Trypanosome Media Preparation Protocols Back to culture introductionHome

This document is also available in pdf but this web page is considered definitive and may have minor corrections that are not in the pdf, although I will attempt to keep the two versions congruent.

Please note that we do not add antibiotics to any media but we do routinely test an aliquot for sterility after filtration.

Modified HMI-9* — for 10 liters for bloodstream forms
8 liter IMDM (Invitrogen/GIBCO Cat. No. 12440)
1 liter FBS (heat-inactivated at 55 °C for 1 h)
1 liter Serum Plus™ (JRH Biosciences Cat. No. 14001)
100 ml Hypoxanthine stock (dissolve 4.0 g of NaOH in 1 liter of water, add 13.6 g hypoxanthine and freeze in 100 ml aliquots).
280 mg Bathocuproine disulfonic acid (final concentration is 50 µM)
1,820 mg Cysteine (add after Bathocuproine) (final concentration is 1.5 mM)
1,100 mg Pyruvic acid
100 mg Uracil**
100 mg Cytosine**
140 µl 2-mercaptoethanol

Filter-sterilize and store in 500 mL bottles at 4 °C.

* Continuous Cultivation of Trypanosoma brucei Blood Stream Forms in a Medium Containing a Low Concentration of Serum Protein without Feeder Cell Layers Hiroyuki Hirumi; Kazuko Hirumi The Journal of Parasitology (1989) 75: 985-989. Available on JSTOR (or here for local use).
** Since January 2007 we routinely omitted Thymidine (previously 39 mg/liter: 0.16 mM). Since August 2009, we routinely added 10 mg/liter each of Uracil and Cytosine because we are using some variants that cannot make pyrimidines. We took out the thymidine because (a) pyrimidines are not required for tryp growth in culture (but it is required in mice: my unpublished data) (b) its omission makes the cells more sensitive to GCV, with which T competes (c) the amount in HMI-9 seems unnecessarily high, although T.brucei does not have a thymidine transporter (mea culpa, the amount of uracil and cytosine are possibly also unnecessarily high as they are actively imported, and cytosine is probably redundant) and other widely used media for Tb culture do not contain it. My enquiries, especially among trypanosome people working on purine and pyrimidine metabolism, have failed to identify why Hirumi originally included it and have not identified any reason to include it (for information on pyrimidine uptake see Gudin S, Quashie NB, Candlish D, Al-Salabi MI, Jarvis SM, Ranford-Cartwright LC & de Koning HP (2006) Trypanosoma brucei: a survey of pyrimidine transport activities. Exp Parasitol 114:118-25. DOI PMID).

SDM-79 minus NaHCO3 — for procyclic forms
By 2009, many labs had exhausted their supplies of SDM-79 from JRH Biosciences. Invitrogen has been willing to prepare it, as described below. SDM was originally made for us by JRH Biosciences Inc, but JRH was incorporated into Sigma in 2005, then Sigma into Invitrogen. A revised formulation (12th November 1998) incorporated all components individually, resulting in some duplications of the previous hybrid formulation (which we should try to reformulate), and includes some undoubtedly unnecessary components, and omits sodium bicarbonate. I do not know how it is formulated by Invitrogen, but colleagues in Europe and USA have found it entirely satsfactory.

This is the latest information (January 2010) I have is from my colleague Samson Obado. Invitrogen UK makes SDM-79 for a tryp consortium. Invitrogen UK said that the US division can retrieve the formula from them. The catalogue number used in the UK for SDM-79 is 07490916N. The USA Invitrogen Cell Systems Customs (Phyllis Penque, Cell System Customs Specialist, Invitrogen Customer Service, 3175 Staley Road, Grand Island, NY 14072. phone: 800-584-0545 email: said their Rapid Research Lab could make SDM-79 (non-GMP) in packages of 1KG ($650), 2KG ($800) or 5KG ($2700). 25.5 grams of powder makes up 1 litre of medium.

Before using, add 1.5 ml hemin stock (final concentration will be 7.5 mg/l: unnecessarily high) and 50 ml heat-inactivated serum to 500 ml medium. The hemin stock is prepared by dissolving 400 mg of NaOH in 200 ml water and adding 500 mg hemin. Autoclave for 20 min and store as 20 ml aliquots at 4 °C.

DTM* — for 1 liter for differentiation of bloodstream to procyclic forms
A key feature of this medium is the absence of glucose. Because MEM minus glucose is not commercially available, DTM is a bit more tedious to prepare. IMDM is available without glucose, but its amino acid composition differs significantly from MEM. DTM calls for adding BME vitamins solution, which GIBCO (Invitrogen) no longer supplies, for lack of demand. The formulation of MEM vitamins (100x, Invitrogen/GIBCO product number 11120) is identical to BME except for the absence of Biotin, which must now be added separately. These changes are indicated in the table below.

* Synchronous differentiation of Trypanosoma brucei from bloodstream to procyclic forms in vitro. Ziegelbauer K, Quinten M, Schwarz H, Pearson TW & Overath P (1990) Eur J Biochem 192:373-8. There is an error in methods in this paper, where modified DTM refers to 10 mM glycine instead of glycerol. The original DTM was further modified (as in the protocol given here) by adding glycerol (in place of glucose), heme, 15% fetal bovine serum, 0.2 mM 2–mercaptoethanol, and extra proline, glutamate, glutamine. Vassella and Boshart further modified DTM by adding 28.2 mg/l bathocuproine and 182 mg/l cysteine (Vassella E, Boshart M. High molecular mass agarose matrix supports growth of bloodstream forms of pleomorphic Trypanosoma brucei strains in axenic culture. Mol Biochem Parasitol 1996;82:91-105). You might want to consider doing this and adding the same two ingredients to SDM79, or any other medium you use.

For 1 liter (nominal final volume), add the following to 900 ml water, adjust the pH to 7.2 with 5 M NaOH and filter-sterilize.

6,800 mg NaCl
400 mg KCl
200 mg CaCl2
140 mg NaH2PO4.H2O
200 mg MgSO4.7H2O
7,940 mg HEPES
2,200 mg NaHCO3
110 mg Sodium pyruvate
10 mg Phenol Red
14 mg Hypoxanthine
1.0 mg Biotin
760 mg Glycerol (1.21 ml of a 50% solution of glycerol)
640 mg Proline
236 mg Glutamic acid
1,340 mg Glutamine
3.0 ml Hemin stock solution at 2.5 mg/ml in 50 mM NaOH. (Dissolve 400 mg NaOH in 200 ml water and add 500 mg hemin. Autoclave for 20 min and store as 20 ml aliquots at 4°C.)
20 ml 50x MEM amino acids solution (Invitrogen/GIBCO Cat. No. 11130051)
10 ml 100x MEM non-essential amino acids solution (Invitrogen/GIBCO Cat. No. 11140050)
10 ml 100x MEM vitamin solution (Invitrogen/GIBCO Cat. No. 11120052)
14 µl 2-mercaptoethanol (final concentration will be 0.2 mM)
150 ml Heat-inactivated FBS
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