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

80 Chapter 6: Storage of Theileria parva stabilates at 4°C and -20°C
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6.2.3. Ice bath storage
Storage on ice meant keeping stabilate vials in a polystyrene box filled with water and pieces of
melting ice (about 3°C). Groups of six vials of each stabilate were thawed at 37ºC for 5 min and
stored on ice for different periods. The K1g stabilate was stored on ice for 3, 8 or 24 h. The C1g
stabilate storage times were 1, 3, 6, 12 or 24 h. Finally, the thawed K2g and K2s stabilates were
kept on ice for 6, 12 or 24 h before the titration. Stabilate for in vivo titration had been stored on ice
for 8, 12, 16, 24 or 32 h as reported by Marcotty et al.(2001).
6.2.4. Storage at -20°C
A group of six vials of each stabilate was transferred from liquid nitrogen storage to a -20ºC freezer
at 4, 2 or 1 week(s) before titration. The freezer temperature was recorded twice daily.
6.2.5. In vitro titrations
The K2g and K2s stabilates stored on ice including a control group (thawed at titration) were
transferred to separate falcon tubes and centrifuged in 25 ml Cellstar ® tubes (Greiner Bio-One,
Frickenhausen, Germany) at 210 g for 10 min. The supernatants, being arbitrarily allocated to
separate rows in a 96-well microtitration plate (12 rows by eight columns), were then diluted
serially eight times (two-fold dilution) in four microtitration plates. PBMC were then added to the
wells and the plates incubated at 37°C in a CO 2 incubator for ten days All stabilate groups were
titrated in parallel in three sessions resulting in a total of 12 plates. A session was taken as a
titration at a given time, sharing a batch of PBMC, culture media and stabilate diluents; thereby
forming a cluster for purposes of statistical analyses (Marcotty et al., 2004). For controls, stabilate
freshly thawed from liquid nitrogen storage was used. C1g and K1g stabilates were titrated in single
sessions. Stabilate was diluted serially 12 times (1.5 fold dilution). Table 7 presents the various
sessions and microtitration plate set up.
For storage at -20ºC, all vials were thawed at 37ºC for 5 min together with a group of controls for
either stabilate, four weeks after the first two groups of stabilates vials had been frozen. Stabilate
from each vial was separately centrifuged. The rest of the procedure was as described for ice
storage. Table 8 illustrates the sessions and numbers of wells read fro the experiment.
On day ten, cytocentrifuged smears of each well were prepared on microscope slides. To avoid
cross contaminations between various stabilates, each row (corresponding to a different stabilate)
was assigned to a particular cyto-centrifuge block and sampling was done from the lowest to the