• 疟原虫的培养方法

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By using increasing volumes of culture medium, one can grow P. falciparum to higher parasitemia (above 10%). This protocol may be especially suited for maintaining high parasitemias of wild isolates, allowing maturation of a maximum number of ring stage parasites to trophozoites.

Procedure
• Set up cultures as described in PARASITES, section I:A, but use the table below as a guide for medium volumes.
• Increase the parasitemia gradually using the indicated volumes until a desired parasitemia is reached, then maintain the culture in the appropriate volume.
For example, for a 200-礚 packed cell culture, use 4 times the volume of medium in the table for any given parasitemia.


% Parasitemia   Volume of medium (mL)/50 礚 of packed culture
10   2.5
12   3
14   3.5
16   4
18   4.5
20   5
24   6
28   7
32   8
36   9
40   10
44   11
48   12
52   13
56   14
60   15
64   16
68   17
72   18
76   19
80   20
84   21
88   22
92   23
96   24
100   25


Reference
KEMRI, CGMRC / Wellcome Trust Research Unit, Kilifi, Kenya
I:E. Arresting Plasmodium falciparum growth at the trophozoite stage with aphidicolin
by Malin Haeggström
Microbiology and Tumor Biology Center (MTC), Karolinska Institutet and Swedish Institute for Infectious Disease Control, Box 280, SE-171 77 Stockholm, Sweden
e-mail: malin.haeggstrom@mtc.ki.se

This is a method to get fresh/frozen isolates or in vitro cultures to arrest in late trophozoite stages by inhibiting DNA synthesis.

Materials and reagents
malaria culture (only young stages! see PARASITES, section I:A) or a vial of frozen malaria parasites
aphidicolin (Sigma-Aldrich)
DMSO

Preparations
• Prepare a 1.5 mg/mL stock-solution of aphidicolin in DMSO.
• Dilute stock-solution 1:1000 into your malaria culture (4 礚 in a 4-mL flask).
• Let the culture continue to grow until the parasites reach trophozoite stage (around 24 h after adding the aphidicolin). See PARASITES, section I:A.


Analyse or use your culture within 12 h after trophozoite stages are reached. Even though the parasites may look fine for some more hours, it is NOT recommended to use them after a longer time in aphidicolin.

Note: If you add the aphidicolin to a culture of later stages, the cells will not arrest.

References
Bull P, KEMRI, CGMRC, Kilifi, Kenya (personal communication)

Inselburg J, Banyal HS. 1984. Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor. Exp Parasitol 57(1):48-54.

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