Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites

Abstract

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome‐cytopharynx complex of Trypanosoma cruzi

“…This ΔMyoF mutant again showed no measurable decrease in parasite growth or viability. We do not know how these parasites are able to survive in the absence of endocytosis but, as stated in the introduction, it may be due to surface membrane transporters that import simple nutrients that are provided in excess in the standard epimastigote l iver i nfusion t ryptose (LIT) media ( Costa et al, 2020 ). This ability to make endocytic-null mutants, it turns out, may be extremely fortuitous, as the lack of a conditional knockdown system in T. cruzi continues to make the study of essential gene function exceedingly difficult ( Chiurillo & Lander, 2021 ).…”

Identification of Sarcocystis spp. in Slaughtered Sheep from Spain and Evaluation of Bradyzoite Viability after Freezing

“…However, when it comes to interpreting results, this technique is much more convenient, as green-stained viable bradyzoites and red-stained non-viable bradyzoites can be distinguished unequivocally. Flow cytometry is commonly employed in parasite viability studies for several different purposes [ 49 , 50 ], and it has proven to be more useful than fluorescence microscopy in those contexts. Whereas fluorescence microscopy is effective for the quantification of cellular components in quantities of dozens or hundreds of cells, flow cytometry excels in the analysis of larger quantities, reaching up to hundreds of thousands of cells in suspension.…”

Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review

“…Of note is a methodology using a colorimetric β-lactamase assay described on intramacrophagic amastigotes of L. donovani [16]. Nevertheless, in the field of cell viability analysis, flow cytometry constitutes an interesting alternative [75,76], which is accurate and largely used to automate the reading of results. Another approach uses the detection and quantification of engineered cells expressing fluorescent gene reporters [77,78] such as green fluorescent protein (GFP) [32,[79][80][81] and bioluminescent gene reporters such as luciferase [82][83][84][85][86], or a combination of both [87][88][89][90].…”