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Ratios of bacterial cells to Entamoeba histolytica trophozoites in cultures
EXPERIMENTAL
PARASITOLOGY 15, 279-283 (1964)
Ratios
of Bacterial
Cells
Trophozoites Richard Department
of Biochemistry,
to Entamoeba in Cultures1 E. Reeves
Louisiana State University New Orleans, Louisiana
(Submitted
histolytica
for publication,
1 April
School of Medicine,
1963)
When relatively fresh bacterial cells were used in MS-F type Entamoeba histolytica cultures, a minimum of 4400 bacterial cells (colony-forming units) sufficed, on the average, to produce each new amebal trophozoite of the F22 strain. Evidence is presented that indicates that this number of bacterial cells reflects a nutritional limit for ameba growth rather than a requirement for the amount of metabolic activity which these cells provide. When suboptimal quantities of fresh bacterial cells were employed together with attenuated bacterial cells, it was found that as few as 2000 fresh cells sufficed to stimulate the production of each new trophozoite. This lower number probably reflects a limiting level of metabolic activity by the associate organism. This required metabolic activity may be expended in the conditioning of the culture fluid rather than in a direct action upon the ameba.
Studies of the cultural requirements of parasitic amebae require an understanding of the stimulation to amebal growth provided by metabolizing (nonmultiplying) bacterial cells. In the MS-F system for the cultivation of ameba (Reeves et al., 1957)) penicillin-inhibited bacterial cells are agglutinated by a component of serum and settle to the lower surface of the culture vessel in the form of a delicate network. As the amebae grow and divide, this network is broken up, and the bacterial cells are ingested in large numbers by the trophozoites. When luxuriant ameba growth occurs, nearly all of the bacterial bodies disappear, and within certain limits, the number of amebae produced is limited by the quantity of available bacterial cells. The present investigation grew from the 1 This investigation was supported by the Surgeon General of the Army, Contract No. DA-49-007-MD756, under the sponsorship of the Commission of Enteric Infections of the Armed Forces Epidemiological Board.
attempt to discover the minimum number of bacterial cells necessary for the production of a new amebal trophozoite. MATERIALS
AND
METHODS
The 200 and F22 strains of Entamoeba histolyticu were obtained from Dr. James Shaffer; the DKB strain, from Dr. Quentin Geiman. All had been cultivated in this laboratory in the MS-F system for several years prior to the experiments. The bacterial stock culture, provisionally designated Bacteroides symbiosus (Stevens, 1956), was originally taken from Shaffer’s “streptobacillus culture no. 2” (Shaffer et al., 1958). It is now recognized that this culture contained several types or strains of similar, gram-negative anaerobes (Sebald, 1962). A stock culture was deposited in the American Type Culture Collection (ATCC No. 12829) at about the time the following experiments were being carried out. The MS-F system for the cultivation of 279
280
REEVES
ameba was used in this work (Reeves et al., 1957). To 12 ml of fluid base medium were added 0.25 ml of horse serum, 5000 units of penicillin, and washed bacterial cells. The inocula were 10,000 trophozoites suspended in small volumes of fluid from a preceding culture. The culture vessels were 16 X 125 mm tubes bearing screw caps with rubber liners. The cultures were incubated at 37” C in a slanting position for 2 or 3 days, after which the amebae were suspended by thorough mixing and counted in a Fuchs-Rosenthal counting chamber. All cultures were examined for bacterial contamination by direct observation and by plating on blood-agar plates; the results are drawn only from cultures free from evident bacterial contamination. However, since this work was completed it has been learned that the ameba cultures now carry an anaerobic mycoplasma, probably a human oral-type pleuropneumonia type organism (PPLO). The base medium contained, per liter: 20 gm Trypticase,’ 10 gm glucose, 2.5 gm thiomalic acid,” 0.79 gm cysteine hydrochloride, 2.0 gm sodium chloride, 2.0 gm dipotassium phosphate and sodium hydroxide to pH 7.0. The composition of the bacterial growth medium was similar, but it lacked the added cysteine and contained 2 gm yeast extract4 per liter. Viable bacterial cells (more accurately, colony-forming units) were assayed in ‘(thioglycollate medium without added dextrose”” in which 2% agar had been dissolved. Oval culture tubes were employed. The bacterial inoculum, 0.1 ml of a suitably diluted bacterial cell suspension, was added to the melted medium at about 51’. After the medium had solidified, it was capped with 2 cm of agar t* Baltimore Biological Laboratory, Inc. Baltimore, Maryland, U.S.A. 3 Difco Laboratories, Inc. Detroit 1, Michigan, u.s..4. 4 Eastman practical grade mercaptosuccinnic acid, twice recrystallized from water. From Distillation Products Industries, Rochester, New York, U.S.A.
medium and incubated at 37”. Colonies were usually visible for counting within 3 days, but all tubes were kept under observation for a total of 7 days. The optical densities of bacterial cultures were measured in 16 X 125 mm tubes in a Coleman model 6 spectrophotometer at a wavelength of 600 mp. RESULTS
Eight-hundred ml of bacterial growth medium was given a 1.55% inoculum from 24 hours growth in a stock B. symbiosus culture. After 18 hours at 37” the culture had just attained the stationary phase, its optical density was 0.54, and its bacterial nitrogen content (Kjeldahl) was 0.101 mg per milliliter. The cells were harvested by centrifugation, washed twice with 100 volumes of saline containing 0.02 iI sodium thiomalate, and
6 b i% ” 0
IO
20
30
Days of Storage of Bacterial Cells FIG. 1. Upper curve: log viable bacterial cells (colony-forming units) per milliliter of stored, lofold concentrated suspension. Lower curves: generations of ameba produced from the standard 10,000 trophozoite inoculum, employing 0.2 ml of the bacterial suspension in each MS-F culture tube.
BACTERIA
The Ratios
of Bacterial
TO
E. histolytica
TABLE I Cells to Trophozoites Produced Entamoeba histolytica
Days of storage of bacterial cells 0 2 5 7 9 12 14 16
Average
TROPHOZOITE
Ratio:
281
RATIO
per Culture,
FZ2 Strain
bacterial cells used ~ trophozoite harvest (-10,000)
of
, using:
1.5 X log cells
7.5 X 108 cells
11,400 9000 6400
4100 6000 6100 4300 5200
5800 5 100 3300 3400
8300
5140
4400
3 X log
cells
12,000 5400 9000 5000 8300
then stored at 2O-4” in suspension with 80 ml of 0.1 A4 sodium thiomalate (pH 7). Viable cell counts were made on this suspension thrice weekly for a period of 40 days. The results of these counts are shown in the upper curve of Fig. 1. The bacterial cells were used for the cultivation of three strains of E. histolytica. Serial transfers of ameba into media containing 0.2, 0.1, or 0.05 ml of the bacterial suspension were made three times weekly for a period of approximately 40 days. Without added bacterial cells, no ameba growth occurred. Some of the results obtained with the greatest quantity of bacterial cells are depicted in the lowest two curves of Fig. 1. Other results, reported in Table I, represent the quotient, i.e., bacterial cells (colony-forming units) divided by trophozoites produced. These last data were taken from results obtained by using bacterial cells less than 17 days old, i.e., before any drop in viable cell counts had occurred. In further experiments, attenuated bacterial cells were employed to supplement suboptimal quantities of the fresh cells. The attenuated cells had been prepared 3 months earlier; the optical density of the culture was then 0.48, and its bacterial nitrogen content was 0.0875 mg per milliliter. The attenuated cells alone would no longer support the multiplication of E. histolytica, and only a negligible proportion
was viable at the time of the experiments. On successive transfer dates, two sets of cultures were prepared: one contained fresh cells only, and the other contained the same quantity of fresh cells plus attenuated cells. Table 11 lists TABLE II F22 Amebic Cultures with 1.5 X 109 Fresh Bacterial Cells plus Attenuated Cells
Trophozoites harvested (thousands)
trophozoites produceda
325 111 137 92 202 202 175 445
2200 3100 4000 4500 2700 2700 3200 2000
690 486 391 346 565 565 475 745
Average a Trophozoite ulum).
Ratio: fresh cells
Increase attributed to attenuated cells (thousands)
211
harvest
minus
10,000
(the
inoc-
some of the results of these experiments: in column 1, total amebic harvests in the attenuated cell-containing cultures; in column 2, the increased number of trophozoites attributed to the addition of the attenuated cells, ans in column 3, the calculated ratio, viable bacterial cells divided by trophozoites produced. In still other experiments in which fewer fresh and more attenuated bacterial
282
REEVES
cells were employed, calculated quotients in the range of 2000 were obtained. DISCUSSION
Experiments on the viability of washed B. symbiosus cells under the specified storage conditions indicate that the number of colonyforming units (loosely designated “viable cells” remains constant for a period of approximately 2 weeks, after which a logarithmic death phase ensues. During the second phase, approximately one half of the bacterial cells lose viability every 3 days. The ability of the bacterial cells (under penicillin inhibition) to support the multiplication of E. histoZytica is not appreciably altered by the onset of this death phase. This observation is in accord with the prior finding of Reeves, Schweinfurth, and Frye (1960) that bacterial cell viability is not essential for amebae growth. In the interval between 25 and 40 days of storage (33 days in the present instance) the bacterial cells rather suddenly lose their ability to support the multiplication of ameba. The evidence implies that some element of bacterial metabolism persists for a brief period after viability is lost, and that it is this element of metabolism which is responsible for the stimulation of ameba growth. Strangely, it seems to be an all or none phenomenon. Instead of a gradually diminishing response to the ageing of the bacterial cells, the multiplication of ameba may continue at a high level through a considerable number of serial transfers and then drop to zero within two successive transfers. The findings seem to suggest a conditioning effect upon the culture medium rather than a direct action upon the amebae, but if so, the effect is not simply due to the attainment of anaerobiosis in the culture, for the bacterial activity, once lost, is not restored by the incubation of the ameba cultures under hydrogen or nitrogen. With 3 X 10” fresh bacterial cells (colonyforming units) per culture, the ratios 18,300, 6100, and 8300 bacterial cells per trophozoite
were obtained for the 200, DKB, and F22 amebal strains, respectively. This quantity of bacterial cells is near the optimum amount for the production of ameba, but fewer bacterial cells per culture lead to lower bacteria to trophozoite ratios. Successively halving the number of bacteria per culture reduced the ratio, for the F22 strain, from 8300 to 5100 and 4400, and a similar decrease occurred with the other strains. To gather evidence as to whether a nutritional or a metabolic aspect of the bacterial cells was the limiting factor in the bacteria to trophozoite ratio, the experiments with attenuated bacterial cells were conducted. Since these cells would no longer support ameba growth, it was assumed that their function would be purely nutritional. The attenuated cells, when employed with 1.5 X 10” fresh cells, reduced the requirement for the latter to as low as 2000 per F22 trophozoite. With half this number of fresh cells per culture, this ratio was also obtained. The added attenuated cells, estimated from the nitrogen analysis to represent 1.3 X 10” original, colony-forming units, produced an average of 211,000 trophozoites per culture. This amounts to 6100 per trophozoite, which is somewhat more than the minimum of 4400 obtained with the fresh cells. But the attenuated cells may have less nutritional value than the fresh cells, and furthermore, a visible residue of unassimilated bacterial cells always remained in the attenuated cell-containing cultures at the time of the ameba harvests. The present findings indicate that studies on the metabolic function of bacterial cells in stimulating the multiplication of E. histolytica (hopefully, a function which may be reproduced axenically) might best be undertaken with slightly attenuated bacterial cells. In cells which have undergone storage for 2-4 weeks, the metabolism will be less extensive, and the essential activities the more conspicuous, than was the case with the freshly harvested cells studied by Bragg and Reeves (1962). Shaffer (1952, 1953) has reported the
BACTERIA
TO
E. histolytica
results of studies on bacterial to trophozoite ratios in cultures of the 200 and F22 strains of E. histolytica. By using the Shaffer-Frye (S-F) medium and freshly harvested bacterial cells in suboptimal amounts, he was able to obtain viable bacterium to trophozoite ratios as low as 500-1000 for the 200 strain of ameba, and approximately 2500 for the F22 strain. Our present findings are in very close agreement with his best results for the latter strain, but in our work the 200 strain of ameba required a greater number of bacterial cells per trophozoite produced. The discovery of a PPLO contaminant in our current ameba cultures raises numerous questions concerning the possible influence of this organism upon ameba growth. We have been unable to determine whether the cultures were so contaminated at the time the reported experiments were carried out. However, it has been found that, in our hands, the contaminant will not support continued growth of either strain of amebae without added bacterial cells. ACKNOWLEDGMENTS
I am indebted to Dr. John F. Kessel, who first pointed out the existence of PPLO organisms in a culture supplied by this laboratory; and to Dr. N. L. Somerson who assisted by making a tentative identification of the type of organism. The technical assistance of Mrs. Dorothy I. Schweinfurth during the course of this work is also gratefully acknowledged.
TROPHOZOITE
RATIO
283
REFERENCES BRAGG, P. D., AND REEVES, R. E.
1962. Studies on the carbohydrate metabolism of a gram-negative anaerobe (Bacteroides symbiosus) used in the culture of Entamoeba histolytica. Journal of Bacteriology 83, 76-84. REEVES, R. E., MELENEY, H. E., AND FRYE, W. W. 1957. A modified Shaffer-Frye technique for the cultivation of E&amoeba histolytica and some observations on its carbohydrate requirements. American Journal of Hygiene 66, 56-62. REEVES, R. E., SCHWEINFURTH, D. I., AND FRYE, W. W. 1960. The cultivation of E&amoeba histolytica with radiation-inactivated bacterial cells. The American Journal of Hygiene 72, 211-217. SEBALD, M. 1962. “Etude sur les batteries anaerobies Gram-negatives asporulees.” Imprimerie Barneoud S. A., Laval, France. SHAFFER, J. G. 1952. Studies on the growth requirements of Endamoeba histolytica. V. Studies on the nature of some of the factors in the Shaffer-Frye medium that affect the propagation of E. histolytica. The American Journal of Hygiene 56, 119-138. SHAFFER, J. G. 1953. Factors affecting the propagation of Endamoeba histolytica in vitro in the S-F medium and in tissue bearing substrate. The Annals of the New York Academy of Sciences 56, 1033-1047. SHAFFER, J. G., SCHULER, R. W., AND KEY, I. D. 1958. Studies on the growth requirements of Entamoeba histolytica. The ingestion of altered bacterial structures by E. histolytica in the Shaffer-Frye medium. American Journal of Tropical Medicine and Hygiene 7, 302-308. STEVENS, W. C. 1956. Taxonomic studies on the genus Bacteroides and similar forms. Ph.D. Thesis, Vanderbilt University, Nashville, Tennessee.
PARASITOLOGY 15, 279-283 (1964)
Ratios
of Bacterial
Cells
Trophozoites Richard Department
of Biochemistry,
to Entamoeba in Cultures1 E. Reeves
Louisiana State University New Orleans, Louisiana
(Submitted
histolytica
for publication,
1 April
School of Medicine,
1963)
When relatively fresh bacterial cells were used in MS-F type Entamoeba histolytica cultures, a minimum of 4400 bacterial cells (colony-forming units) sufficed, on the average, to produce each new amebal trophozoite of the F22 strain. Evidence is presented that indicates that this number of bacterial cells reflects a nutritional limit for ameba growth rather than a requirement for the amount of metabolic activity which these cells provide. When suboptimal quantities of fresh bacterial cells were employed together with attenuated bacterial cells, it was found that as few as 2000 fresh cells sufficed to stimulate the production of each new trophozoite. This lower number probably reflects a limiting level of metabolic activity by the associate organism. This required metabolic activity may be expended in the conditioning of the culture fluid rather than in a direct action upon the ameba.
Studies of the cultural requirements of parasitic amebae require an understanding of the stimulation to amebal growth provided by metabolizing (nonmultiplying) bacterial cells. In the MS-F system for the cultivation of ameba (Reeves et al., 1957)) penicillin-inhibited bacterial cells are agglutinated by a component of serum and settle to the lower surface of the culture vessel in the form of a delicate network. As the amebae grow and divide, this network is broken up, and the bacterial cells are ingested in large numbers by the trophozoites. When luxuriant ameba growth occurs, nearly all of the bacterial bodies disappear, and within certain limits, the number of amebae produced is limited by the quantity of available bacterial cells. The present investigation grew from the 1 This investigation was supported by the Surgeon General of the Army, Contract No. DA-49-007-MD756, under the sponsorship of the Commission of Enteric Infections of the Armed Forces Epidemiological Board.
attempt to discover the minimum number of bacterial cells necessary for the production of a new amebal trophozoite. MATERIALS
AND
METHODS
The 200 and F22 strains of Entamoeba histolyticu were obtained from Dr. James Shaffer; the DKB strain, from Dr. Quentin Geiman. All had been cultivated in this laboratory in the MS-F system for several years prior to the experiments. The bacterial stock culture, provisionally designated Bacteroides symbiosus (Stevens, 1956), was originally taken from Shaffer’s “streptobacillus culture no. 2” (Shaffer et al., 1958). It is now recognized that this culture contained several types or strains of similar, gram-negative anaerobes (Sebald, 1962). A stock culture was deposited in the American Type Culture Collection (ATCC No. 12829) at about the time the following experiments were being carried out. The MS-F system for the cultivation of 279
280
REEVES
ameba was used in this work (Reeves et al., 1957). To 12 ml of fluid base medium were added 0.25 ml of horse serum, 5000 units of penicillin, and washed bacterial cells. The inocula were 10,000 trophozoites suspended in small volumes of fluid from a preceding culture. The culture vessels were 16 X 125 mm tubes bearing screw caps with rubber liners. The cultures were incubated at 37” C in a slanting position for 2 or 3 days, after which the amebae were suspended by thorough mixing and counted in a Fuchs-Rosenthal counting chamber. All cultures were examined for bacterial contamination by direct observation and by plating on blood-agar plates; the results are drawn only from cultures free from evident bacterial contamination. However, since this work was completed it has been learned that the ameba cultures now carry an anaerobic mycoplasma, probably a human oral-type pleuropneumonia type organism (PPLO). The base medium contained, per liter: 20 gm Trypticase,’ 10 gm glucose, 2.5 gm thiomalic acid,” 0.79 gm cysteine hydrochloride, 2.0 gm sodium chloride, 2.0 gm dipotassium phosphate and sodium hydroxide to pH 7.0. The composition of the bacterial growth medium was similar, but it lacked the added cysteine and contained 2 gm yeast extract4 per liter. Viable bacterial cells (more accurately, colony-forming units) were assayed in ‘(thioglycollate medium without added dextrose”” in which 2% agar had been dissolved. Oval culture tubes were employed. The bacterial inoculum, 0.1 ml of a suitably diluted bacterial cell suspension, was added to the melted medium at about 51’. After the medium had solidified, it was capped with 2 cm of agar t* Baltimore Biological Laboratory, Inc. Baltimore, Maryland, U.S.A. 3 Difco Laboratories, Inc. Detroit 1, Michigan, u.s..4. 4 Eastman practical grade mercaptosuccinnic acid, twice recrystallized from water. From Distillation Products Industries, Rochester, New York, U.S.A.
medium and incubated at 37”. Colonies were usually visible for counting within 3 days, but all tubes were kept under observation for a total of 7 days. The optical densities of bacterial cultures were measured in 16 X 125 mm tubes in a Coleman model 6 spectrophotometer at a wavelength of 600 mp. RESULTS
Eight-hundred ml of bacterial growth medium was given a 1.55% inoculum from 24 hours growth in a stock B. symbiosus culture. After 18 hours at 37” the culture had just attained the stationary phase, its optical density was 0.54, and its bacterial nitrogen content (Kjeldahl) was 0.101 mg per milliliter. The cells were harvested by centrifugation, washed twice with 100 volumes of saline containing 0.02 iI sodium thiomalate, and
6 b i% ” 0
IO
20
30
Days of Storage of Bacterial Cells FIG. 1. Upper curve: log viable bacterial cells (colony-forming units) per milliliter of stored, lofold concentrated suspension. Lower curves: generations of ameba produced from the standard 10,000 trophozoite inoculum, employing 0.2 ml of the bacterial suspension in each MS-F culture tube.
BACTERIA
The Ratios
of Bacterial
TO
E. histolytica
TABLE I Cells to Trophozoites Produced Entamoeba histolytica
Days of storage of bacterial cells 0 2 5 7 9 12 14 16
Average
TROPHOZOITE
Ratio:
281
RATIO
per Culture,
FZ2 Strain
bacterial cells used ~ trophozoite harvest (-10,000)
of
, using:
1.5 X log cells
7.5 X 108 cells
11,400 9000 6400
4100 6000 6100 4300 5200
5800 5 100 3300 3400
8300
5140
4400
3 X log
cells
12,000 5400 9000 5000 8300
then stored at 2O-4” in suspension with 80 ml of 0.1 A4 sodium thiomalate (pH 7). Viable cell counts were made on this suspension thrice weekly for a period of 40 days. The results of these counts are shown in the upper curve of Fig. 1. The bacterial cells were used for the cultivation of three strains of E. histolytica. Serial transfers of ameba into media containing 0.2, 0.1, or 0.05 ml of the bacterial suspension were made three times weekly for a period of approximately 40 days. Without added bacterial cells, no ameba growth occurred. Some of the results obtained with the greatest quantity of bacterial cells are depicted in the lowest two curves of Fig. 1. Other results, reported in Table I, represent the quotient, i.e., bacterial cells (colony-forming units) divided by trophozoites produced. These last data were taken from results obtained by using bacterial cells less than 17 days old, i.e., before any drop in viable cell counts had occurred. In further experiments, attenuated bacterial cells were employed to supplement suboptimal quantities of the fresh cells. The attenuated cells had been prepared 3 months earlier; the optical density of the culture was then 0.48, and its bacterial nitrogen content was 0.0875 mg per milliliter. The attenuated cells alone would no longer support the multiplication of E. histolytica, and only a negligible proportion
was viable at the time of the experiments. On successive transfer dates, two sets of cultures were prepared: one contained fresh cells only, and the other contained the same quantity of fresh cells plus attenuated cells. Table 11 lists TABLE II F22 Amebic Cultures with 1.5 X 109 Fresh Bacterial Cells plus Attenuated Cells
Trophozoites harvested (thousands)
trophozoites produceda
325 111 137 92 202 202 175 445
2200 3100 4000 4500 2700 2700 3200 2000
690 486 391 346 565 565 475 745
Average a Trophozoite ulum).
Ratio: fresh cells
Increase attributed to attenuated cells (thousands)
211
harvest
minus
10,000
(the
inoc-
some of the results of these experiments: in column 1, total amebic harvests in the attenuated cell-containing cultures; in column 2, the increased number of trophozoites attributed to the addition of the attenuated cells, ans in column 3, the calculated ratio, viable bacterial cells divided by trophozoites produced. In still other experiments in which fewer fresh and more attenuated bacterial
282
REEVES
cells were employed, calculated quotients in the range of 2000 were obtained. DISCUSSION
Experiments on the viability of washed B. symbiosus cells under the specified storage conditions indicate that the number of colonyforming units (loosely designated “viable cells” remains constant for a period of approximately 2 weeks, after which a logarithmic death phase ensues. During the second phase, approximately one half of the bacterial cells lose viability every 3 days. The ability of the bacterial cells (under penicillin inhibition) to support the multiplication of E. histoZytica is not appreciably altered by the onset of this death phase. This observation is in accord with the prior finding of Reeves, Schweinfurth, and Frye (1960) that bacterial cell viability is not essential for amebae growth. In the interval between 25 and 40 days of storage (33 days in the present instance) the bacterial cells rather suddenly lose their ability to support the multiplication of ameba. The evidence implies that some element of bacterial metabolism persists for a brief period after viability is lost, and that it is this element of metabolism which is responsible for the stimulation of ameba growth. Strangely, it seems to be an all or none phenomenon. Instead of a gradually diminishing response to the ageing of the bacterial cells, the multiplication of ameba may continue at a high level through a considerable number of serial transfers and then drop to zero within two successive transfers. The findings seem to suggest a conditioning effect upon the culture medium rather than a direct action upon the amebae, but if so, the effect is not simply due to the attainment of anaerobiosis in the culture, for the bacterial activity, once lost, is not restored by the incubation of the ameba cultures under hydrogen or nitrogen. With 3 X 10” fresh bacterial cells (colonyforming units) per culture, the ratios 18,300, 6100, and 8300 bacterial cells per trophozoite
were obtained for the 200, DKB, and F22 amebal strains, respectively. This quantity of bacterial cells is near the optimum amount for the production of ameba, but fewer bacterial cells per culture lead to lower bacteria to trophozoite ratios. Successively halving the number of bacteria per culture reduced the ratio, for the F22 strain, from 8300 to 5100 and 4400, and a similar decrease occurred with the other strains. To gather evidence as to whether a nutritional or a metabolic aspect of the bacterial cells was the limiting factor in the bacteria to trophozoite ratio, the experiments with attenuated bacterial cells were conducted. Since these cells would no longer support ameba growth, it was assumed that their function would be purely nutritional. The attenuated cells, when employed with 1.5 X 10” fresh cells, reduced the requirement for the latter to as low as 2000 per F22 trophozoite. With half this number of fresh cells per culture, this ratio was also obtained. The added attenuated cells, estimated from the nitrogen analysis to represent 1.3 X 10” original, colony-forming units, produced an average of 211,000 trophozoites per culture. This amounts to 6100 per trophozoite, which is somewhat more than the minimum of 4400 obtained with the fresh cells. But the attenuated cells may have less nutritional value than the fresh cells, and furthermore, a visible residue of unassimilated bacterial cells always remained in the attenuated cell-containing cultures at the time of the ameba harvests. The present findings indicate that studies on the metabolic function of bacterial cells in stimulating the multiplication of E. histolytica (hopefully, a function which may be reproduced axenically) might best be undertaken with slightly attenuated bacterial cells. In cells which have undergone storage for 2-4 weeks, the metabolism will be less extensive, and the essential activities the more conspicuous, than was the case with the freshly harvested cells studied by Bragg and Reeves (1962). Shaffer (1952, 1953) has reported the
BACTERIA
TO
E. histolytica
results of studies on bacterial to trophozoite ratios in cultures of the 200 and F22 strains of E. histolytica. By using the Shaffer-Frye (S-F) medium and freshly harvested bacterial cells in suboptimal amounts, he was able to obtain viable bacterium to trophozoite ratios as low as 500-1000 for the 200 strain of ameba, and approximately 2500 for the F22 strain. Our present findings are in very close agreement with his best results for the latter strain, but in our work the 200 strain of ameba required a greater number of bacterial cells per trophozoite produced. The discovery of a PPLO contaminant in our current ameba cultures raises numerous questions concerning the possible influence of this organism upon ameba growth. We have been unable to determine whether the cultures were so contaminated at the time the reported experiments were carried out. However, it has been found that, in our hands, the contaminant will not support continued growth of either strain of amebae without added bacterial cells. ACKNOWLEDGMENTS
I am indebted to Dr. John F. Kessel, who first pointed out the existence of PPLO organisms in a culture supplied by this laboratory; and to Dr. N. L. Somerson who assisted by making a tentative identification of the type of organism. The technical assistance of Mrs. Dorothy I. Schweinfurth during the course of this work is also gratefully acknowledged.
TROPHOZOITE
RATIO
283
REFERENCES BRAGG, P. D., AND REEVES, R. E.
1962. Studies on the carbohydrate metabolism of a gram-negative anaerobe (Bacteroides symbiosus) used in the culture of Entamoeba histolytica. Journal of Bacteriology 83, 76-84. REEVES, R. E., MELENEY, H. E., AND FRYE, W. W. 1957. A modified Shaffer-Frye technique for the cultivation of E&amoeba histolytica and some observations on its carbohydrate requirements. American Journal of Hygiene 66, 56-62. REEVES, R. E., SCHWEINFURTH, D. I., AND FRYE, W. W. 1960. The cultivation of E&amoeba histolytica with radiation-inactivated bacterial cells. The American Journal of Hygiene 72, 211-217. SEBALD, M. 1962. “Etude sur les batteries anaerobies Gram-negatives asporulees.” Imprimerie Barneoud S. A., Laval, France. SHAFFER, J. G. 1952. Studies on the growth requirements of Endamoeba histolytica. V. Studies on the nature of some of the factors in the Shaffer-Frye medium that affect the propagation of E. histolytica. The American Journal of Hygiene 56, 119-138. SHAFFER, J. G. 1953. Factors affecting the propagation of Endamoeba histolytica in vitro in the S-F medium and in tissue bearing substrate. The Annals of the New York Academy of Sciences 56, 1033-1047. SHAFFER, J. G., SCHULER, R. W., AND KEY, I. D. 1958. Studies on the growth requirements of Entamoeba histolytica. The ingestion of altered bacterial structures by E. histolytica in the Shaffer-Frye medium. American Journal of Tropical Medicine and Hygiene 7, 302-308. STEVENS, W. C. 1956. Taxonomic studies on the genus Bacteroides and similar forms. Ph.D. Thesis, Vanderbilt University, Nashville, Tennessee.