Purification protocols

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Tissue Culture Media:

Frank Hinks modification of Grace's Medium, bought in powder and mixed in the lab, Sigma's TNM-FH.

Mixing the media:

1. Measure out 90% of the final volume of media
2. Slowly mix in powdered media with constant gentle stirring, no heat
3. Add 0.35g of Sodium bicarbonate
4. Bring the solution to its final volume and adjust the pH to 6.6

Fetal Bovine Serum (5%) was added as well as 50 mg/ml gentomycin and the media was filter sterilized using a Corning Sterile Filter Set with a 0.45 µm pore size.

Transferring Cultures:

Cultures were kept in Corning Costar Tissue Culture Flasks. In transferring the cultures 5 ml of media was dispensed into the tissue culture flask using a sterile serological pipette. Then a small amount of culture was added to the flask using a sterile pipette, and also a small amount was placed on a slide to check for contamination. Cultures were transferred twice a week.

Purifying In Vitro Cell Cultures:

In order to separate vegetative cells from pellicles a Percoll gradient solution was used. To make the gradient a gradient maker and a range of concentration from 10-50% was used. The Percoll must be prepared with a final concentration of 10% 1.5M NaCl. The volume of the gradients is determined by how much sample there is. The volume of the gradients were 6 ml and this was able to support sample loads of up to 3x10⁸ cells in 1 ml. The gradient is then centrifuged in a swinging bucket rotor at around 700-1000g for 20 minutes. Pellicles are collected using a syringe, and usually occurred 1-2 ml down from the top of the gradient after centrifugation. This Percoll solution was then diluted down at least 1:5 and spun at 1000g for 15 minutes. This wash step was repeated three times to remove the Percoll.

Separating Cysts from In Vivo blood samples or In Vivo preparations:

Ludox is best for separating cysts from vegetative cells, or from insect matter. In order to make this gradient the Ludox was diluted into a 10% solution and a 75% solution, which was the range for the gradient. A gradient maker was used to actually form the gradient, and the samples were centrifuged in a swinging bucket rotor for however long it takes to get clear bands (depending on sample usually 90-120 min). Then the bands were harvested using a syringe and the sample was washed 5 times in plenty of water. At least a 1:10 dilution of Ludox to water was typically used.