Toxoplasma gondii oocyst purification. Protocol: MJerome060601 Ó2001 Michael W. White-Department of Veterinary Molecular Biology || mjerome@montana.edu
WhiteProtocols2001
1.0 Toxoplasma gondii oocyst purification. 1.1 Reagents. 2.2 M Sucrose: 1180 g + 820 ml Milli-Q, dissolve overnight. 0.9 M Sucrose: 360 ml 2.2 M sucrose + 520 ml Milli-Q 0.7 M Sucrose: 280 ml sucrose + 600 ml Milli-Q. 1.2 Assemble the following items in a fume hood: ·Large autoclavable funnel ·Squares of cheesecloth to line funnel approx. 10” X 10” ·Tongue Depressors ·Autoclave Bag ·Pan for boiling waste ·250 ml polypropylene centrifuge bottles ·Pipet aid ·Cat fecals in small disposable cups with lids ·Water 2.0 Soak fecal collections in water in cup for a couple of hours or overnight. When ready the mixture should have the consistency of light cream. Stir with the tongue depressor to assure the proper consistency. 3.0 Line funnel with cheesecloth. Place the funnel into the top of a centrifuge bottle and pour the fecal slurry through the cheesecloth. Gently press the fecal through the cheesecloth with the tongue depressor. To do this pick up the corners of the cheesecloth and form a pouch and press with the depressor. 4.0 Place the cheesecloth with the fecal particulate into the cup, cover, and dispose of it into the autoclave bag. The slurry in the centrifuge bottle should then be mixed one to one with 2.2 M sucrose. 5.0 Using the pipet aid layer 40 mls of the 0.9 M sucrose on top of the slurry and then carefully layer 40 mls of the 0.7 M sucrose on the 0.9 M sucrose. 6.0 Centrifuge in the tabletop for 20 minutes at 20 degree Celsius at 2500 RPM. The oocysts should be in the top 20 –40 mls. Carefully remove this layer by pipetting and putting it into 50 ml tubes. Mix this layer with water at a 1:1 concentration and spin in centrifuge for 20 minutes at 2000 RPM. Oocysts are in the pellet. 7.0 Pellets from the 50 ml tubes may be combined in 2% sulfuric acid. Count oocysts using a hemacytometer. 8.0 Remaining liquid and pellet should be mixed and poured into the boil pan and boiled heavily. After boiling, the waste may be disposed of.
Department of Veterinary Molecular Biology, Montana State University, Bozeman MT