Protozoal Enzyme Problem

[Heather Martin]

I am PhD student studying the protozoal enzyme(s) responsible for the 
degradation of bacterial cell walls in the rumen. My problem is the fact that 
the protozoa are impossible to culture without bacteria, and there is an 
indigenous population of endosymbionts always present inside them. So far, I 
have detected 3 bacteriolytic enzymes from a population of the rumen ciliate 
Entodinium caudatum. Unfortunately, I have also seen the same three enzymes 
from protozoa-free rumen fluid, suggesting that the enzyme activity from my 
protozoa, could be due to bacterial contamination. In the preparation of my 
protozoal extract, I include an overnight incubation with an antibiotic 
cocktail, to try and eliminate as many of the bacteria as possible. I then 
sonicate the protozoa, spin them, and the supernate is my extract which I then 
run on SDS polyacrylamide gels with Micrococcus lysodeiticus incorporated into 
them, so bacteriolytic activity shows up as clear bands in the opaque gel.