A New Liquid Medium without Blood and Serum for Culture of Hemoflagellates

Sadigursky, Moysés; Brodskyn, Cláudia

doi:10.4269/ajtmh.1986.35.942

Cited by 43 publications

(29 citation statements)

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“…The strain was continuously maintained by subculture at 7-to 8-day intervals in liver infusion tryptose (LIT)-hemin medium. 7 Mass culture of the same strain that was raised in LIT-medium without the addition of hemin, but supplemented with fetal calf serum (10%), penicillin (100 IU), and streptomycin (100 μg/mL) was harvested 7 days later. After treatment with β-mercaptoethanol and fixation in 2% (w/v) formaldehydeLocke's solution, the antigen was stained with Coomassie Brilliant Blue (Sigma Aldrich Co Ltd., Ayshire, United Kingdom) and finally resuspended in 1.2% (w/v) formaldehyde-citrate saline solution.…”

Section: Methodsmentioning

confidence: 99%

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

Osman¹,

Mahamoud²,

Abass³

et al. 2016

The American Journal of Tropical Medicine and Hygiene

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Abstract. A prerequisite for the control of visceral leishmaniasis (VL) is the accessibility to reference diagnostics. The high price of the freeze-dried direct agglutination test (FD-DAT) and the short shelf-life time of the rK39 strip test (rK39) have limited the application of these tests in Sudan. An original liquid DAT (LQ-DAT) with high reproducibility compared with the FD-DAT and rK39 has been routinely produced in our laboratory since 1999. In this study, a 3.4-year-old batch (of more than 90 test batches produced to date) was chosen to validate the diagnostic performance of this test against microscopy, FD-DAT, and rK39 in 96 VL and 42 non-VL serum samples. Relatively higher sensitivity (95/96, 99.0%) was recorded for the LQ-DAT than for the FD-DAT (92/96, 95.8%) and rK39 (76/96, 79.2%), probably because of the use of the endemic autochthonous Leishmania donovani isolate as the antigen. Experience with the LQ-DAT, its low cost of production, ease of providing this test, and diagnostic reliability compared with the FD-DAT suggest that widescale implementation of the LQ-DAT can contribute to sustainable VL control in Sudan.

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Section: Methodsmentioning

confidence: 99%

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

Osman¹,

Mahamoud²,

Abass³

et al. 2016

The American Journal of Tropical Medicine and Hygiene

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“…Attempts to replace serum by bovine serum albumin 20,21 or mixture of purine bases, vitamins, and bovine albumin fraction IV 22 were also made for cultivating L. donovani promastigote forms. An easily prepared, nearly defined medium containing salts, glucose, and tryptose (i.e., an LIT medium), to which was added concentrated RPMI 1640 and 199 media, was shown to be adequate for the cultivation of L. chagasi and L. amazonensis promastigotes 23 and other Leishmania species. 24 Media developed for serum-free growth of mammalian cells were adapted for the serial cultivation of different Leishmania species.…”

mentioning

confidence: 99%

Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

Merlen

Sereno²,

Brajon³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.Parasites from the genus Leishmania cause a variety of disease states in humans and other mammals in tropical and subtropical areas, which include cutaneous, mucocutaneous, and visceral leishmaniasis. The parasite undergoes a digenic life cycle between a nonmotile intracellular amastigote stage parasitizing the mammalian phagocytic cells and a flagellated, motile promastigote stage in the midgut of its sandfly vector. 1 A similar promastigote form develops when parasites are cultured in cell-free medium. 2

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“…The concentrated RPMI-1640 medium and medium 199 containing salts, glucose, and tryptose were observed satisfactory for the cultivation of L. chagasi and L. amazonensis promastigotes (Sadigursky and Brodskyn, 1986). Beef extract and yeast extract along with inorganic salts have also been used for the cultivation of Leishmania promastigotes to replace FBS (Ali et al 1998).…”

Section: Resultsmentioning

confidence: 99%

Soy protein isolate: A substitute of fetal bovine serum for the in vitro cultivation of Leishmania donovani

Sidana

Alam

Farooq

2017

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Fetal Bovine Serum (FBS) is an expensive source of macronutrients which are required for proper nutrition of Leishmania parasite in the culture medium. An alternative, cost effective source of macronutrients which can replace the use of FBS in tissue culture medium is required. The potential of Soy Protein Isolate (SPI) to replace FBS in RPMI-1640 medium for the in vitro cultivation of Leishmania donovani was evaluated. Commercially available SPI powder was used in RPMI-1640 medium as a substitute of FBS to cultivate L. donovani promastigotes. The growth, multiplication and morphology of cultivated parasites was observed in conventional RPMI-1640 with 10% FBS (v/v) and RPMI-1640 containing 10% SPI (v/v) by using light microscopy, measurement of absorbance and cell counting. The growth of Leishmania promastigotes in the medium containing 10% SPI was slower in initial phase; however, the parasites were morphologically larger as compared to those in RPMI-1640 medium containing 10% FBS. Cell count in the SPI-containing RPMI-1640 medium was 2.3 × 10 8 cells/ml whereas it was 1.9 × 10 7 cells/ml in RPMI-1640 with 10% FBS. This study concludes that RPMI-1640 may be supplemented with SPI instead of FBS for the in vitro cultivation of Leishmania donovani promastigotes to decrease the culture maintenance cost in developing countries.

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A New Liquid Medium without Blood and Serum for Culture of Hemoflagellates

Cited by 43 publications

References 0 publications

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

Local Production of a Liquid Direct Agglutination Test as a Sustainable Measure for Control of Visceral Leishmaniasis in Sudan

Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

Soy protein isolate: A substitute of fetal bovine serum for the in vitro cultivation of Leishmania donovani

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