration for which the microbiome stays reduced. Initial sample priming will involve the reduction of the microbiome through keeping the organisms in Seawater filtered with a .22 micron filtered, and feeding with Artemia that have had their microbiome knocked down through the use of antibiotics.
Parameters to be monitored throughout this experiment:
- CFU counts
- microbial knockdown through 16s PCR
- growth rate through change in wet weight
- pedal laceration rate
- morphological changes through photo monitoring;
Note the last three parameters will be monitored in individuals kept in 6 well plates for the duration of the experiment; all other individuals will be kept in 12 well plates
Protocol
Initial priming: individuals will be kept in 12 well plates for 1 month, in Artificial seawater which has been filtered through a 0.22 micron filter (FSW) and fed twice weekly with Artemia treated with ABS 1 ( see entry on antibiotic preparations)
Wet weights for anemones in the Physiology plates were taken by transfering the anemone using a transfer pipette to a clean petri dish, pipetting off any excess water and then transferring to a second petri dish and weighed on the scale
| Plate | Well | Weight (g) |
|---|---|---|
| control | 1 | 0.0049 |
| control | 2 | 0.0059 |
| control | 3 | 0.0036 |
| control | 4 | 0.0040 |
| control | 5 | 0.0057 |
| control | 6 | 0.0047 |
| ABS 1 | 1 | 0.0043 |
| ABS 1 | 2 | 0.0045 |
| ABS 1 | 3 | 0.0053 |
| ABS 1 | 4 | 0.0043 |
| ABS 1 | 5 | 0.0039 |
| ABS 1 | 6 | 0.0051 |
| ABS 2 | 1 | 0.0054 |
| ABS 2 | 2 | 0.0034 |
| ABS 2 | 3 | 0.0078 |
| ABS 2 | 4 | 0.0040 |
| ABS 2 | 5 | 0.0049 |
| ABS 2 | 6 | 0.0058 |
3 samples were taken to obtain time 0 data for comparison. 1 anemone was taken from each of the three 12-well control plates for this purpose. the corresponding sample numbers are SAM_133- SAM_135.
For each of the samples:
- a handheld pestle was used to homogenixe the anemone in 300ul of filtered ASW
- 50 ul was used to plate serial dilutions ( 10 -1, 10-2, 10-3). for each dilution 50 ul was plated o a marine agar plate using roller beads
- plates were incubated at 30 degrees for approximately 24 hours
- 250 ul was transfered to an mp biomedicals lysing matrix E tube with the glass Bead removed
- 500 ul of DNA/RNA shield was added to each of the samples
- samples were bead beat for two rounds of 60 sec at 8 m/s with a five minute pause between rounds and frozen at -80 degrees.