Parasitology
Novel culture medium for the axenic growth of Balamuthia mandrillaris

https://doi.org/10.1016/j.diagmicrobio.2015.04.007Get rights and content

Highlights

  • A new medium for axenic culture of Balamuthia mandrillaris is proposed.

  • This culture medium is easier to prepare and cheaper than those existing already.

  • It can be used also as preservation medium for B. mandrillaris trophozoites for extended periods without changing the media at room temperature.

Abstract

Until now, for axenic cultivation of Balamuthia mandrillaris, the BM-3 culture medium and the Modified Chang's special medium have been the only ones recommended, but they have some disadvantages, as both require many components and their preparations are laborious. Therefore, we developed a novel culture medium for B. mandrillaris axenic cultivation. Each one of the 11 components of BM-3 was combined with Cerva's medium as basal culture medium. Ten strains of B. mandrillaris including the reference strain CDC:V039 and 9 environmental isolates were used during trials. After testing all combinations, the basal medium complemented with 10× Hank's balanced salt solution was the only one that supported confluent growth of B. mandrillaris. Cell shape and motility of trophozoites were normal. This developed medium is as useful as BM-3 for axenization. The development of a cheaper and easy-to-prepare medium for B. mandrillaris opens the possibility of increasing its study.

Introduction

To isolate and cultivate Gymnamoebae from the environment including pathogenic free-living amoebae Naegleria and Acanthamoeba, several culture media have been developed, and some of recipes can be find in Page (1988). According to Dykova and Kostka (2013), there are some “difficult” strains, e.g., Mayorella gemmifera, Saccamoeba, and several Naegleria strains, which need well-grown monolayers of cell cultures for subculturing them, as Balamuthia mandrillaris needs them for its isolation from the environment; thus, it has been scanty (Lares-Jiménez et al., 2014).

B. mandrillaris is an opportunistic protist pathogen causative of necrotizing hemorrhagic granulomatous encephalitis among other infections. More than 200 cases have been reported worldwide with its majority within the Americas (Lorenzo-Morales et al., 2013). Infections due to B. mandrillaris have been estimated with a fatality rate approaching 98%, and no specific successful treatment was reported (Siddiqui and Khan, 2015). For advances in the study of free-living amoebae infections, it is primordial to grow those organisms in the laboratory. B. mandrillaris, unlike most of other free-living amoebae, does not feed on Gram-negative bacteria; thus, the use of nonnutrient agar coated with bacterial cultures has resulted to be ineffective for its growth. Until now, complex media such as the BM-3 culture medium and the modified Chang's special medium are the only ones that have been recommended for axenic cultivation of B. mandrillaris (Kiderlen et al., 2006, Schuster and Visvesvara, 1996). BM-3 medium is excellent for massive growth of B. mandrillaris due to its richness in nutrients, but it has some disadvantages, as it requires 11 components plus newborn calf serum and its preparation is laborious for it has to be prepared and sterilized separately, by different methods, and then put them together. The modified Chang's special medium was created by Kiderlen et al. (2006) to sustain the axenic culture of B. mandrillaris based on another liquid medium developed to cultivate and differentiate pathogenic and nonpathogenic Naegleria fowleri strains (De Jonckheere, 1977); it is a good medium to cultivate B. mandrillaris as the BM-3 medium, but it is also complex in composition and preparation. Cerva's medium is a simpler medium based on Bacto™ Casitone and designed originally for Acanthamoeba (Hartmannella) castellanii (Cerva, 1966), modified later to grow N. fowleri by adding sterile fresh horse serum and antibiotics, and now is used for axenic cultivation of most of free-living amoebae (Cerva, 1969). The objective of this study was to find an easy-to-prepare culture medium for axenic growth of B. mandrillaris using Cerva's medium as basal component.

Section snippets

Materials and methods

Ten strains of B. mandrillaris, including the original isolate (CDC:V039), upon which the description of the amoeba as a new genus and species was based (Visvesvara et al., 1990, Visvesvara et al., 1993), and 9 environmental isolates from water and soil (Lares-Jiménez et al., 2014), were used during trials. Stock cultures of the amoebae were maintained in axenic conditions at 37 °C in BM-3 medium by serial subcultivation (Schuster and Visvesvara, 1996). For assays, Cerva's medium, consisted in

Results and discussion

After the first week of daily observations of the tested combinations of BM-3 components and Cerva's medium, amoebae numbers diminished in every flask. At the end of the second week of incubation, no trophozoites nor cysts were seen in any modification, except for the one consisting of the basal medium complemented with 10× Hank's balanced salt solution (34.0 mL/500 mL) (Table 1) where growth was recovered. Although Hank's balanced salt solution is a very common component of several culture

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