- Email: [email protected]
Studies on the nutrition of Endamoeba histolytica
Studies
on the Nutrition
of Endumoeba
I. Amino Herbert
Blumenthal, James
Acid
histolytica.
Buffers
Joseph B. Michaelson, N. DeLamater’
and
Department of Biochemistry and Departlnent of Medicine, University of Southern California School of Medicine, Los .Angeles ‘7, California (Submitted
for publication,
4 August
1953)
In reviewing the literature
on culture methods and media for the in it was noted that where buffers were used, they have invariably been phosphate buffers, (Balamuth, 1946, Anderson and Hansen, 1947, and Cleveland and Collier, 1930). Since phosphate plays an important part in the mechanisms of both energy storage and transfer (Meyerhof, 1951) in all animals and plants previously studied, and since an active acid phosphatase has been demonstrated in trophozoites of E. histolytica (Carrera and Changus, 1948, and Balamuth, 1950), we were interested in the sources of phosphate available to amebae. It has been shown that there is a definite pH optimum for amebic growth in vitro (Chang, 1912), and the importance of hydrogen ion concentration per se was not in doubt. There was the question, however, whether phosphate buffers were also important in providing a source of inorganic phosphate, or whether other buffers might serve equally well.
vitro growth of E. histolytica,
MATERIALS
AND METHODS
A modified Boeck-Drbohlav medium (1925) was prepared in which the following buffer systems were substituted for the phosphate system of the Stone’s buffer (1935) that is normally used as an overlay in this laboratory, all other components being held constant: 1 This work was sponsored by the Commission on Enteric Infections of the Armed Forces Epidemiological Board and supported by the Office of the Surgeon General, Department of the Army, Washington, D. C. The use of certain facilities of the Allan Hancock Foundat,ion of the University of Southern California is gratefully acknowledged. Grateful acknowledgement is made to Dr. John W. Mehl for his encouragement and support during the course of this work, and 60 Mr. Peter J. Rennie for his technical assistance. 321
322
BLUMENTHAL,
MICHAELSON
AND
DELAMATER
a. 0.1 M dh-alanine b. 0.1 M I-glutamic acid c. 0.1 M dl-aspartic acid These buffer systems were prepared by titration of a 0.1 M solution of the amino acids with cu. 2 N sodium hydroxide so that the total volume was altered less than 1%. These three amino acids were chosen because they are normal physiological constituents of the system used, being present in egg (Calvery, 1944) and being interrelated metabolically as the three important portals of entry of amino acids into the tricarboxylic acid cycle via trasamination (Cohen, 1939; 1940). It is recognized that their buffering capacity is not great near neutrality, but this consideration seemed outweighed by their physiological nature. The egg slants were then overlaid with 3 ml of these modified amino acid buffers, which contained the salts of Stone’s buffer. Approximately 20 mg of sterile Bacto Rice powder was added just before use. Transfers of cultures were made at 4%hour intervals except on weekendswhen 72-hour transfers were made. In all cases the cultures were run in duplicate for a
total of 21-23 days. The Denton* strain of E. histol&a, grown with its normal flora, and monobacterially with Aerobacter aerogenes, was used. Parallel control cultures containinq flora alone were simultaneously run. RESULTS
The results of these experiments are summarized in Table I. In all three experimental buffer systems it was found that growth was more plentiful in the more alkaline pH range, the amebae being motile and containing ingested starch granules. As the buffering capacity of these systems is maximum at lower hydrogen ion concentrations, this maximum growth of amebae is expected because a greater degree of homeostasis of the medium results. However, where the pH of the culture tubes was checked after growth it was found that there was little correlation between the starting pH of the medium and the pH of the medium after growth. Table II shows the effect of amebic and bacterial growth on the pH of the glutamic-acid system after 72 hours of culture on the 23rd day of transfer. It will be noted that the mixed flora cultures tended toward the more acid range of the buffers, approaching an average value of pH 7.6, while the Aerobacter cultures tended toward the alkaline range, with an average pH of 8.5. It cannot, therefore, be inferred that the best growth occurred in ranges of maximum buffering. It is interesting that the pH of * Obtained from Dr. F. Ryden, Vanderbilt Nashville, Tenn., June 6, 1949.
Universit,y
School of Medicine,
NUTRITION
OF
ENDAMOEBA
TABLE Growth of Amebae Buffer
Phosphate dla-Alanine dla-Alanine dh-Alanine dla-Alanine I-Glutamic I-Glutamic l-Glutamic I-Glutamic dl-Aspartic dl-Aspartic dl-Aspartic
cont.rol
acid acid acid acid acid acid acid
I
after $1 Days in Modified
PH
7.5 7.6 7.9 8.2 8.5 7.6 7.9 8.2 8.5 8.1 8.4 8.7
323
HISTOLYTICA
Amebae plus flora
Whole EQQ Media
Amebae plus A. acrogcnes
++* + + + ++ + + + ++ +
Mixed
++ + + + ++ + + + ++ + + ++
flora
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
l Growth WBB estimated from observations of sediment from cultures. (++) = greater than 2 amebae per higher powered field (+) = 1 to 2 amebae per high powered field (-) = no amebae found; growth was considered positive only if motile
amebae
++ ++ ++ ++ ++ ++ ++ ++ ++ f+ ++ ++
were
present.
the mixed flora cultures tended toward the accepted pH optimum of 7.5, while the Aerobacter-E. histolytica cultures become more alkaline, a result characteristic of cultures of A. aerogeneswhen this organism is grown in pure culture containing fermentable substrates (Wilson and Miles, 1946; Clark and Lubs, 1915). This could indicate that in E. histolytica cultures, changes in pH of the media after growth are a function of the flora. Phillips (1953) has suggested the dependence of E. histoZytica on the metabolism of its associates,a finding in line with our data. It should also be noted that in the aspartic acid buffer system the Aerobatter-ameba culture grew much better than the mixed flora-ameba culture, a situation not, in line with our usual experience. TABLE change in pH of Glutamic pH at start
7.6 7.9 8.2 8.5
II
Acid EQQ Medium Denton-mixed
7.6 7.9 7.7 7.4
After
7.9 Hours
Growth
pH at end of 72 hours Bora Denton-Acrobaclw
8.5 8.5 8.3 8.5
324
BLUMENTHAL,
MICHAELSON AND DELAMATER SUMMARY
1. It has been shown that E. histolytica will grow in media buffered by systems other than phosphate. 2. E. hislolylica, under the conditions described, obtains sufficient phosphate for growth from coagulated whole egg, accompanying flora, rice powder, or a combination of any of these. 3. In the amino acid buffer systems used, regardless of the pH at the start of grou-th, there is a change in pH at 48 to 72 hours, which appears characteristic of the accompanying bacterial flora. 4. A system has been demonstrated in whch amebse in a monobacterial culture show better growth than a culture of amebae containing mixed bacterial flora. REFERENCES ANDERSON,H. H., AND HANREN, E. I,. 1947. Cultivation of Endamoeba histolytica. Ann. sot. helge mbd. trop. 27, 1-15. BALAMIJTH, W. 1946. Improved egg yolk infusion for cultivation of Entamoeba histol&a and other intestinal protozoa. Am, J. Clin. Pathol. 16, 380-384. BALAMUTH, W. 1950. Acid phosphatase staining reactions in intestinal amehae. J. Parasitol. 36, (suppl.), 37. BOECK, W. C., AND DRBOHLBV, J. 1925. The cultivation of Endanloeba histolytica. Am. J. Hyg. 6, 371-407. CALVERY, H. 0. 1944. In Chemistry of Amino Acids and Proteins. (C. L. A. Schmidt, ed.) Charles C. Thomas, Baltimore, 2nd Ed. CARRERA, G. M., AND CNANGUS, G. W. 1948. Demonstration of acid phosphatsse in Endamoeba histolytica. Proc. Sot. Ezptl. Sol. Med. 63, 610-611. CHANG, S. L. 1942. Studies on Endamoeba histolytica. I. Effect of hydrogen-ion concentration on encystat,ion of Endamoeba histolytica in culture. Am. J. Trap.
dfed. 22, 471-484.
CLARK, W. M., AND Luss, H. A. 1915. The differentiation of bacteria of the colonaerogenes family by use of indicators. J. Infectious Diseases 1’7, 160-173. CLEVELAND, L. R., ANI) COLLIER, J. 1930. Various improvements in the cultivation of Endamoeba histolytica. Am. J. Hyg. 12, 606-613. COHEN, P. P. 1939. Transaminat,ion in pigeon breast muscle. Biochem. J. 33.147% 1487. COHEN, P. 1’. 1940a. Transamination with purified enzyme preparations (transaminase). J. Biol. Chem. 136, 565-584. CONE?*', 1’. P., 1940b. Kineticsof trsnsaminaseactivity. J. Viol. Chem. 136,585-601. MEYERHOF, 0. 1951. In. Phosphorus Metabolism, A Symposium on the Role of Phosphorus in the Metabolism of Plants and Animals. (W. D. McElroy and B. Glass, eds.), Vol. 1, 3-10, Johns Hopkins Press, Baltimore. PHILLIPS, B. P. 195.7.The effects of various treatment procedures on t,he metabolism of ‘I’rvparzosoma crr~zi and its ability to support growth of h’ndamoeba histolytica.
Am. J. 7’rop. Med. Hz/g. 2, 47-53.
STONE, W. S. 1935. A method of producing encystment in cult,ures of Endamoeba histolytica. Am. J. Trap. Med. 16, 681-684. WILRON, G. S., AND MILES, A. A. 1946. In Principles
of Bacteriology
and Immunity
(Topley and Wilson, eds.), Vol. I, 660-663, 3rd ed., Williams Baltimore.
& Wilkins,
on the Nutrition
of Endumoeba
I. Amino Herbert
Blumenthal, James
Acid
histolytica.
Buffers
Joseph B. Michaelson, N. DeLamater’
and
Department of Biochemistry and Departlnent of Medicine, University of Southern California School of Medicine, Los .Angeles ‘7, California (Submitted
for publication,
4 August
1953)
In reviewing the literature
on culture methods and media for the in it was noted that where buffers were used, they have invariably been phosphate buffers, (Balamuth, 1946, Anderson and Hansen, 1947, and Cleveland and Collier, 1930). Since phosphate plays an important part in the mechanisms of both energy storage and transfer (Meyerhof, 1951) in all animals and plants previously studied, and since an active acid phosphatase has been demonstrated in trophozoites of E. histolytica (Carrera and Changus, 1948, and Balamuth, 1950), we were interested in the sources of phosphate available to amebae. It has been shown that there is a definite pH optimum for amebic growth in vitro (Chang, 1912), and the importance of hydrogen ion concentration per se was not in doubt. There was the question, however, whether phosphate buffers were also important in providing a source of inorganic phosphate, or whether other buffers might serve equally well.
vitro growth of E. histolytica,
MATERIALS
AND METHODS
A modified Boeck-Drbohlav medium (1925) was prepared in which the following buffer systems were substituted for the phosphate system of the Stone’s buffer (1935) that is normally used as an overlay in this laboratory, all other components being held constant: 1 This work was sponsored by the Commission on Enteric Infections of the Armed Forces Epidemiological Board and supported by the Office of the Surgeon General, Department of the Army, Washington, D. C. The use of certain facilities of the Allan Hancock Foundat,ion of the University of Southern California is gratefully acknowledged. Grateful acknowledgement is made to Dr. John W. Mehl for his encouragement and support during the course of this work, and 60 Mr. Peter J. Rennie for his technical assistance. 321
322
BLUMENTHAL,
MICHAELSON
AND
DELAMATER
a. 0.1 M dh-alanine b. 0.1 M I-glutamic acid c. 0.1 M dl-aspartic acid These buffer systems were prepared by titration of a 0.1 M solution of the amino acids with cu. 2 N sodium hydroxide so that the total volume was altered less than 1%. These three amino acids were chosen because they are normal physiological constituents of the system used, being present in egg (Calvery, 1944) and being interrelated metabolically as the three important portals of entry of amino acids into the tricarboxylic acid cycle via trasamination (Cohen, 1939; 1940). It is recognized that their buffering capacity is not great near neutrality, but this consideration seemed outweighed by their physiological nature. The egg slants were then overlaid with 3 ml of these modified amino acid buffers, which contained the salts of Stone’s buffer. Approximately 20 mg of sterile Bacto Rice powder was added just before use. Transfers of cultures were made at 4%hour intervals except on weekendswhen 72-hour transfers were made. In all cases the cultures were run in duplicate for a
total of 21-23 days. The Denton* strain of E. histol&a, grown with its normal flora, and monobacterially with Aerobacter aerogenes, was used. Parallel control cultures containinq flora alone were simultaneously run. RESULTS
The results of these experiments are summarized in Table I. In all three experimental buffer systems it was found that growth was more plentiful in the more alkaline pH range, the amebae being motile and containing ingested starch granules. As the buffering capacity of these systems is maximum at lower hydrogen ion concentrations, this maximum growth of amebae is expected because a greater degree of homeostasis of the medium results. However, where the pH of the culture tubes was checked after growth it was found that there was little correlation between the starting pH of the medium and the pH of the medium after growth. Table II shows the effect of amebic and bacterial growth on the pH of the glutamic-acid system after 72 hours of culture on the 23rd day of transfer. It will be noted that the mixed flora cultures tended toward the more acid range of the buffers, approaching an average value of pH 7.6, while the Aerobacter cultures tended toward the alkaline range, with an average pH of 8.5. It cannot, therefore, be inferred that the best growth occurred in ranges of maximum buffering. It is interesting that the pH of * Obtained from Dr. F. Ryden, Vanderbilt Nashville, Tenn., June 6, 1949.
Universit,y
School of Medicine,
NUTRITION
OF
ENDAMOEBA
TABLE Growth of Amebae Buffer
Phosphate dla-Alanine dla-Alanine dh-Alanine dla-Alanine I-Glutamic I-Glutamic l-Glutamic I-Glutamic dl-Aspartic dl-Aspartic dl-Aspartic
cont.rol
acid acid acid acid acid acid acid
I
after $1 Days in Modified
PH
7.5 7.6 7.9 8.2 8.5 7.6 7.9 8.2 8.5 8.1 8.4 8.7
323
HISTOLYTICA
Amebae plus flora
Whole EQQ Media
Amebae plus A. acrogcnes
++* + + + ++ + + + ++ +
Mixed
++ + + + ++ + + + ++ + + ++
flora
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
l Growth WBB estimated from observations of sediment from cultures. (++) = greater than 2 amebae per higher powered field (+) = 1 to 2 amebae per high powered field (-) = no amebae found; growth was considered positive only if motile
amebae
++ ++ ++ ++ ++ ++ ++ ++ ++ f+ ++ ++
were
present.
the mixed flora cultures tended toward the accepted pH optimum of 7.5, while the Aerobacter-E. histolytica cultures become more alkaline, a result characteristic of cultures of A. aerogeneswhen this organism is grown in pure culture containing fermentable substrates (Wilson and Miles, 1946; Clark and Lubs, 1915). This could indicate that in E. histolytica cultures, changes in pH of the media after growth are a function of the flora. Phillips (1953) has suggested the dependence of E. histoZytica on the metabolism of its associates,a finding in line with our data. It should also be noted that in the aspartic acid buffer system the Aerobatter-ameba culture grew much better than the mixed flora-ameba culture, a situation not, in line with our usual experience. TABLE change in pH of Glutamic pH at start
7.6 7.9 8.2 8.5
II
Acid EQQ Medium Denton-mixed
7.6 7.9 7.7 7.4
After
7.9 Hours
Growth
pH at end of 72 hours Bora Denton-Acrobaclw
8.5 8.5 8.3 8.5
324
BLUMENTHAL,
MICHAELSON AND DELAMATER SUMMARY
1. It has been shown that E. histolytica will grow in media buffered by systems other than phosphate. 2. E. hislolylica, under the conditions described, obtains sufficient phosphate for growth from coagulated whole egg, accompanying flora, rice powder, or a combination of any of these. 3. In the amino acid buffer systems used, regardless of the pH at the start of grou-th, there is a change in pH at 48 to 72 hours, which appears characteristic of the accompanying bacterial flora. 4. A system has been demonstrated in whch amebse in a monobacterial culture show better growth than a culture of amebae containing mixed bacterial flora. REFERENCES ANDERSON,H. H., AND HANREN, E. I,. 1947. Cultivation of Endamoeba histolytica. Ann. sot. helge mbd. trop. 27, 1-15. BALAMIJTH, W. 1946. Improved egg yolk infusion for cultivation of Entamoeba histol&a and other intestinal protozoa. Am, J. Clin. Pathol. 16, 380-384. BALAMUTH, W. 1950. Acid phosphatase staining reactions in intestinal amehae. J. Parasitol. 36, (suppl.), 37. BOECK, W. C., AND DRBOHLBV, J. 1925. The cultivation of Endanloeba histolytica. Am. J. Hyg. 6, 371-407. CALVERY, H. 0. 1944. In Chemistry of Amino Acids and Proteins. (C. L. A. Schmidt, ed.) Charles C. Thomas, Baltimore, 2nd Ed. CARRERA, G. M., AND CNANGUS, G. W. 1948. Demonstration of acid phosphatsse in Endamoeba histolytica. Proc. Sot. Ezptl. Sol. Med. 63, 610-611. CHANG, S. L. 1942. Studies on Endamoeba histolytica. I. Effect of hydrogen-ion concentration on encystat,ion of Endamoeba histolytica in culture. Am. J. Trap.
dfed. 22, 471-484.
CLARK, W. M., AND Luss, H. A. 1915. The differentiation of bacteria of the colonaerogenes family by use of indicators. J. Infectious Diseases 1’7, 160-173. CLEVELAND, L. R., ANI) COLLIER, J. 1930. Various improvements in the cultivation of Endamoeba histolytica. Am. J. Hyg. 12, 606-613. COHEN, P. P. 1939. Transaminat,ion in pigeon breast muscle. Biochem. J. 33.147% 1487. COHEN, P. 1’. 1940a. Transamination with purified enzyme preparations (transaminase). J. Biol. Chem. 136, 565-584. CONE?*', 1’. P., 1940b. Kineticsof trsnsaminaseactivity. J. Viol. Chem. 136,585-601. MEYERHOF, 0. 1951. In. Phosphorus Metabolism, A Symposium on the Role of Phosphorus in the Metabolism of Plants and Animals. (W. D. McElroy and B. Glass, eds.), Vol. 1, 3-10, Johns Hopkins Press, Baltimore. PHILLIPS, B. P. 195.7.The effects of various treatment procedures on t,he metabolism of ‘I’rvparzosoma crr~zi and its ability to support growth of h’ndamoeba histolytica.
Am. J. 7’rop. Med. Hz/g. 2, 47-53.
STONE, W. S. 1935. A method of producing encystment in cult,ures of Endamoeba histolytica. Am. J. Trap. Med. 16, 681-684. WILRON, G. S., AND MILES, A. A. 1946. In Principles
of Bacteriology
and Immunity
(Topley and Wilson, eds.), Vol. I, 660-663, 3rd ed., Williams Baltimore.
& Wilkins,