Folic acid the essential supplement to brain heart infusion broth for cultivation and cloning of Leishmania donovani promastigotes

Abstract

Multiplication of virulent and avirulent Leishmania donovani promastigotes was inhibited by brain heart infusion broth (BHI) (3.7%) supplemented with haemolysed or whole blood (2.5-10%) or heat-inactivated foetal bovine serum (HIFBS) (10-20%). Peptone, glutamine, tryptophan, purine bases, ribonucleosides, deoxyribonucleosides, B-vitamins and vitamin C were evaluated for the reversal of this growth inhibition. Supplementation of BHI with folic acid further supplemented with haemolysed rabbit blood or HIFBS and haemin supported propagation in serial subpassages of 2 primary isolates and 3 other virulent strains of L. donovani and 1 strain of L. major. Folic acid supplemented BHI with 3% rabbit blood in solid phase with 0.7% agar also supported the colony formation of L. donovani. This manuscript documents the development of high efficiency media for the cultivation of L. donovani promastigotes in liquid phase and as colonies on agar plates using BHI as a basal medium.

An Integrated Morphological and Molecular Approach to a New Species Description in the Trypanosomatidae: the Case of Leptomonas podlipaevi n. sp., a Parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

“…Antibiotics were omitted from the medium after several passages. Cells were cloned by plating 200 ml of diluted (10 À 5 to 10 À 6 ) cultures onto BHI-agar medium supplemented with hemin (10 mg/ ml) and folic acid (80 mg/ml) (Cruz, Coburn, and Beverley 1991;Kar 1997).…”

In Vitro Cultivation of Promastigotes of Iraqi Leishmanial Species in Serum-Free Liquid Medium Containing Human Urine

“…However L. donovani shows a more rapid growth and a higher final density (3.8×107 cell/ml; G 16.65) than L. tropica (2.4×107 cells/ml; G 18.21) and L. major (1.9×107 cells/ml; G 19.02). Beef extract and BHI serves as major source for heme and amino acids repectively, which are essential for the survival of the parasite (Kar, 1997, Ali et al, 1998. It is also noted that the supplementation of peptone, a source of amino acids, supported the propagation of the promastigotes.…”

“…Cellular viability was assessed, before and after incubations, by motility as well as by light microscopy observation using Trypan blue cell dye exclusion for promastigote forms. The generation time (G) of the parasite was determined as described by Kar (1997).…”

“…A variety of culture media have been designed for cultivating the promastigote forms of different leishmanial species (Berens et al, 1976, McCarthy -Burke et al, 1991, Kar, 1997, Bagrova et al, 2012. There are three basic types of culture media: biphasic, semisolid and liquid.…”

Enzymes of the De novo Pyrimidine Biosynthetic Pathway in Leishmania tropica

“…These are, the de novo biosynthesis from non-pyrimidine precursors ( Fig.1) and salvage synthesis utilizing preformed exogenous pyrimidines. Much of the evidence to suggest the presence of the de novo pyrimidine biosynthesis in parasites has come mainly from studies of the composition of minimal defined media for the growth of organism (Kar et al, 1990, Kar, 1997 and the incorporation of radioactive biocarbonate and orotate into nucleic acid pyrimidine rings (3)(4)(5). Preliminary investigations have demonstrated the existence of a number of de novo enzymes in Leishmania mexicana amazonensis, L. m. mexicana and L. major (Hassan and Coombs, 1988;Berens et al, 1995;Carter et al, 2003;Hill et al, 1981;Hammond and Gutteridge, 1982;French et al, 2011;Wilson et al, 2012), vis, orotate phosphoribosyltransferase and orotidylate decarboxylase.…”