Mar 03, 2020
Ectoplasmic Net (EN) formation in Aurantiochytrium limacinum (ATCC MYA-1381)
- 1University of Illinois at Urbana-Champaign;
- 2State University of New York at Stony Brook
- Collier Lab
Protocol Citation: Anbarasu Karthikaichamy, Jackie Collier 2020. Ectoplasmic Net (EN) formation in Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.bc7hizj6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 03, 2020
Last Modified: March 03, 2020
Protocol Integer ID: 33737
Keywords: Aurantiochytrium, protists, ectoplasmic net
Abstract
Protocol for ectoplasmic net (EN) formation in Aurantiochytrium limacinum (ATCC MYA-1381).
Before start
Grow cells in GPY media for 3 days
d-GPY plating
d-GPY plating
- Take 150 µL of 3 day old cells grown on GPY media and plate it on d-GPY plates.
- The recipe for d-GPY meida is as follows,
| Components | Quantity (%) | |
| Yeast extract | 0.05 | |
| Peptone | 0.1 | |
| Glucose | 0.2 | |
| Prepare 1/2 saline media by adding appropriate amount of instant ocean and water. | ||
| For agar plates | 1.5% agar | |
- Incubate the plates at 25 °C Overnight
Note
Add appropriate antibiotics to minimize bacterial contamination.
Zoospore production
Zoospore production
- After overnight incubation (~24 h), add just enough artificial sea water ((with appropriate antibiotics) to cover the entire plate.
- Incubate the plates for 03:00:00 25 °C
Note
Do not disturb the plates!
- After the incubation, take a small aliquot from the sea water layer and observe for the presence of zoospore.
- The zoospore production starts in about 90 mins, but it peaks around 3 h.
Settlement and EN formation
Settlement and EN formation
- Take an empty sterile petridish and place a sterile microscopic slide.
- Add just enough A1 media (with appropriate antibiotics) on to the petridish so that the microscopic slide is immersed.
- Alternatively, a cover slip can also be used.
- Aspirate arround 50 µL to 100 µL of zoospores without disturbing the plate.
- Carefully drop the zoospores all over the microscopic slide.
- Cover the petridish and incubate at 25 °C Overnight
- Observe the EN the next day.
Note
EN starts forming after 90 mins incubation in the slides. If the experiment demands to observe EN formation, the zoospores can be allowed to settle on a glass bottom petridish for overnight timelapse microscopy.