Question
My pond (20'x12'x4'deep) was full of stink grass/musk-grass chara algae
until I raked out most of it. I then drained the water and removed most
minnows, platys, shrimp, 2 perch, and a gar. Next I added water and
added copper sulfate incrementally until filled. I'm sure I used too high a
dose but worried it may affect live oak and bald cypress roots. My main
question is whether 24-36 hour exposure enough to kill what little chara is
left along with spores if I drained it tonight or morning? After draining
completely is it necessary to treat the fish I removed to kill spores directly
before putting in pond?  

Answer
It depends on the hardness of the water.  In very hard waters, Copper Sulfate only has a reaction time of about ten minutes.  So the question isn't so much 'how long to allow the Copper Sulfate to work,' but 'how long the copper sulfate will work before becoming an inactive compound; and is this long enough to eradicate the algae.'  This is why you usually have to treat several times in a row.  Check your water's hardness and refer to a water hardness and Copper Sulfate treatment dose chart to be safe.  At the end, spot-treat any remaining concentrated areas.

In a clay bottom pond, Copper Sulfate is known to cause a sterile bottom, meaning that the beneficial bacteria cannot settle there.  There is a fairly recent chelated copper compound (sold as cutrine) that is more gentle on the pond and will also biodegrade.  If you find that you need to treat again I recommend that you use this instead.