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Awhile ago I posted looking for advice on a poor stuffy Bb below the stave on my Conn 26K asking if it was a common trait on this model.
Most of the replies suggested looking at the valve alignment I finally got around to doing this and have some video of the valves in action.
Bb is played first and third and is extremely difficult to get to sound nice.
The valves appear to be aligned to my untrained eye but I may be wrong.
http://youtu.be/7N0HWSv88M8" target="_blank" target="_blank" target="_blank
first valve at rest and depressed.

http://youtu.be/vykM-tceEW0" target="_blank" target="_blank" target="_blank
second valve at rest and depressed

http://youtu.be/GfUimgV17t4" target="_blank" target="_blank" target="_blank
third valve at rest and depressed

The sousaphone is going in the bath tonight and getting a good snaking now that I can see the loose flaps of gunk in the pipes.

If anybody can see a problem with the alignment please let me know the valve are original to the cluster as all serial numbers match.
All other notes using 1 and 3 sound easily and well.

A low Bb on an Eb tuba/sousa is '2nd from the bottom' and not always a note that really 'sings'. You're gonna need all the help you can get... including getting all the 'gunk' out of the horn.

I am assuming you are using the borescope to look into the tuning slides. The alignment there looks OK. But... how do the ports in the open bugle ('up' ports) line up? I seldom use the tuning slide ports to check alignment. I prefer to go in through either the leadpipe or up from the main slide.