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[stacker]

I took 1 mL of Aggregatibacter actinomycetemcomitans inoculated broth and placed it into a 1.5 mL microcentrifuge tube and centrifuged it. I removed the supernatant and left the bacteria pellet at the bottom of the tube. I then put 1 mL of glycerol into the tube but forgot to vortex the bacteria and the glycerol together and put them into the freezer. So instead of the tube looking like a cup of water thoroughly mixed with salt, it looked more like a cup of water with solid sediments at the bottom before I put it into the freezer.
Could this be the reason why my freezer-stored bacteria will not grow?

[bob1]

It certainly could be.  You should have a narrow layer of cells that will be OK, but you would probably be better off starting the culture again.