A Simple and Reliable Method for Cloning Naegleria fowleri

Abstract

As part of a study aimed at gaining a clearer understanding of the pathogenesis of Naegleria fowleri, we have developed a simple and reliable technique for cloning trophozoites. Although cloning of N. fowleri has been reported using limiting dilution (Jonckheere, 1979, Path. Biol. 27: 453-458) and by micropipette technique (Wong et al., 1977, J. Parasit. 63: 872-878), the degree of success is difficult to assess since no data were provided on plating efficiency for either method. We have modified a previously described procedure for cloning Naegleria gruberi (Fulton, 1970, Meth. Cell Physiol. 4: 341-475) for use with N. fowleri which has proved to be highly reproducible and provides excellent efficiency of plating. Naegleria fowleri Lee (ATCC-30894) is routinely maintained in Nelson medium containing 2% calf serum as previously described (Weik and John, 1977, J. Parasit. 63: 868-871). Amebae used in cloning experiments were grown in tissue culture flasks (75 cm2, Falcon Plastics, Oxnard, California) containing 50 ml of Nelson medium supplemented with 2% calf serum. Flasks are inoculated with 104 amebae/ml and allowed to incubate for 72 hr at 37 C. The amebae were dislodged by placing the flasks on ice for 10 min and the resulting suspension of cells was vortexed to dissociate any remaining clumps of organisms. After quantitation by hemacytometer count and in some experiments by Coulter counter (Weik and John, 1977, J. Parasit. 63:150-151) the amebae were diluted to a final concentration of 1030 amebae/ml in Nelson medium without calf serum. The Escherichia coli (hospital isolate) used in cloning N. fowleri were first grown for 3 hr at 37 C with agitation in 50 ml of Nutrient Broth (Difco). The bacteria were diluted 1/10, 1/ 100, and 1/1,000 in soft nutrient agar (Difco, 0.5% agar w/v) and maintained at 55 C in a heated waterbath. The dilution was made in the following manner: 9.0 ml of bacteria in soft nutrient agar are placed in the heated waterbath and then 1.0 ml of Nelson medium containing the diluted amebae was added. The tube is then immediately vortexed and poured onto petri plates containing a non-nutrient agar base (1.5% agar, 10 ml per plate). Control plates containing no amebae were included with each experiment to assure that the observed plaques were not due to bacteriophage. The soft agar plates were allowed to harden at room temperature for 5 hr and then placed upright in a 37 C incubator stacked in sets of 3. After 24 hr incubation, parafilm was placed around the petri plates to prevent drying of the medium. The results from a representative cloning exp riment are shown in Table I. Plaques containing amebae are regularly observed only after 5 to 6 days of incubation with the presence of amebae being confirmed by phase-contrast microscopy. It is especially important to inspect plates carefully after this period since plaques will merge or disappear within the succeeding 24-48 hr. Amebae in the plaque can be observed growing in a concentric manner, a characteristic feature of Naegleria that has been previously described (Carter, 1970, J. Pathol. 100: 217-244). For amplification of clones, amebal plaques are aseptically cut out of the agar plates and placed in Falcon flasks containing Nelson medium supplemented with 2% calf serum and 100 ,tg/ml streptomycin. Two of the amebal clones tested so far have maintained their infectivity for mice. A notable feature of these experiments is the high efficiency of plating (100%) obtained when 3,000 bacteria/ml are initially diluted into the overlay whereas no plaques are observed when 300 or 30 bacteria/ml are used. We have had success using bacterial concentrations between 3,000 and 9,000 bacteria/ml in the initial overlay. The cloning method described in this report differs from previous methods in the use of the soft agar overlay. The observed high efficiency of plating may be due in part to the more effective separation of the individual amebae during the plating procedure. A previous cloning method for N. gruberi employed a glass spreading bar to