126.96.36.199.1.3. Density gradient
The most commonly used density gradient substrates used for microsporidian spore purification are sucrose, sodium chloride, cesium chloride, and two silica colloids: Ludox HS40 and Percoll. The density gradient can be continuous or discontinuous (i.e. stratified), heaviest at the bottom and lightest at the top, thus allowing purified particles to be located at specific regions of the density gradient. Among all density gradient media, Percoll is frequently used for Nosema spore purification because it offers many advantages, including: 1. Ease of preparation; 2. Low viscosity, permitting rapid sedimentation at low speed centrifugation; 3. Low osmolarity and no toxicity; 4. Excellent stability under any autoclaving sterilization conditions (Fig. 6).
Purification procedure with Percoll substrate
- Gently overlay the Nosema spore suspension on a discontinuous Percoll (Sigma-Aldrich; St. Louis, USA) gradient consisting of 10 ml each of 25, 50, 75, and 95 % Percoll solution from top to bottom, respectively.
- Centrifuge the column at 10,000 G for 20 minutes at 4 oC.
- Discard the supernatant
- Re-suspend the pellet in sterile water.
- Repeat the process 3 times,
- Overlay the spore suspension on 20 ml 100 % Percoll solution
- Centrifuging the column at 3,000 G for 2-3 minutes to pellet the lighter cellular debris and leave the spores on the top of Percoll solution.
- Dilute the 100 % Percoll solution by adding the equal amount of water and pellet the spores by centrifugation at 10,000 G for 15 minutes.
- Re-suspend the spore pellet in 2-5 ml sterile water to produce a spore suspension with a purity of greater than 99 % (Chen et al., 2009).
Fig. 6. The Percoll gradient will be constructed by layering 95 %, 75 %, 50 % and 25 % Percoll solution from bottom to top in a 50 ml ultracentrifuge tube. The spore suspension will be overlayed onto the gradient and centrifuged. After centrifugation, the supernatant will be removed and the spore pellet will be suspended in sterile water. After two or three times purification on Percoll gradient, the resulting pellet will contain very pure spores.