Adapted from Hirumi & Hirumi (1994) Parasitology Today 10:80

HMI-9 powder is custom made by Life Technologies and is generally purchased on a consortium basis

One pot is sufficient to make up 5 litres after addition of the following:

Sodium bicarbonate	15 g
Pen-Strep (Life Technologies)	50 ml
ß-mercaptoethanol	70 µl

Make up to 4.5 litres with distilled water, filter sterilise, store at 4°C in 450 ml volumes.

Prior to use, add 50 ml fetal bovine serum (FBS; Sigma), giving a final concentration of 10% FBS and store at 4°C for up to two months.

NB: Some early publications indicate that FBS should be heat inactivated at 56°C for 30 minutes prior to use in bloodstream-form T. brucei culture – this is not necessary; in fact our unpublished data suggests that using non-inactivated FBS leads to faster, more robust cell growth.

Cultures should be grown in 5% CO₂ incubator at 37°C, and should not exceed a density of 2.5×10⁶ cells per ml.