EXPERIMENTAL
46, 108-112 (1978)
PARASITOLOGY
Trypanosoma
cruzi:
Cultivation in Macromolecule-Free and Synthetic Media
Semisynthetic
S. CHIA-TUNG PAN Department
of Tropical
Public Health, Huruard School of Public Health, Boston, Massachusetts 02115, U.S.A.
(Accepted for publication
2 May 1978)
PAN, S. C.-T. 1978. Trypanosoma cruzi: Cultivation in macromolecule-free semisynthetic and synthetic media. Experimental Parasitology 46, 108-112. A macromolecule-free semisynthetic medium (F-81) was devised to culture Trypanosoma cruzi serially at room temperature. F-81 contains only one undefined substance, trypticare, which consists primarily of short-chain polypeptides. In F-81 medium T. cruzi will grow to a density of 35 to 43 x 10’ organisms/ml, a density comparable to that obtainable in a serum-containing medium such as F-69. High concentrations of water-soluble vitamins appear to have a serumreplacing effect in the F-81 medium. A completely synthetic medium (F-84) was prepared by replacing trypticase in F-81 with Trager’s amino acid mixture. T. cruzi epimastigotes could be serially cultured in F-84, with a maximum yield of 9.2 X 10’ organisms/ml of medium after 3 to 4 weeks of incubation at 27 C. INDEX DESCRIPTORS: Tqpanosoma CTUZ~; Hemoflagellate; Protozoa parasitic; Cultivation, in c&o; Medium, semisynthetic and synthetic.
Trypanosoma cruzi, the cause of Chagas’ disease, has been successfully cultured in vitro for nearly 70 years (Bishop 1967; Pan 1976; Tobie 1964). With few exceptions, most culture media contain one or more chemically undefined substances (Anderson and Krassner 1975); and the yield when cultured at room temperature consists of essentially epimastigotes (“culture forms”) (Pan 1968, 1971, 1976). Since “culture” forms of T. crud are readily grown in vitro, it seems reasonable that synthetic or semisynthetic media for these organisms could be prepared more easily and that these media could become a basis for preparing synthetic media for the cultivation of the vertebrate stages. In this study, we describe a macromolecule-free semisynthetic and a synthetic media for serial cultivation of T. crud epimastigotes.
MATERIALS
0014-4894/78/0461-0108$02.00/O 0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
The Brazil strain of Typanosoma cruxi (Pan 1968) and a Colombian strain (Federici et al. 1964) were used. The strains were routinely maintained in NNN medium (Pan 1968) at room temperature by transfer at 4-week intervals. The starting inocula for experiments with semisynthetic media were obtained by inoculating each of several tubes of F-69 medium (Pan 1978) with 1 ml of a l-week-old NNN culture (479th passage for the Brazil strain and 29th passage for Colombian strain), and incubating at 27 C. Cultures in F-69 were harvested 4 to 6 days later, washed three times with phosphate-buffered saline, pH 7.2, containing 0.4% glucose (PBS-G), resuspended in PBS-G, and counted in a hemacytometer.
108 Copyright All rights
AND
TTyf3U?WSO??UZ
TABLE Composition
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
C?UXi:
CULTIVATION
IN
DEFINED
I
of F-81 (Semisynthetic) for Trypanosoma cruzi
Distilled water Medium 199 (10 X concentrate)” Glucose (0.8%) Sodium pyruvate (0.1 M) Trypticase (50/,)b Vitamin solution (see Table III) Biotin (2 mg/lOO ml of HzO) Folic acid (10 mg/lOO ml of HzO) Vitamin B12 (1 mg/lOO ml of H&) Methyl cellulose (4%) Hemin (50 mg/lOO ml of 0.01 N NaOH)” 12. Antibiotic mixture (penicillin G, lo5 U/ml and streptomycin, lo5 pg/ml) 13. ATP, ADP, and AMP solution (see Table IV) 14. NaHC03 (4.2%)
TABLE Medium
20.0 ml 10.0 25.0 2.0 10.0 2.0 2.0 5.0 1.0 5.0 5.0 0.1 10.0 3.0
a Morgan et al. (1950), Medium 199, obtained from Microbiological Associates, Inc., Maryland. * Baltimore Biological Laboratories, Maryland. c Teichmann’s crystals.
The starting inocula for experiments with synthetic media were derived from established cultures in semisynthetic medium ( F-81, see below). Before inoculation, cultures were washed three times with PBS-G. For experiments, cultures in duplicate (screw-cap tubes, 16 x 125 mm, each containing 5 ml of fresh medium) were inoculated with 1 ml of culture from the previous passage and incubated at 27 + 1 C. After incubation, organisms from both tubes were pooled, 1 ml of the pool was used for counting, 2 ml was used for subinoculation, and the remainder was used for preparing duplicate stained smears as described previously (Pan 1968, 1978). Five hundred organisms were counted at 1000X on each of two smears per passage and classified as to morphologic type (for details, see Pan 1968, 1978). The composition of one (F-81) of the two semisynthetic media (Tables I, III, IV) and of one (F-84) of the two synthetic media (Tables II, V, VI) is summarized in
Composition
109
MEDIA
II
of the F-84 (Synthetic) for Trypanosoma‘ cruzi
Medium
1. Trager’s amino acid solution minus tyrosine (see Table V) 2. L-Tyrosine (40 mg/lOO ml) 3. Glucose (0.8%) 4. Medium 199 (10X) 5. Sodium pyruvate (0.1 M) 6. Vitamin solution (see Table III) 7. Biotin (2 mg/lOO ml of HZO) 8. Folic acid (10 mg/lOO ml of HzO) 9. Vitamin B12 (1 mg/lOO ml of HzO) 10. Metal solution (see Table VI) 11. Methyl cellulose (4y0) 12. Hemin (50 mg/lOO ml of 0.01 N NaOH)a 13. L-Glutamine (200 m&f) 14. Antibiotic mixture (penicillin G, lo6 U/ml and streptomycin, 106 rg/ml) 15. ATP, ADP, and AMP solution (see Table IV) 16. NaHC03 (4.2y0) a Teichmann’s
15.0 ml 10.0 25.0 10.0 2.0 2.0 2.0 5.0 1.0 1.0
4.0 4.0 4.0 0.1 10.0 5.0
crystals.
Tables I through VI. F-81 medium is essentially F-69 medium (Pan 1978) minus fetal bovine serum but with a higher concentration of water-soluble vitamins. F-84 medium is essentially F-81 minus trypticase but with a higher concentration of amino acids. All solutions were sterilized by filtration through a Millipore filter (pore size, 0.45 pm) except that trypticase, methyl cellu-
TABLE Composition 1. 2. 3. 4. 5. 6. 7. 8. 9.
III
of Vitamin
p-Aminobenzoic acid n-Calcium pantothenate Choline chloride i-Inositol Nicotinamide Nicotinic acid Pyridoxal-HCl Pyridoxine-HCl Pyridoxaminee2HCl 10. Riboflavin-5-phosphate-Na 11. Thiamine 12. Distilled water
Solution 30 mg 40 30 30 50 20 20 20 20 2 20 100 ml
110
S. CZIIA-TUNG
lose and metallic salt solutions were autoclaved at 15 psi pressure for 15 min.
RESULTS
Attempts to culture Trypanosoma cruzi in semisynthetic and synthetic media at 33, 35, and 37 C were unsuccessful. However, when the temperature of incubation was at 27 C, both strains of T. cruzi could be cultured serially. Table VII summarizes the growth characteristics of T. cruzi (the Brazil strain) in semisynthetic medium F-81 at selected passages. Throughout the experiment (over 63 passages in 13 months), the organisms grew well, and on the average increased eight-fold during an incubation period of 6 to 11 days. During the first six passages, practically all organisms were epimastigotes. Thereafter, relatively large numbers of trypomastigotes (metacyclic types) werk usually present in the culture at the end of exponential growth. The growth characteristics of T. cruzi in F-81 medium are remarkably similar to those observed in experiments with F-69 medium at room temperature (Pan 1978). The organisms (95% epimastigotes) cultured in F-81 (19th passage) can infect human skin-muscle cells in vitro at 35 C. However, culturing of organisms in F-81 appeared to alter their ability to invade skin-muscle cells in that there was a delay of 2 to 3 days when compared with epimastigotes cultured in F-69 medium (containing 10% fetal bovine serum) (Pan 1978 ) .
TABLE The ATP, ADP,
PAN TABLE
Composition without
of Trager’s (1957) Amino Acid Solution Tyrosine for Leischn~ania tar&&e
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
I,-Arginine-HCl L-Histidine L-Isoleucine L-Leurine r,-Lysine HCI L-Methionine L-Phenylalanine L-Threonine L-Tryptophnne L-JTaline r,-Alanine I,-Aspartic acid Glycine L-Proline I,-Serine I,-Glutamic acid Iktilled water
120 mg 60 240 600 500 120 160 200 80 200 280 480 40 200 160 760 100 ml
The results of experiments with synthetic medium F-84 are summarized in Table VIII. The inocula originally derived from the 13th passage in F-81 and contained 92.6% epimastigotes and 7.470 trypomastigotes. Although T. crud could be serially subcultured at 27 C in F-84 for 6 months, it was apparent that (1) the medium was not optimal in terms of growth, (2) the generation time was prolonged, and (3) practically all organisms were epimastigotes after the second passage. However, on examination by light microscopy the epimastigotes showed no morphologic abnormalities. DISCUSSION
Several investigators (Little and Oleson 1951; Citri and Grossowicz 1955; O’Daly
IV
and AMP
TABLE
Sollltion
Composition 1. 2. 3. 4. 5. 6. 7.
V
I-CysteineeHCl Glutathione (reduced) Ascorbic acid ATP ADP AMP Distilled water
5mg 20 5 50 10 10 100 ml
1. 2. 3. 4. 5. 6.
VI
of Metal
ZnS04.7HzO H,BOs CLISO~ MnS04.4HxO CoSOa.7HsO Distilled water
Solution 220 mg 1 3; 4 100 ml
~?Yj~CZ?lO~OWlxZ CTUZi:
CULTIVATION TABLE
.
Subculture No.
1 2 3 4 5 6 12 16
Growth
of Trypanosoma
Incubation Days
9 8 7 6 7 8 10 11
IN DEFINED
VII
cruzi (the Brazil Strain) F-81 Medium at 27C
Inoculum x 106
Increaseb (n-fold)
7.6” 10.3 21.0 26.0 27.1 23.2 22.8 35.2
8.2 12.2 7.4 6.3 5.1 10.6 9.1 7.5
111
MEDIA
in Semisynthetic
Distribution Epimastigotes
by morphologic Trypomastigotes
98.4 99.6 99.4 98.8 98.2 97.6 91.2 91.4
types (%)
Promastigotes
Amastigotes
1.2 0.2 0.2 0 0 0.6 0 0.2
0 0 0 0 0 0.2 0 0
0.4 0.2 0.4 1.2 1.8 1.6 8.8 8.4
Inoculum contained over 99.9% epimastigotes. b Calculated increase over specified incubation period.
1975) have described serial cultivation of Typanosoma c~uzi in semisynthetic media at room temperature. However, their media included at least one undefined blood product containing macromolecules which could not be replaced by simpler substances. Recently, a defined medium was devised for the cultivation of T. brucei at room temperature (Cross and Manning 1973). This medium was subsequently applied to culture a strain of T. cruzi (Anderson and Krassner 1975). Although the authors reported that T. cruzi could be serially cultured for six passages, no supTABLE Growth
Subculture No.
1 2 3 4 5 6
of Trypanosoma
porting data on growth were presented. Our synthetic medium F-84 is relatively easy to prepare, and is less complex in composition than Cross an,d Mating’s (1973) medium HX-25 which contains two coenzymes, several fatty acids, and linoleic acid-albumin complex. Our experimental results with semisynthetic medium F-81 indicate that T. CTUZi epimastigotes require no macromolecules for growth and that the fetal bovine serum in F-69 medium (Pan 1978) may be replaced by the addition of augmented amounts of water soluble vitamins. VIII
cruzi (the Brazil Strain) F-84 Medium at 27C
Incubation (days)
Inoculum (X 106)
Increes@ (n-fold)
21 24 29 30 28 28
24.5” 14.6 6.3 5.5 4.6 5.6
4.4 2.6 5.3 5.0 7.3 9.9
in Synthetic
Distribution Epimastigotes
by morphologic Trypomastigotes
93.4 99.8 99.6 100.0 99.9 99.8
a Inoculum contained 92.6% epimastigotes and 7.4yc trypomastigotes. b Calculated incrswe over specified incubation period,
6.6 0.2 0.4 0.0 0.1 0.2
types (70)
Promastigotes 0 0 0 0 0 0
Amastigotes 0 0 0 0 0 0
112
S. CHIA-TUNC
The only undefined nutritional component in F-81 medium is trypticase, a peptone. Trypticase consists of short-chain polypeptides containing 16 amino acids and small amounts of vitamins (BBL 1973). Thus, F-81 may become an important starting point for the development in the future of a highly efficient synthetic medium. One problem encountered in using F-81 was that hemin may precipitate as crystals when the medium becomes acid after a period of good growth of T. cruzi. There was a tendency for the epimastigotes to form rosettes around hemin crystals. Crystallization of hemin was even more pronounced in synthetic medium F-84, perhaps to the point of becoming unavailable. This may be one reason why T. cruxi did not grow in F-84 as well as in F-81. Although epimastigotes could be propagated serially in synthetic medium F-84, the density of organisms at peak growth did not exceed 9.2 x 106/ml of medium, and the incubation period was extended. Additional reasons for the reduced growth rate may be: (1) Trypticase may contain small amounts of an impurity supportive of growth; and (2) short-chain polypeptides may be utilized by the organisms more efficiently in protein synthesis than amino acids. ACKNOWLEDGMENTS These studies were supported in part by the U.S. Army Medical Research and Development Washington, D.C., Contract No. Command, DAMD-17-74-C4064, and by Training Grant AI00177 from the U.S. Public Health Service. REFERENCES ANDERSON, S. J., AND KRASSNER, S. M. 1975. Axenic culture of T~t~panosoma cluzi in a chemically defined medium. Journal of Parasitology 61, 144-145. Baltimore Biological Laboratory. 1973. “BBL
PAN
Manual of Products and Laboratory Procedures,” 5th ed. Baltimore Biological Laboratory, Baltimore, Md. BISHOP, A. 1967. Problems in the cultivation of some parasitic protozoa. Advances in Parasitology 5, 93-138. CITHI, N., AND G~ossow~cz, N. 1955. A partially defined culture medium for Trypanosoma cruzi and some other haemoflagellates. Journal of General Microbiology 13, 273-278. CROSS, G. A. M, AND MANNING, J. C. 1973. Cultivation of Trypunosoma brucei spp. in semidefined and defined media. Parasitology 67, 315-331 FEDERICI, E. E., ABEL~~ANN, W. H., AND NEVA, F. A. 1964. Chronic and progressive myocarditis an d myositis in C,H mice infected with Trypanosoma crud. American Journal of Tropical Medicine and Hygiene 13, 272-280. LIKE, P. A., AND OLESON, J. J. 1951. The cultivation of Trypanosoma crud Journal of Bacteriology 61, 709-714. MORGAN, J. F., MORTON, H. J., AND PARKER, R. C. 1950. Nutrition of animal cells in tissue culture: I, Initial studies on a synthetic medium. PTOceedings of the Society of Experimental Biology and Medicine 73, 1-S. O’DALY, J. A. 1975. .4 new liquid medium for Trypanosoma (Schizotrypanum) cruzi. Journal of Protozoology 22, 265-270. PAN, C. 1968. Cultivation of the leishmaniform stage of Trypanosoma crzczi in cell-free media at different temperatures. American Journal of Tropical Medicine and Hygiene 17, 823-832. PAN, C. 1971. Cultivation and morphogenesis of Trypanosoma cruzi in improved liquid media. Journal of Protozoology 18, 556-560. PAN, S. C. 1976. In oitro cultivation of amastigotes of Trypanosomu cruzi in cell-free media, American Trypanosomiasis Research, Pan American Health Organization Scientific Publication No. 318, pp. 121-126. PAN, S. C. 1978. Trypanosoma cruzi: Intracellular stages grown in a cell-free medium at 37 C. Experimental Parasitology 45, 215-224. TOBIE, E. J. 1964. Cultivation of mammalian trypanosomes. Journal of Protozoology 11, 418423. TRAGER, W. 1957. Nutrition of a hemoflagellate ( Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. Journal of Protozoology 4, 269-276.
46, 108-112 (1978)
PARASITOLOGY
Trypanosoma
cruzi:
Cultivation in Macromolecule-Free and Synthetic Media
Semisynthetic
S. CHIA-TUNG PAN Department
of Tropical
Public Health, Huruard School of Public Health, Boston, Massachusetts 02115, U.S.A.
(Accepted for publication
2 May 1978)
PAN, S. C.-T. 1978. Trypanosoma cruzi: Cultivation in macromolecule-free semisynthetic and synthetic media. Experimental Parasitology 46, 108-112. A macromolecule-free semisynthetic medium (F-81) was devised to culture Trypanosoma cruzi serially at room temperature. F-81 contains only one undefined substance, trypticare, which consists primarily of short-chain polypeptides. In F-81 medium T. cruzi will grow to a density of 35 to 43 x 10’ organisms/ml, a density comparable to that obtainable in a serum-containing medium such as F-69. High concentrations of water-soluble vitamins appear to have a serumreplacing effect in the F-81 medium. A completely synthetic medium (F-84) was prepared by replacing trypticase in F-81 with Trager’s amino acid mixture. T. cruzi epimastigotes could be serially cultured in F-84, with a maximum yield of 9.2 X 10’ organisms/ml of medium after 3 to 4 weeks of incubation at 27 C. INDEX DESCRIPTORS: Tqpanosoma CTUZ~; Hemoflagellate; Protozoa parasitic; Cultivation, in c&o; Medium, semisynthetic and synthetic.
Trypanosoma cruzi, the cause of Chagas’ disease, has been successfully cultured in vitro for nearly 70 years (Bishop 1967; Pan 1976; Tobie 1964). With few exceptions, most culture media contain one or more chemically undefined substances (Anderson and Krassner 1975); and the yield when cultured at room temperature consists of essentially epimastigotes (“culture forms”) (Pan 1968, 1971, 1976). Since “culture” forms of T. crud are readily grown in vitro, it seems reasonable that synthetic or semisynthetic media for these organisms could be prepared more easily and that these media could become a basis for preparing synthetic media for the cultivation of the vertebrate stages. In this study, we describe a macromolecule-free semisynthetic and a synthetic media for serial cultivation of T. crud epimastigotes.
MATERIALS
0014-4894/78/0461-0108$02.00/O 0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
The Brazil strain of Typanosoma cruxi (Pan 1968) and a Colombian strain (Federici et al. 1964) were used. The strains were routinely maintained in NNN medium (Pan 1968) at room temperature by transfer at 4-week intervals. The starting inocula for experiments with semisynthetic media were obtained by inoculating each of several tubes of F-69 medium (Pan 1978) with 1 ml of a l-week-old NNN culture (479th passage for the Brazil strain and 29th passage for Colombian strain), and incubating at 27 C. Cultures in F-69 were harvested 4 to 6 days later, washed three times with phosphate-buffered saline, pH 7.2, containing 0.4% glucose (PBS-G), resuspended in PBS-G, and counted in a hemacytometer.
108 Copyright All rights
AND
TTyf3U?WSO??UZ
TABLE Composition
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
C?UXi:
CULTIVATION
IN
DEFINED
I
of F-81 (Semisynthetic) for Trypanosoma cruzi
Distilled water Medium 199 (10 X concentrate)” Glucose (0.8%) Sodium pyruvate (0.1 M) Trypticase (50/,)b Vitamin solution (see Table III) Biotin (2 mg/lOO ml of HzO) Folic acid (10 mg/lOO ml of HzO) Vitamin B12 (1 mg/lOO ml of H&) Methyl cellulose (4%) Hemin (50 mg/lOO ml of 0.01 N NaOH)” 12. Antibiotic mixture (penicillin G, lo5 U/ml and streptomycin, lo5 pg/ml) 13. ATP, ADP, and AMP solution (see Table IV) 14. NaHC03 (4.2%)
TABLE Medium
20.0 ml 10.0 25.0 2.0 10.0 2.0 2.0 5.0 1.0 5.0 5.0 0.1 10.0 3.0
a Morgan et al. (1950), Medium 199, obtained from Microbiological Associates, Inc., Maryland. * Baltimore Biological Laboratories, Maryland. c Teichmann’s crystals.
The starting inocula for experiments with synthetic media were derived from established cultures in semisynthetic medium ( F-81, see below). Before inoculation, cultures were washed three times with PBS-G. For experiments, cultures in duplicate (screw-cap tubes, 16 x 125 mm, each containing 5 ml of fresh medium) were inoculated with 1 ml of culture from the previous passage and incubated at 27 + 1 C. After incubation, organisms from both tubes were pooled, 1 ml of the pool was used for counting, 2 ml was used for subinoculation, and the remainder was used for preparing duplicate stained smears as described previously (Pan 1968, 1978). Five hundred organisms were counted at 1000X on each of two smears per passage and classified as to morphologic type (for details, see Pan 1968, 1978). The composition of one (F-81) of the two semisynthetic media (Tables I, III, IV) and of one (F-84) of the two synthetic media (Tables II, V, VI) is summarized in
Composition
109
MEDIA
II
of the F-84 (Synthetic) for Trypanosoma‘ cruzi
Medium
1. Trager’s amino acid solution minus tyrosine (see Table V) 2. L-Tyrosine (40 mg/lOO ml) 3. Glucose (0.8%) 4. Medium 199 (10X) 5. Sodium pyruvate (0.1 M) 6. Vitamin solution (see Table III) 7. Biotin (2 mg/lOO ml of HZO) 8. Folic acid (10 mg/lOO ml of HzO) 9. Vitamin B12 (1 mg/lOO ml of HzO) 10. Metal solution (see Table VI) 11. Methyl cellulose (4y0) 12. Hemin (50 mg/lOO ml of 0.01 N NaOH)a 13. L-Glutamine (200 m&f) 14. Antibiotic mixture (penicillin G, lo6 U/ml and streptomycin, 106 rg/ml) 15. ATP, ADP, and AMP solution (see Table IV) 16. NaHC03 (4.2y0) a Teichmann’s
15.0 ml 10.0 25.0 10.0 2.0 2.0 2.0 5.0 1.0 1.0
4.0 4.0 4.0 0.1 10.0 5.0
crystals.
Tables I through VI. F-81 medium is essentially F-69 medium (Pan 1978) minus fetal bovine serum but with a higher concentration of water-soluble vitamins. F-84 medium is essentially F-81 minus trypticase but with a higher concentration of amino acids. All solutions were sterilized by filtration through a Millipore filter (pore size, 0.45 pm) except that trypticase, methyl cellu-
TABLE Composition 1. 2. 3. 4. 5. 6. 7. 8. 9.
III
of Vitamin
p-Aminobenzoic acid n-Calcium pantothenate Choline chloride i-Inositol Nicotinamide Nicotinic acid Pyridoxal-HCl Pyridoxine-HCl Pyridoxaminee2HCl 10. Riboflavin-5-phosphate-Na 11. Thiamine 12. Distilled water
Solution 30 mg 40 30 30 50 20 20 20 20 2 20 100 ml
110
S. CZIIA-TUNG
lose and metallic salt solutions were autoclaved at 15 psi pressure for 15 min.
RESULTS
Attempts to culture Trypanosoma cruzi in semisynthetic and synthetic media at 33, 35, and 37 C were unsuccessful. However, when the temperature of incubation was at 27 C, both strains of T. cruzi could be cultured serially. Table VII summarizes the growth characteristics of T. cruzi (the Brazil strain) in semisynthetic medium F-81 at selected passages. Throughout the experiment (over 63 passages in 13 months), the organisms grew well, and on the average increased eight-fold during an incubation period of 6 to 11 days. During the first six passages, practically all organisms were epimastigotes. Thereafter, relatively large numbers of trypomastigotes (metacyclic types) werk usually present in the culture at the end of exponential growth. The growth characteristics of T. cruzi in F-81 medium are remarkably similar to those observed in experiments with F-69 medium at room temperature (Pan 1978). The organisms (95% epimastigotes) cultured in F-81 (19th passage) can infect human skin-muscle cells in vitro at 35 C. However, culturing of organisms in F-81 appeared to alter their ability to invade skin-muscle cells in that there was a delay of 2 to 3 days when compared with epimastigotes cultured in F-69 medium (containing 10% fetal bovine serum) (Pan 1978 ) .
TABLE The ATP, ADP,
PAN TABLE
Composition without
of Trager’s (1957) Amino Acid Solution Tyrosine for Leischn~ania tar&&e
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
I,-Arginine-HCl L-Histidine L-Isoleucine L-Leurine r,-Lysine HCI L-Methionine L-Phenylalanine L-Threonine L-Tryptophnne L-JTaline r,-Alanine I,-Aspartic acid Glycine L-Proline I,-Serine I,-Glutamic acid Iktilled water
120 mg 60 240 600 500 120 160 200 80 200 280 480 40 200 160 760 100 ml
The results of experiments with synthetic medium F-84 are summarized in Table VIII. The inocula originally derived from the 13th passage in F-81 and contained 92.6% epimastigotes and 7.470 trypomastigotes. Although T. crud could be serially subcultured at 27 C in F-84 for 6 months, it was apparent that (1) the medium was not optimal in terms of growth, (2) the generation time was prolonged, and (3) practically all organisms were epimastigotes after the second passage. However, on examination by light microscopy the epimastigotes showed no morphologic abnormalities. DISCUSSION
Several investigators (Little and Oleson 1951; Citri and Grossowicz 1955; O’Daly
IV
and AMP
TABLE
Sollltion
Composition 1. 2. 3. 4. 5. 6. 7.
V
I-CysteineeHCl Glutathione (reduced) Ascorbic acid ATP ADP AMP Distilled water
5mg 20 5 50 10 10 100 ml
1. 2. 3. 4. 5. 6.
VI
of Metal
ZnS04.7HzO H,BOs CLISO~ MnS04.4HxO CoSOa.7HsO Distilled water
Solution 220 mg 1 3; 4 100 ml
~?Yj~CZ?lO~OWlxZ CTUZi:
CULTIVATION TABLE
.
Subculture No.
1 2 3 4 5 6 12 16
Growth
of Trypanosoma
Incubation Days
9 8 7 6 7 8 10 11
IN DEFINED
VII
cruzi (the Brazil Strain) F-81 Medium at 27C
Inoculum x 106
Increaseb (n-fold)
7.6” 10.3 21.0 26.0 27.1 23.2 22.8 35.2
8.2 12.2 7.4 6.3 5.1 10.6 9.1 7.5
111
MEDIA
in Semisynthetic
Distribution Epimastigotes
by morphologic Trypomastigotes
98.4 99.6 99.4 98.8 98.2 97.6 91.2 91.4
types (%)
Promastigotes
Amastigotes
1.2 0.2 0.2 0 0 0.6 0 0.2
0 0 0 0 0 0.2 0 0
0.4 0.2 0.4 1.2 1.8 1.6 8.8 8.4
Inoculum contained over 99.9% epimastigotes. b Calculated increase over specified incubation period.
1975) have described serial cultivation of Typanosoma c~uzi in semisynthetic media at room temperature. However, their media included at least one undefined blood product containing macromolecules which could not be replaced by simpler substances. Recently, a defined medium was devised for the cultivation of T. brucei at room temperature (Cross and Manning 1973). This medium was subsequently applied to culture a strain of T. cruzi (Anderson and Krassner 1975). Although the authors reported that T. cruzi could be serially cultured for six passages, no supTABLE Growth
Subculture No.
1 2 3 4 5 6
of Trypanosoma
porting data on growth were presented. Our synthetic medium F-84 is relatively easy to prepare, and is less complex in composition than Cross an,d Mating’s (1973) medium HX-25 which contains two coenzymes, several fatty acids, and linoleic acid-albumin complex. Our experimental results with semisynthetic medium F-81 indicate that T. CTUZi epimastigotes require no macromolecules for growth and that the fetal bovine serum in F-69 medium (Pan 1978) may be replaced by the addition of augmented amounts of water soluble vitamins. VIII
cruzi (the Brazil Strain) F-84 Medium at 27C
Incubation (days)
Inoculum (X 106)
Increes@ (n-fold)
21 24 29 30 28 28
24.5” 14.6 6.3 5.5 4.6 5.6
4.4 2.6 5.3 5.0 7.3 9.9
in Synthetic
Distribution Epimastigotes
by morphologic Trypomastigotes
93.4 99.8 99.6 100.0 99.9 99.8
a Inoculum contained 92.6% epimastigotes and 7.4yc trypomastigotes. b Calculated incrswe over specified incubation period,
6.6 0.2 0.4 0.0 0.1 0.2
types (70)
Promastigotes 0 0 0 0 0 0
Amastigotes 0 0 0 0 0 0
112
S. CHIA-TUNC
The only undefined nutritional component in F-81 medium is trypticase, a peptone. Trypticase consists of short-chain polypeptides containing 16 amino acids and small amounts of vitamins (BBL 1973). Thus, F-81 may become an important starting point for the development in the future of a highly efficient synthetic medium. One problem encountered in using F-81 was that hemin may precipitate as crystals when the medium becomes acid after a period of good growth of T. cruzi. There was a tendency for the epimastigotes to form rosettes around hemin crystals. Crystallization of hemin was even more pronounced in synthetic medium F-84, perhaps to the point of becoming unavailable. This may be one reason why T. cruxi did not grow in F-84 as well as in F-81. Although epimastigotes could be propagated serially in synthetic medium F-84, the density of organisms at peak growth did not exceed 9.2 x 106/ml of medium, and the incubation period was extended. Additional reasons for the reduced growth rate may be: (1) Trypticase may contain small amounts of an impurity supportive of growth; and (2) short-chain polypeptides may be utilized by the organisms more efficiently in protein synthesis than amino acids. ACKNOWLEDGMENTS These studies were supported in part by the U.S. Army Medical Research and Development Washington, D.C., Contract No. Command, DAMD-17-74-C4064, and by Training Grant AI00177 from the U.S. Public Health Service. REFERENCES ANDERSON, S. J., AND KRASSNER, S. M. 1975. Axenic culture of T~t~panosoma cluzi in a chemically defined medium. Journal of Parasitology 61, 144-145. Baltimore Biological Laboratory. 1973. “BBL
PAN
Manual of Products and Laboratory Procedures,” 5th ed. Baltimore Biological Laboratory, Baltimore, Md. BISHOP, A. 1967. Problems in the cultivation of some parasitic protozoa. Advances in Parasitology 5, 93-138. CITHI, N., AND G~ossow~cz, N. 1955. A partially defined culture medium for Trypanosoma cruzi and some other haemoflagellates. Journal of General Microbiology 13, 273-278. CROSS, G. A. M, AND MANNING, J. C. 1973. Cultivation of Trypunosoma brucei spp. in semidefined and defined media. Parasitology 67, 315-331 FEDERICI, E. E., ABEL~~ANN, W. H., AND NEVA, F. A. 1964. Chronic and progressive myocarditis an d myositis in C,H mice infected with Trypanosoma crud. American Journal of Tropical Medicine and Hygiene 13, 272-280. LIKE, P. A., AND OLESON, J. J. 1951. The cultivation of Trypanosoma crud Journal of Bacteriology 61, 709-714. MORGAN, J. F., MORTON, H. J., AND PARKER, R. C. 1950. Nutrition of animal cells in tissue culture: I, Initial studies on a synthetic medium. PTOceedings of the Society of Experimental Biology and Medicine 73, 1-S. O’DALY, J. A. 1975. .4 new liquid medium for Trypanosoma (Schizotrypanum) cruzi. Journal of Protozoology 22, 265-270. PAN, C. 1968. Cultivation of the leishmaniform stage of Trypanosoma crzczi in cell-free media at different temperatures. American Journal of Tropical Medicine and Hygiene 17, 823-832. PAN, C. 1971. Cultivation and morphogenesis of Trypanosoma cruzi in improved liquid media. Journal of Protozoology 18, 556-560. PAN, S. C. 1976. In oitro cultivation of amastigotes of Trypanosomu cruzi in cell-free media, American Trypanosomiasis Research, Pan American Health Organization Scientific Publication No. 318, pp. 121-126. PAN, S. C. 1978. Trypanosoma cruzi: Intracellular stages grown in a cell-free medium at 37 C. Experimental Parasitology 45, 215-224. TOBIE, E. J. 1964. Cultivation of mammalian trypanosomes. Journal of Protozoology 11, 418423. TRAGER, W. 1957. Nutrition of a hemoflagellate ( Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. Journal of Protozoology 4, 269-276.
