Table 1 Conversion rates for newly transformed schistosomula (NTS) of Schistosoma mansoni obtained using a modified vortex transformation procedure
Experiment | Description | Cercarial suspension (ml) | No. of cercariae | No. of schistosomula | Conversion rate (%) | Mean ± SD | Observations |
|---|---|---|---|---|---|---|---|
1 | Reduced motility: 30 min on ice; centrifugation: 800× rpm, 5 min, 4 °C; purification: 3 steps (15 min on ice) | 35 | 3500 | 1750 | 50.0 | a | Experiment 1 and 2: lower conversion rate; considerable number of tails and cercariae detected |
2 | Reduced motility: 30 min on ice; centrifugation: 800× rpm, 5 min, 4 °C; purification: 5 steps (15 min on ice) | 38 | 6080 | 2800 | 47.0 | 61.4 ± 10.2 | |
3 | 22.5 | 3825 | 2900 | 75.8 | Despite an increase in the schistosomula conversion rate, several tails were observed in wells | ||
4 | Reduced time between purification steps (15–10 min) (centrifuged 2× in order to obtain a solid pellet); slight increase of vortexing time | 45 | 5850 | 3240 | 55.0 | a | Alteration of culture supplemented media M199, 20 mM HEPES, 10% iFBS. It is probable that the time of initial suspension on ice was insufficient to decrease parasite motility, which led to a loss of cercariae after supernatant removal |
5 | 25 | 10,000 | – | – | Due to higher number of cercariae in initial suspension, it is probable that the time on ice was not enough to reduce motility. Consequently, it did not form a solid pellet after centrifugation leading to release of cercariae after removal of supernatant | ||
6 | Experiments 6–12: reduced motility: 60 min on ice; centrifugation: 1000× rpm, 10 min, 4 °C; purification similar to experiments 4 and 5; introducing checkpoints during the transformation method | 50 | 5250 | 3720 | 70.9 | 68.6 ± 4.8 | Fewer cercariae and tails detected. Good parasite fitness. Increased of concentration of iFBS to 15%. Final culture media established as supplemented M199 with HEPES and 10% FBS |
7 | 100 | 11,750 | 7380 | 62.8 | Due to a great number of cercariae, mechanical transformation was performed on separated tubes | ||
8 | 40 | 12,000 | 8640 | 72.0 | Experiment 8 and 9: lower number of tails and cercariae. Good parasite fitness | ||
9 | 50 | 3000 | 2250 | 75.0 | |||
10 | 27.5 | 1640 | 1040 | 63.4 | Lower number of cercariae because several infected snails had died, thus only a few snails were infecting and shedding cercariae. In this case, we only performed 4 steps for purification in order to reduce loss of schistosomula during the process | ||
11 | 50 | 4625 | 3000 | 64.8 | Lower number of tails and cercariae, as well good parasite fitness | ||
12 | 50 | 7375 | 5265 | 71.4 |