Table 2. Synopsis of tests with various media used for growing C. bombi in culture (see Information S1) for details on the composition of media), after [38].

Medium	Modifications, additions1 , 2	Finding
Established media	- Grace's medium	- None of these media did lead to satisfactory growth of C. bombi.
	- SM medium
	- SDM-79-medium
Mattei medium	- Standard medium	Cultivation possible with some success [37].
	- Pollen (autoclaved): 10 g/l, 30 g/l; 10% FBS added.	- Pollen known to increase parasitaemia in living hosts. No improvement in growth compared to standard medium observed. Parasites become deformed and perish.
	- Honey: 2 g/l, 6 g/l; 10% FBS added	- No improvement in growth compared to standard medium observed.
	- Bicarbonate buffer (pH 7.6): NaHCO3 2.2 g/l, plus Na2HPO4.H2O: 5 g/l to keep osmolarity. This buffer additionally modified by adding L-arginine, or yeast extract.	- No improvement in growth compared to standard medium observed; neither for the bicarbonate-enriched medium, nor for the modifications thereof.
	- FBS: 10%, 15%, 25%	- 10% found to promote growth.
Standard medium	- Mattei-medium, modified by adding 10% FBS, and 2 µg/l haemin	- Used as the reference medium for the following modifications
	- addition of citric cycle intermediates (fumaric acid, citric acid, pyruvic acid, α-ketoglutarate:10 mM each).	- Only minimal effects on growth.
	- addition of sugars (D− fructose, maltose, D+ mannose: 10 mM each).	- Faster growth. Maximum effect with 10 and 20 mM fructose (40 mM is inhibiting).
Modified standard medium (10 mM fructose added)	- “Mix 2”, several compounds added.	- Mix 2 promoted growth. The added compounds were therefore tested separately.
	- 10 µM folic acid added	- Same positive effect as Mix2. Therefore adopted as improved medium.
Standard-FF	Standard medium, modified by adding 10 mM fructose, and 10 µM folic acid.	- Improved growth compared to Standard medium alone.
	- BME-Vitamin mix added.	- Marginal improvement in growth compared to Standard-FF.
	- PABA/Biotin-stock solution added.	- No improvement in growth compared to Standard-FF.
	- Vitamin B added	- Slight improvement in growth compared to Standard-FF.
	- Haemin added: 2, 4, 8, 16, and 32 µg/l	- Addition of 2 µg/l sufficient to promote growth compared to Standard-FF.
Haemin-enriched Standard-FF	Standard-FF with 2 µg/l haemin
	- addition of amino acids: taurin (10 mM); L-ornithine-monhydrochloride (2.5 mM); mix of isoleucine-valine- L-ornithine-monhydrochloride (1.5 mM, 1.7 mM, 1.2 mM); L-proline (2.5 mM).	- No effects, except for L-proline.

¹FBS: Fetal Bovine Serum (Biological Industries, K Beth Haemek, Israel). FBS could not be replaced by horse serum, or calf serum, as cells agglutinated and perished in these alternatives.

²Composition of media can be found in the following references.

- Grace (Gibco 11605) [68], developed for insect cell cultures.

- SDM-79 [69], used for cultivation of pro-cyclic forms of Trypanosoma brucei.

- SM [70], used for cultivation of trypanosomatids.