TITLE: Injection of algae into Ambystomid (salamander) embryo
AUTHORS: John  Burns, Hui  Yang, Joost S Mansour

[DESCRIPTION]
This protocol describes the steps taken to artificially introduce algal cells into salamander tissues by microinjection for the study of salamander-alga endosymbiosis. Microinjection allows experimental manipulation of the separate partners before the establishment of symbiosis. For instance, algae can be grown with a heavy nitrogen (15N) or carbon (13C) label prior to injections to trace nutrient transfer.

[BEFORE_START]
Salamander egg clutches need to be collected as close to laying as possible (i.e. very young). Collected egg clutches can be cleaned following the same steps as the first section of this protocol. Once washed the egg clutches should be incubated in the dark to prevent algal growth. This way there should be no algae other than the ones injected during this protocol. Leave the egg clutches to incubate and grow to the desired stage.

[GUIDELINES]
Embryos (A. maculatum) for injection should be between stages 26-31. Injections of younger embryos (~stage 19) ended with the death of all embryos. Injection of embryos at stage 33+ can be problematic because as the embryos flex, they can tear the injection site, preventing the wound from healing and causing eventual death.
Injection of embryos at stages 26-30 has good survival: 95% survival at 24 hours, >50% after 5 days 

[STEPS]
SECTION: Prepare the embryos
1. Load whole eggs containing embryos into a strainer.
SECTION: Prepare the embryos
2. Wash eggs with 70% ethanol for ~20 seconds.
SECTION: Prepare the embryos
3. Wash eggs with Milli-Q water for ~20 seconds.
 

SECTION: Prepare the embryos
4. Following the water rinse, transfer eggs to a 100 mm petri dish containing sterile 20% Holtfreter's solution.

  
 


SECTION: Prepare the embryos
5. Embryos are now ready for injection. 
SECTION: Prepare the algae
6. Pour 40 mL of algal culture into a 50 mL falcon tube.
SECTION: Prepare the algae
7. To pellet the algae centrifuge at 300 x g, 8 min 

SECTION: Prepare the algae
8. Remove supernatant, and place tube upside-down on a kimwipe to remove excess liquid.
SECTION: Prepare the algae
9. Add 20 mL sterile 20% Holtfreter's solution to the algal pellet and resuspend it.
SECTION: Prepare the algae
10. Repeat centrifuge  at 300 x g, 8 min 
SECTION: Prepare the algae
11. Remove supernatant, remove excess liquid as before (  ) 

 

SECTION: Prepare the algae
12. Resuspend the pellet in 400 uL of 20% Holtfreter's solution. 
This is the 100x concentrated algal solution that will be used for injections.
 

SECTION: Prepare the algae
13. Fix a sample of the concentrated algal culture in 0.1% glutaraldehyde for cell counts. This can be used to verify the concentration of the culture finally used.
 

SECTION: Inject the embryos with algae
14. Prepare an individual embryo in a well in plastic mold (e.g., Fisherbrand™ Disposable Base Molds or any kind of well that will hold the egg snug). 
Eggs are typically kept and injected underwater (20% Holtfreter's)
SECTION: Inject the embryos with algae
15. Place the embryo in its mold/well on the dissecting microscope.
SECTION: Inject the embryos with algae
16. Prepare the syringe by backfilling the syringe with the concentrated algal solution. Close the syringe by adding the needle.
SECTION: Inject the embryos with algae
17. Prep the needle by evacuating air, and push out some solution to ensure that algae are coming out during injections.
SECTION: Inject the embryos with algae
18. Carefully penetrate the egg and then break into the embryo's skin surface with the needle (beveled). It is not necessary to push the needle deep into the embryo. 
 

 

SECTION: Inject the embryos with algae
19. Inject algae.
 

SECTION: Inject the embryos with algae
20. Carefully withdraw the needle straight back.
SECTION: Inject the embryos with algae
21. Transfer the embryo to an individual well in a 24 well plate containing 1.5 mL 20% Holtfreter's solution.     
SECTION: Prepare the embryos
1.1.   

SECTION: Prepare the embryos
2.1.   

SECTION: Prepare the embryos
3.1.    *wash steps can be repeated if desired.  
SECTION: Inject the embryos with algae
21.1. Repeat until all embryos have been injected (  ). 


 
Video of algal injection. Example of algal injection into an early (21-23) stage A. maculatum embryo. Later stages such as 25-28 are recommended.
SECTION: Inject the embryos with algae
22. Place the 24-well plate containing the injected embryos in an incubator with a 12/12 light-dark cycle.