IIARTMANNELLA CULBERTSONI AS REVEALED IN SCANNING ELECTRON MICROSCOPY1

DELMAS J. ALLEN AND L. ]. A. DIDIO, Department of Anatomy, Medical College of Ohio, Toledo, Ohio 43614 Abstract. An axenic culture of culbertsoni obtained from the American Type Culture Collection was used in this study. maintained in a trypticase soy broth medium were concentrated, fixed and dehydrated before being transferred drop-wise to round microscope cover classes for critical point drying in liquid CO2. Dried specimens were coated with carbon and palladium-gold and studied with an SEM. H. culbertsoni showed extreme polymorphism. Surface specializations or microappendages, not previously demonstrated in the light microscope, were observed. Some of the amoebas possessed smooth cellular surfaces with scattered bleb-like and bulbous ectoplasmic projections and others had irregular surface contours with thread- like processes of varying lengths. Other cells exhibited finger-like projections which formed a fringe over most of the surface. Such advanced pleomorphism, under identical environmental conditions, depends on the phase of growth, differential adhesion and locomotion of cells. OHIO J. SCI. 76(4): 167, 1976

Free-living soil amoebas or Acanth- as Harlmannella by Singh and Das (1970). (Butt, 1966) have been difficult After finding several other hartmannellid to describe by light microscopy, and as a amoebas which were identical, they pro- result disagreement and confusion con- posed the new species Hartmannella cerning their prevail in the culbertsoni. voluminous literature. The disagreement The discovery of the pathogenicity of is due mainly to different views about some free-living amoebas (Butt et al, taxonomic criteria (F. C. Page, personal 1968; Callicott, 1968; Callicott et al, communication). According to Page 1968; Cerva et al, 1969; Culbertson, 1971) (1967b), the erosion of the taxonomic resulted in the division of the superclass line between the free-living and parasitic Sarcodina into two groups based on their amoebas has been responsible for the dif- habitats (Cotter, 1973). One group is ficulty encountered in dealing with the made up of free-living amoebas which in- subclass Rhizopoda (phylum Protozoa). crease in number at the expense of other The evolution of the taxonomic prob- small organisms, usually bacteria. The lem began when Jahnes, Fullmer and Li other group exists in some type of sym- (1957) found a small amoeba classified as biotic relationship with other eukaryotic Acanthamoeba in monkey tissue culture organisms. The fact that the so-called cells. Later Culbertson, Smith and Min- free-living group includes members which ner (1958) observed a similar amoeba in are pathogenic under certain conditions tissue culture fluid suspected of having has further complicated the taxonomy of an unknown simian virus. When this amoebas. tissue culture fluid was injected into the The two schemes of classification found brains of mice and monkeys, only free- in the literature (Singh, 1952; Page, living amoebas identified as Acanth- 1967a, 1967b) overlap, and as a result it amoeba were found in resulting lesions of is difficult to assign names to the amoebas. severe primary meningoencephalitis. Singh (1952) suggested a scheme of classi- These motile amoebas were designated fication based upon the type of mitosis. He proposed two large families, Hartman- Manuscript received March 22, 1976 and in nellidae and Schizopyrenidae, the main revised form April 20, 1976 (#76-29). difference being the dissolution of the 167 168 DELMAS J. ALLEN AND L. J. A. DIDIO Vol. 76 nucleolus in the former, and the per- tion (300-400 rpm), separated from the super- natant nutrient medium, re-suspended in a sistence and division of it in the latter sodium cacodylate-sucrose rinse (Sabatini el al, family. Page (1967a, 1967b) modified 1963) at pH 7.3, before being fixed with a this scheme with a greater emphasis upon cacody late-buffered paraformaldehyde-gluta- morphological features. According to raldehydc fixative (Karnovsky, 19(15). The fixed samples were dehydrated through a Page's criteria, the A-l Strain is Acanth- graded series of ethanols by alternately concen- amoeba, while by Singh's it is Hart- trating the amoebas by centrifugation and re- mannella. suspending in the next higher grade of ethanol. Amoebas in absolute ethanol were transferred Page (1967a, 1967b) described these drop-wise to chemically clean, round cover pathogenic amoebas in detail but an ac- glasses before being dried in a Denton Critical curate demonstration of their surface Point Drying System using liquid CO2. The morphology was beyond the resolving specimen cover glasses were mounted on SEM stubs with an electrically conductive adhesive power and depth of field of the light (Electrodag) and coated with carbon and pal- microscope. Two scanning micrographs ladium-gold in a Denton DV-502 Vacuum high (SEM) of H. culberisoni, without a de- vacuum evaporator. Specimens were viewed tailed interpretation, were included in the in an ETEC Autoscan scanning electron micro- scope using 20 kV and photographed on 4 x 5 study on cystforming filose amoebas by Kodak Commercial Estar Thick Base Film Sawyer and Griffin (1975). Since no other (Xo. 4127). description of H. culbertsoni has been re- ported using this technique, it was RESULTS deemed worthwhile to examine the sur- Observations were made on 8 mi- faces of H. culbertsoni, regarded as one of croscope slide cover glasses containing a the etiological agents in primary amoebic total of approximately 30,000 H. cul- meningoencephalitis, to better depict its bertsoni amoebas (30,000/ml). It was morphological features during active (log apparent immediately that this H-A phase) growth. organism exhibited polymorphism. The METHODS AXD MATERIALS cell surface and the micro- and filiform An axcnic, concentrated suspension of H. extensions of ectoplasm (pseudopodia) as culbertsoni was obtained from the American revealed in light microscopy (fig. 1) ap- Type Culture Collection for use in this study. pear more complex and varied in the Stock suspension transfers to a trypticasc soy scanning electron microscope. Surface broth nutrient medium as suggested by Cul- bertson (1971) resulted in more satisfactory specializations, not previously seen in the maintenance of the amoebas when kept at 37° C light microscope, characterize //. cul- and pH of 7.2. Harlnmnnella was selected over bertsoni (figs. 2-6). These include short Naeglerid, the other well known amoebic patho- microvillous-like, filiform, bleb-like and gen, primarily because the H-A amoebas do not require living cells for satisfactory growth finger shaped appendages. The amoeba from small inocula. in figure 2 has several long filiform Amoebas were concentrated by centrifuga- (thread-like) microappendages (arrows)

EXPLANATION OF PLATE FIGURE 1. A light micrograph of an unstained, fixed H-A (Harlnmnnella culbertsoni) amoeba. X1,250. FIGURES 2-6. Hartmannella culbertsoni SEMs studied from above so as to be directly comparable with the light photomicrograph (viewed at an angle of 45°). FIGURE 2. Spherical amoeba with several long filiform microappendages (arrows) and a com - paratively smooth surface with small bleb-like ectoplasmic projections. X2,300. FIGURE 3. Ovoid cell which appears to have been in locomotion when fixed. The long filiform processes projecting primarily from one area suggest locomotion in that direction. Some of the long processes possess terminal swellings (arrow). X2,800. FIGURE 4. Slug-shaped amoeba with finger-like projections that form a fringe over most of the surface, and probably represents the more active locomotive form. X2,300. FIGURE 5. Two rounded, non-locomotive forms of H. culbertsoni. These two cells are morpho- logically different from each other and from the other amoebas (figs. 2-4). Neither possess long filiform extensions. The cell on the right possesses lower, bleb-like sur- face contours; the one on the left is covered with microvillous and short filiform pro- jections. X2,800. FIGURE 6. Higher magnification of an area of the cell in fig. 5 (left). Note irregular surface with numerous short microappendages. Most of which have terminal swellings. One finger-like extension is also discernible (arrow). X8,400. No. 4 SEM OF HARTMANNELLA CULBERTSONI 169

Figures 1-0 170 DELMAS J. ALLEN AND L. J. A. DIDIO Vol. 76 and a comparatively smooth cellular sur- rapidly from the cell surface and disap- face with scattered bleb-like and bulbous pear into the cell as demonstrated for ectoplasmic projections. Figure 3 shows microvilli on cultured cells (Gey, 1955), an ovoid cell with irregular contours and and as suggested for certain bleb-like long filiform processes projecting pri- structures on the ependymal lining of in- marily from one area. Most of the long tact mammalian tissues (Allen, 1975). processes possess terminal swellings. The Culbertson (1971), in his study of patho- cell in figure 4 exhibits finger-like (fim- genic soil amoebas, observed that after briae) projections which form a fringe certain stimuli the Naegleria could gene- over most of the surface. These pro- rate a varying number of temporary cesses are stouter and do not possess flagellate forms (trophozoite). These bulbous terminations. Figure 5 shows flagella were, in turn, usually lost after a two cells that are also morphologically few hours, and the amoebic form assumed different from each other and from the again. Possibly the polymorphism is re- other amoebas (figs. 2-4). Neither of lated to efforts of recently suspended and these amoebas possess long filiform ex- centrifugally washed amoebas to re-estab- tensions. The cell on the right side pos- lish surface contacts as indicated by sesses short microappendages, most of Rajaraman et al (1974) when studying which appear bulbous, while the one on the processes of cell adhesion and spread- the left is covered with microvillous-like ing in human WI-38 tissue culture cells. and filiform projections of ectoplasm. A higher magnification of an area of the Scanning electron microscopic observa- latter amoeba (fig. 6) shows an irregular tions present us with the realization that cell surface having numerous microap- this pathogenic protozoan is not only slug- pendages with terminal swellings and one shaped (limax) with numerous ecto- finger-like extension (arrow) is discernible plasmic extensions, but that its surface in this micrograph. has a variety of specializations. The observed polymorphism may depend DISCUSSION upon conditions of growth, cell adhesion, Hartmannella culbertsoni exhibits pleo- cell spreading, locomotion and parasitic morphism, a characteristic of most amoe- nature as affected by the surrounding bic cells. The number, shape and size environment. There is no doubt that H. and arrangement of its processes vary culbertsoni taken from the same culture from cell to cell even under identical con- under identical osmotic conditions ex- ditions (age of culture, method of prepa- hibits polymorphism. The application ration, etc.). Because of the pleomorphic of SEM to protozoa is of demonstrated nature of these cells and their processes, value and should be exploited fully. a classification scheme based on external Currently we are refining our techniques morphology alone is not practical. The to use SEM combined with transmission observations reported herein confirm Cul- electron microscopy and cytochemistry bertson's (1971) light microscopic findings as both an experimental and a diagnostic in which he concluded that morphological tool for identifying pathogenic soil amoe- features were rather inconstant in both bas in cerebro-spinal fluid from animals the H-A and N (Naegleria) groups. The having been subjected to them. conclusion that morphology alone is not sufficient for taxonomic distinctions does Acknowledgments. The authors express their not appear to be premature on the basis gratitude to Mrs. Doris Sabo for her help with the final manuscript. This work was supported of previous work (Wilkins and Thompson, in part by General Research Grant 2-1-3-93315 1974; Rajaraman et al, 1974; Sawyer and from the Medical College of Ohio at Toledo. Griffin, 1975) and current work in our laboratory. LITERATURE CITED Some of the microappendages (micro- Allen, D. J. 1975. Scanning electron micro- villi, blebs, etc.) appear to be emerging scopy of epiplexus macrophages (Kolmer or forming while others appear fully cells) in the dog. J. Comp. Neur. 161: 197- formed or in the process of pinching off 214. Butt, C. G. 1966. Primary amoebic meningo- from the surface. These microappend- encephalitis. New Eng. J. Med. 274: 1473- ages may have the ability to emerge 1476. No. 4 SEM OF HARTMANNELLA CULBERTSONI 171

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