Description of a Naegleria sp. isolated from two cases of primary amoebic meningo-encephalitis, and of the experimental pathological changes induced by it.

SYNOPSIS. Axenic and monobacterial cultures were utilized to study the ultrastructure of the amoeboid and flagellate stages in the life-cycle of Naegleria gruberi. The cytoplasm of the amoeboid stage is poor in electron-dense components; a loosely organized endoplasmic reticulum is present. The absence of cytoplasmic inclusions in the ectoplasm is the only detectable difference between it and the endoplasm. The nucleus of Naegleria is typically vesicular. The mitochondria are of the type commonly found in protozoa. Food and contractile vacuoles are described. No Golgi apparatus has been found.



The general ultrastructure of the flagellate resembles that of the amoeba. The kinetic apparatus consists of flagella and their associated kinetosomes, a “spur” made up of isodiametric fibrils (14 mμ in diameter) attaching to the base of the kinetosome, a cross-striated rhizoplast, and thin-walled fibrils (22 mμ in diameter) with a center of attachment in the kinetosomal complex.



Neither centrioles nor kinetosomes have been found in the amoeba. Complex arrangements of endoplasmic reticulum have been seen in the cytoplasm of transforming individuals which may be points of origin of the kinetosomes and/or flagella. The nuclei in metamorphosing individuals frequently show unusual plasticity.



Some speculations on possible orgins and affinities of Naegleria are included.

An Electron Microscope Study of the Amoebo-flagellate, Naegleria gruberi (Schardinger). I. The Amoeboid and Flagellate Stages*

A systematic study of small free-living amoebae has been made under standardized and reproducible cultural conditions, and their pathogenicity has been tested in mice. Naegleria aerobia, Hartmannella culbertsoni and H. rhysodes are pathogenic; H. castellanii, H. astronyxis, H. palestinensis, H. glebae, H. exundans, H. vermiformis, Schizopyrenus russelli, Didascalus thorntoni and Tetramitus rostratus are non-pathogenic. Strains of H. culbertsoni and H. rhysodes are present in Indian soils. A classification of the order Amocbida Kent into families Schizopyrenidae Singh, Hartmannellidae Volkonsky, 1931 emend. Singh, 1952, and Endamoebidae (Calkins), based on nuclear division, is proposed, and the relation of this classification to previously defined families and genera of amoebae and its bearing on phylogeny are discussed. Metronidazole and other anti-amoebic drugs are ineffective against N. aerobia and H. culbertsoni in vitro and in meningo-encephalitis in mice.

Studies on pathogenic and non-pathogenic small free-living amoebae and the bearing of nuclear division on the classification of the order amoebida.

Duplicate cultures of 53 strains representing 9 species of Neisseria were analyzed by gas-liquid chromatography for cellular fatty acids N sicca, N mucosa, N flava, N flavescens, N perflava, N subflava, and the several serotypes of N meningitidis examined were found to comprise a fairly homogeneous group on the basis of the percentages of individual fatty acids present Lactose-fermenting Neisseria also were in this group N catarrhalis, however, contained decanoic acid in addition to the acids occurring in the other species Moreover, the 18 C-saturated and monoenoic acids together constituted 36% of the total fatty acid composition for N catarrhalis, while the comparable mean value for the other species was less than 13%

Fatty Acid Composition of Neisseria Species as Determined by Gas Chromatography

Primary amoebic meningo-encephalitis: clinical, pathological and epidemiological features of six fatal cases.

A flagellate amoeba, isolated from Ohio River water and identified as Naegleria gruberi (Schardinger), has been maintained in permanent cultures on a buffered sucrose nitrate agar with its original bacterial associate, identified as Proteus mirabilis. On Czapek agar plates, the growth curve of the amoeba at 25-27° was found to consist of a lag phase of about 4 hr., a log growth phase of about 60 hr., and a growth-encystment phase of 2-3 weeks. At 37° the lag phase was shortened by half and the log growth phase by about a third. The generation time was 4 1/2 hr. at 25-27° and 2 1/2 hr. at 37°. Continuous cultivation at 37°, however, resulted in occurrence of abnormal forms. Very poor growth of amoeba on nutrient and tryptose agars was attributed to their high organic nitrogen content, poor growth in liquid media to the difficulty of amoebae to engulf bacteria. Attempts to grow the amoeba on heat-killed bacteria were failures. Redox potential and pH determinations revealed that the mixed fluid cultures were aerobic and the amoebae perished at pH values more acid than 5·6. Replacement of Proteus mirabilis by other bacteria by Oehler's technique was unsuccessful because of swarming but was successful with a Flavobacterium sp., which produced a yellow pigment whose antibiotic effect prevented swarming. Permanent cultures of the amoeba with the new bacterial associate were maintained after the amoeba overcame the antibiotic effect of the pigment. Replacement of the Flavobacterium sp. in the mixed culture by new species of bacteria was easily accomplished, and permanent cultures were maintained with Aerobacter aerogenes, Escherichia coli, 8 species of Salmonella and 2 of Shigella. The amoebae encysted earlier on all these species than on Proteus mirabilis.
Cytological observations include: (a) appearance of the chromatin material as short rods; (b) no evidence of thread-like structures radiating from the karyosome or chromatin patches in resting nucleus, nor the centrioles in mitosis; (c) karyosome origin of both the ‘interzonal body’ and the polar caps.

S. L. Chang

Papers

Cultural, Cytological and Ecological Observations on the Amoeba Stage of Naegleria gruberi