GROWTH OF ACANTHAMOEBA CASTELLANI WITH THE

JOURNAL OF BACTRIOLOGY Vol. 87, No. 1, pp. 220-225 January, 1964 Copyright 1964 by the American Society for Microbiology Printed in U.S.A. GROWTH OF ACANTHAMOBA CASTLLANI WITH TH YAST TORULOPSIS FAMATA L. C. NRO, MA G. TARVR, AND L. R. HDRICK Biology Department, Illinois Institute of Technology, Chicago, Illinois Received for publication 13 September 1963 ABSTRACT NRO, L. C. (Illinois Institute of Technology, Chicago), MA G. TARVR, AND L. R. HDRICK. Growth of Acanthamoeba castellani with the yeast Torulopsisfamata. J. Bacteriol. 87:220-225. 1964.- Acanthamoeba castellani and Torulopsis famata cells were cultured together for 10 days at three different temperatures: 18, 25, and 32 C. In the 18- and 25-C series, the yeast population reached a peak within 3 to 5 days and then declined for the next 5 days. This decrease in yeast population corresponded with an increase in the development of amoebae, which first appeared as vegetative cells and later transformed themselves into cysts. The changes in population were determined by counts in a hemocytometer. The vegetative amoeba population seldom exceeded 4 X 107 cells per bottle culture, but the encysted population approached 16 X 107 cysts per bottle culture within 10 days of incubation. The amoebae actively ingested the yeasts and used these cells as their principal source of energy. At 18 C each amoeba consumed 70 yeast cells per day; at 25 C each amoeba ingested 35 yeast cells per day. In the 32-C series, both the yeast and the amoebae had slow growth rates. The development of cysts corresponded with the growth of the yeast population without the rapid increase in yeast population prior to the growth of the amoebic cells. Acanthamoeba was first reported as living together with Cryptococcus pararoseus castellani and bacteria by Castellani (1930). He predicted that the disappearance of the yeasts was due to a lytic enzyme produced by the amoebae and active upon yeast, but he could not detect any lytic effect from the extracts of the mixed culture. Douglas (1930) discovered an enzyme of these amoebae which could liquify gelatin. Van Rooyen (1932) studied the relationship of the growth of 56 different bacteria with A. castellani, and concluded that the clearing of bacteria from the agar surface was caused by ingestion of bacteria rather than by extracellular lvtic enzymes. Neff (1957) reported that an Acanthamoeba species could not use living cells of Saccharomnyces cerevisiae, S. ellipsoideus, or Schizosaccharomyces pombe as food, but that the amoeba could grow on autoclaved cells of these yeast species. This paper is a report on the study of the rate of growth of A. castellani with the yeasts Torulopsis famata or Candida parapsilosis at 18, 25, and 32C. MATRIALS AND MTHODS The amoeba was isolated in conjunction with the yeast C. parapsilosis by Helen Maclean from soil associated with clover-root nodules. The amoeba was identified as A. castellani by. C. Bovee of the University of Florida. arly experiments were performed with the amoeba plus the yeast C. parapsilosis. However, this yeast produces many filaments, singly and in clusters. Therefore, by the use of a hot-water bath (56 C for 20 min), the vegetative cells of the yeast were killed, and the resistant amoeba cysts were transferred to a nonfilamentous species of yeast, T. famata NNRL Y-548. Freshly isolated amoeba plus yeast cultures were transferred four successive times upon YM agar, to which were added 100,ug of penicillin and 100,ug of chlortetracycline per ml of medium. The composition of YM agar in grams per liter was: glucose, 10; yeast extract, 3; malt extract, 3; peptone, 5; and agar, 20. Addition of antibiotics inhibited growth of contaminatory bacteria. Both cultures, the amoeba plus Candida and the amoeba plus Torulopsis, were transferred weekly upon YM-agar slants and incubated at 28 C for 7 days during the experimental period of about 2 years. At the end of the incubation period, most of the amoebae had become eneysted. In the preparation of cultures for an experiment, 5 ml of sterile 0.001 M P04 buffer (ph 7.0) were added to each of ten agar tubes of yeasts with eneysted amoebae. The cells were suspended in this buffer with the aid of a sterile loop. Any tubes with large clumps of eneysted amoebae were discarded. The amoeba-yeast suspension was diluted with nine parts of sterile phosphate 220

V()L. 87, 1964 GROWTH OF A. CASTLLANI WITH T. FAMATA 221 buffer, and the amoeba cysts were counted in a hemocytometer with a depth of 0.2 mm. The amoeba-yeast suspension was adjusted by diluting with sterile phosphate buffer, or by adding fluid from other amoeba-rich suspensions until there were approximately 300,000 amoeba cysts per ml. The adjusted amoeba-yeast suspension was heated for 5 min in a 58-C water bath to kill the vegetative cells; 0.5 ml of this heated suspension was then delivered to each of 80 bottle slants. A bottle slant is a 150-ml milk-dilution screwcapped Pyrex bottle in which 20 ml of YM agar had solidified on one side. A suspension of a separate culture of the yeast T. famata was diluted so that the cell density was about 6 X 106 cells per ml. To each bottle slant, 0.1 ml of this suspension was added, giving a ratio of approximately four yeast cells per amoeba cyst. The inocula were mixed upon the surface of the agar by tilting the bottle backward and forward several times. The inoculated bottle slants were placed in incubators for the length of the experiment (approximately 10 days). The incubators maintained the desired temperatures within -0.5 C. The three experimental temperatures were 18, 25, and 32 C. Because of the rapid growth of yeast cells, counts were made every 12 hr for the first 48 hr in the 18- and 25-C experiments. For the next 8 days, readings were made at 24-hr intervals. In the 32-C experiment, the 12-hr interval was maintained for 108 hr in a vain attempt to catch the rise in yeast numbers. After 108 hr, readings were at every 24 hr until the end of the tenth day. ach sample unit consisted of five bottleslants, selected at random. After the desired incubation period, 20 ml of sterile 0.001 M P04 buffer were added to each bottle, and the yeasts and amoebae were suspended in the buffer; 10 ml of this suspension were then added to separate test tubes, and the optical density was determined with a Coleman spectrophotometer at a wavelength of 610 m,u. The average optical densities at the end of the incubation period for the respective temperatures were: 1.52 for 18 C; 1.52 for 25 C, and 0.45 for 32 C. These densities served as an aid in the preparation of dilutions with an appropriate number of cells for accurate counting. The number of vegetative and encysted amoebae was determined in a hemocytometer with a depth of 0.2 mm. The number of yeasts was counted in a hemocytometer with a depth of 0.1 mm. Duplicate determinations were made for each of the five bottles for every sampling period. The precision of the duplicate counts was checked by plotting the average values of duplicate counts vs. the range between duplicate counts. This check was made to determine whether duplicates conformed to a chi-square distribution. RSULTS The relative rates of development of yeast cells, vegetative amoebae, and encysted amoebae at the three different experimental temperatures of 18, 25, and 32 C are given in Fig. 1, 2, and 3. At both 18 and 25 C, there was a great increase in the number of yeast cells, followed by a decrease in cells. This decrease was accompanied by a rise in the number of vegetative amoebae and later of encysted amoebae. In the 18-C series, the yeast population decreased within 5 days from 14.2 X 109 cells to 2.2 X 109 cells, an average decrease of 2.2 X 109 cells per day. The average number of amoebae converted to cysts per day was approximately 3 X 107. Therefore, each amoeba consumed an average of 70 yeasts per day. If we assume that at this temperature the vegetative stage existed o 0 a, o xo x) 0 O 0 z (D I a Z 0 40 80 120 160 200 240 FIG. 1. Growth of Acanthamoeba castellani with Torulopsis famata at 18 C.

222 NRO, TARVR, AND HDRICK J. BACTRIOL. for 2 days, then each vegetative amoeba ingested 140 yeast cells. In the 25-C series, the decrease in yeast population per day was 8 X 108; the average number of amoebae converted to cysts per day was 2.3 X 107. Therefore, each amoeba consumed 35 yeasts per day. If we assume that each amoeba was in the vegetative stage for 1.5 days, then each amoeba engulfed approximately 53 yeast cells. In the 32-C series, there was, in general, a parallel development of encysted amoebae and yeast cells. Both the yeast cells and the amoebae were delayed in growth, and there was no initial in- r- 0 x mcl CD 4 0 z 8-7. 6-5- 4-3. 2- I- 0-0 40 80 120 160 200 240 FIG. 2. Growth of Acanthamoeba castellani with Torulopsis famata at 25 C. 5 Key 0 Yeast a ncysted 32C/ Amoebae 0_, Vegetative Amoebae 4- x 0 4>- 0 08 0 0 0. z z 07 0 40 80 120 160 200 FIG. 3. Growth of Acanthamoeba castellani with Torulopsis famata at 32 C. crement of yeast cells followed by a decrease due to the engulfment of yeast cells by vegetative amoebae. The probable reason for this parallelism of yeast population and encysted amoebae is that the vegetative stage of the amoebae exists only for a short time prior to the development of cysts. The growth and survival of T. famata cells in the absence of amoebae indicated that, after the cells reached the stationary growth phase, the population remained steady through the tenth day (Table 1). Information about the percentage of encystment of A. castellani is of interest. The data (Fig. 4) clearly indicate that the length of the vegetative stage is temperature-dependent; the higher the temperature, the shorter the vegetative period, and the more rapid is encystment. The differences in per cent encystment at the TABL 1. Growth and survival of Torulopsis famata in the absence of amoebae (i Adgea) 18 C 25 C 32 C Temp Total count* Viable count Total count Viable count Total count Viable count 2 2.8 X 109 2.7 X 109 6.8 X 109 6.3 X 109 6.7 X 107 6.6 X 107 4 7.0 X 109 6.0 X 109 1.5 X 1010 1.4 X 1010 7.2 X 107 7.0 X 108 6 10 x 109 8.2 X 109 1.6 X 1010 1.4 X 1010 9.4 X 108 9.0 X 108 8 1.12 X 1010 9.6 X 109 1.6 X 1010 1.2 X 1010 1.4 X 109 1.1 X 109 10 1.12 X 1010 8.6 X 109 1.6 X 1010 1.2 X 1010 1.4 X 109 1.0 X 109 * Counts are expressed as number of cells per bottle.

VOL. 87, 1964 GROWTH OF A. CASTLLANI WITH T. FAMATA 223 minimum were proportional to the temperature increments; thus, there was a 30% difference for each of the two 7-C temperature differences (18 to 25 C, and 25 to 32 C). For each 7-degree increase in temperature, there was a decrease of about 48 hr in the appearance of the minimum of encystment. The ratios of the number of yeast cells to vegetative amoebae are given in Table 2. The pattern is somewhat different for each temperature. At 18 C the peak number of yeasts with reference to vegetative amoebae was attained in 72 hr, and this was followed by a gradual decrease, except for a slight increase on the ninth and tenth days. At 25 C there was no single large peak but several smaller ones distributed at several time periods. At 32 C the ratio of yeasts to vegetative amoebae was rather constant, with an average of about 1,500 yeasts per amoeba. In Table 3 are presented the ratios of the number of T. famata cells per encysted amoeba. At both 18 and 25 C, the ratio peak was reached at 72 hr; this was followed by a more or less gradual decrease. For the 32-C series there were only an average of about 100 yeasts per amoeba cyst at any one time period. The chi-square test was used as a tool for making a decision as to the experimental design. Because of the variable nature of the data, five bottles were selected for each sampling period in a w 1001 80-60- u40-0 40 80 120 160 200 240 FIG. 4. Per cent encystment of Acanthamoeba castellani grown with Torulopsis famata at various temperatures. TABL 2. Ratio of Torulopsis famata to the vegetative phase of Acanthamoeba castellani at various times and temperatures Age of Ratio at culture 18 C 25 C 26 C hr 0 - _ 12 7,410 3,980 4,940 24 1,620 3,310 614 36 4,520 9,790 235 48 11,800 15,900 477 60-349 72 30,200 9,340 1,420 84-1,440 96 14,400 279 1,640 108 - - 1,270 120 2,550 8,270-144 992 11,100-156 - - 2,000 168 401 7,450 180 - - 1,060 204 691 216 1,510 4,080 228 1,280 240 2,840 5,800 TABL 3. Ratio of Torulopsis famata to the encysted phase of Acanthamoeba castellani at various times and temperatures Age of Ratio at culture 18 C 25 C 32 C hr 0 3.40 5.40 4.18 12 706 483 544 24 493 1,110 361 36 1,820 7,520 90.9 48 10,700 16,800 55.1 60 22.0 72 79,500 20,700 118 84 114 96 77,800 364 71.8 108-110 120 48,000 340-132 - 80.2 144 5,950 136 156 237 168 292 79.8-180 143 192 158 522 204 57.6 216 168 66.9 228 70.1 240 18.9 106 -

994 NRO, TARVR, AND) HDRICK J. BA\CT'RIO)L. TABL 4. Occu7rrence of chi-squiares in the count of the vegetative and encysted amt,oebic stages in first half of growth versus the second half of the growth Stage Values higher Values lower than P =.01* than P =.01* 18 C 25 C 32 C 18 C 25 C 32 C Vegetative First half 30 29 35 0 1 0 Second half 25 30 35 5 0 0 ncysted First half 29 30 33 1 0 2 Second half 23 18 25 7 12 10 * Measured at the indicated temperatures. first half, in experiments with vegetative and encysted amoebae, the agreement was good within the lprobabilities of 0.01. In the second half, there was considerably more variation, especially for the encysted-amoeba studies. This increased variation Nwas due to the clumping of the encysted amoebae. The aggregates of cysts could not be dispersed with 20% maltose solution, or with surface-active agents of the nonionic, cationic, or anionic types. Therefore, to reduce this bias so that the counts were in the desired range of variation, cultures which upon suspension formed a few aggregates rather than a uniform turbidity were not counted, but were discarded. In Fig. a are shown two bottles upon which cultures have grown. In the bottle on the left, the surface is covered with wzhite yeast colonies. The bottle on the right shows clearing of yeast colonies due to engulfment of yeast cells by the vegetative amoebae. A fewv white colonies are on the agar surface; these consist of large numbers of yeast cells and vegetative amoebae. The areas "cleared" of most of the yeast cells w-ere covered with a yellow-brown colored film of amoebic cysts. FIG. 5. Growth in bottle-slants showing zones of clearing duie to the amoeba-consuming yeast. Left bottle, growth of T'orulopsis famtata only. Right bottle, growth of yeasts and A4canthamoeba castellani. series. The results indicated that the counts followed the Poisson distribution (Table 4). For the studies with both the vegetative and the encysted amoebae, results are presented for the first and second halves of the incubation period. In the A4k. FIG. 6. lani. Vegetative phase of A carnthaunoeba castel-

VOL. 87, 1964 GROWTH OF A. CASTLLANI WITH T. FAMATA 225 At the end of 7 days of incubation, there was a relatively small amount of agglutination of the amoebic cysts. It was for this reason that the 7-day cyst cultures rather than older cultures were used for inocula. During the vegetative phase of the amoebae, yeast cells could be observed within the vacuole of the amoebae (Fig. 6). Prior to cyst formation, the yeasts were completely digested because yeast cells were never found within an encysted amoeba. DISCUSSION vidence presented in this report supports the thesis that the cleared zones in the yeast growth were due to engulfment of the fungal cells by the amoebae. No evidence of the external lysis of the yeast cells by amoebic enzymes was ever observed over at least a 2-year period of frequent microscopic study. Furthermore, the direct correspondence of increment in amoebae with the decrease in yeast cells, especially in the 18-C series, gives credence to the theory that clearing is due to devouring of the yeast. cells by the amoebae. The finding that these amoebae eat living cells of T. famata and C. parapsilosis differs from results reported by Neff (1957) for other species of yeasts. In our studies, A. castellani grew quite rapidly upon T. famata and less rapidly upon C. parapsilosis. This slower growth is due to the fact that amoebae had difficulty in engulfing the numerous filaments present in the cultures. In unpublished work, we obtained a rapid growth and encystment of the amoebae within 3 days with Bacillus sp. At the end of the period, the agar surface appeared to be clear of growth except for dark-brown clusters of amoebic cysts. A word of caution should be introduced with regard to the experimental use of these soil amoebae, because they may be pathogenic. Culbertson et al. (1959) found Acanthamoeba in brain tumors of monkeys which had been exprimentally infected with unfiltered tissue-culture fluid. LITRATUR CITD CASTLLANI, A. 1930. An amoeba growing in cultures of a yeast. J. Trop. Med. Hyg. 33:160, 188-191. CULBRTSON, C. G., J. W. SMITH, H. K. COHN, AND J. R. MINNR. 1959. xperimental infection of mice and monkeys by Acanthamnoeba. Am. J. Pathol. 35:185-197. DOUGLAS, M. 1930. Notes on the classification of the amoeba found by Castellani in cultures of a yeast-like fungus. J. Trop. Med. Hyg. 33: 258-259. NFF, R. J. 1957. Purification, exenic cultivation and description of a soil amoeba, Acanthamoeba. J. Protozool. 4:176-182. VAN ROOYN, C.. 1932. The effect of amoeba (Hartmannella) castellani on bacterial cultures. J. Trop. Med. Hyg. 35:118-123.