In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

Chapter 5: Snap freezing of T. parva stabilates 73
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Snap
2-Step
Programmed
A
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
B
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-8 -7 -6 -5 -4 -3
1
1
1
0.9
0.9
0.9
0.8
0.8
0.8
0.7
0.7
0.7
C
0.6
0.5
0.6
0.5
0.6
0.5
0.4
0.4
0.4
0.3
0.3
0.3
0.2
0.2
0.2
0.1
0
-8 -7 -6 -5 -4 -3
0.1
0
-8 -7 -6 -5 -4 -3
0.1
0
-8 -7 -6 -5 -4 -3
Stabilate dilution (ln t.e.)
Figure 11: Titration curves (logistic prediction) of Theileria parva stabilates cryoprotected
with (A) glycerol, (B) sucrose, (C) trehalose and frozen by snap-freezing, 2-step slow
freezing and programmable freezer (control). Y-axis is the proportion of positive wells. The
points are observed averages
5.4. Discussion
Snap-freezing of stabilates in the cryoprotectant formulations used induced significant infectivity
losses, especially in the glycerol stabilates. It is speculated that the low recovery of viable
sporozoites could be due to the packaging of the stabilates in 1.5 ml vials. This may have resulted in
a non uniform cooling due to the diameter of the column with the material at the periphery of the
tube freezing first and protecting the deeper material, that cooled more slowly from outside in.
Ideally, straws should be used to increase the surface area to volume ratio. Also, a limited range of