Figure 1.
Diagram of digital flow control box with flow controllers.
A diagram showing the set up of the digital control box and gas flow controllers. Inset diagrams the principle of the flow controls as adapted from the manual. Individual parts are detailed in Table 1 in addition to as follows: A: Model 954 Digital Flow Box, B: 840L Mass Flow Controller –10 SLPM (N2) capacity, C: 840L Mass Flow Controller –1000 SCCM (CO2) capacity, D: 840L Mass Flow Controller –1000 SCCM (O2) capacity, E: 840-CDCL (15 ft) Cable, F: Compression fitting Union Cross, G: Compression threaded adapter, H: Barbed fittings, I: Copper tubing, J: Brass tubing, K: Clear PVC tubing, L: Pall Acro 0.2 µm PTFE vent filter, M: Modular Incubator Chamber, N: N2/CO2 Gas regulator, O: O2 Gas regulator, P: (Optional) 840L Mass Flow Controller –1000 SCCM (Air). Gas flow rates shown for B, C, and D correspond to a total flow rate of 10 SLPM at the appropriate gas percentages for parasite culture (94% N2, 5% CO2, 1% O2).
Table 1.
Necessary supplies and useful accessories for assembling an adjustable gas mixer.
Figure 2.
Gas concentration comparisons for field applicable culture methods.
A. Graph of time and oxygen percentage as measured through modular incubator chamber output nozzle. Inset shows the time to reach 1% O2 for each method. Four independent experiments were performed and error bars represent standard deviation. B. Concentrations of CO2 and O2 for field applicable culture methods: Candle Jar, Pre-mixed gas cylinder (CYL), and gas-mixing (GM) device, compared to ambient atmospheric percentages. Three independent measurements were made (with five measurements for the gas-mixing device) and error bars represent standard deviation.
Figure 3.
Quantitative 4-cycle growth assays.
A & B. Laboratory adapted isolate 4-cycle growth rate comparisons: A. 3D7 and B. Dd2. C & D. Short-term adapted Senegalese isolate 4-cycle growth rate comparisons: C. P19.04 and D. Th32.09. Results from individual experiments are shown, conducted in triplicate, with error bars representing standard error.
Figure 4.
Semi-quantitative comparisons of long-term routine culture.
A. Laboratory adapted (decades) isolate (3D7) routine growth comparisons. Cultures were split 1∶10 every cycle. B & C. Short term adapted (months) Senegalese isolates routine growth comparisons. Cultures were split 1∶10 every cycle, or media change only as appropriate, as indicated by action on odd days. Results from individual experiments are shown, conducted in triplicate, with error bars representing standard error.
Figure 5.
Cryopreserved field isolate recovery time.
Time to positive growth from field prepared cryopreserved parasites without previous culture adaptation: A. isolate Th29.09. B. isolate Th33.09. Results from individual experiments are shown, conducted in triplicate, with error bars representing standard error.