- Email: [email protected]
A modification of Diamond's medium for the axenic culture of Entamoeba histolytica
746
TRANSACTIONS OFTHEROYALSOCIETY OFTROEWAL
A modification histolytica C. J. Marinkelle,
of Diamond’s
MEDICINE
medium
Apartado
Akreo
4976,
Bogotci,
D. E.,
Abstract The use of casein hydrolysate in Diamond’s axenic culture medium TPS-1 in replacement of trypticase allowed good growth of the trophozoites of Entamoeba histolytica. This modified medium also supported growth of trophozoites preserved for 16 months in liquid nitrogen. Considerable labour and cost of serum can be saved by using 5% instead of 10% bovine serum in combination with this modified medium. , Introduction Until 1980, trypticase (BBL) was used in most parts of the world m media for axenic growth of Entamoeba histolytica. Recently this substance has not supported good growth and most investigators have replaced it with casein digest peptone (BBL). However, production of casein digest peptone was unfortunately stopped in 1987. The objective of the present investigation was, therefore, to find a medium which allowed good growth of E. histolytica and also permitted recovery of growth after long-term preservation in liquid nitrogen. Materials and Methods E. histolytica strain HM-1 (IMSS) was grown and subcultured in Nunc 12 ml flat-bottomed screwcanned tubes bv standard nrocedures (DIAMOND. 1968).
-
Growth in the following 6 different media was CornDared. Medium I: modified TPS-1 (DIAMOND. 198d), in which trypticase (BBL, 11921) Gas replaced by the now-discontinued casein digest peptone (BBL, 97023). Medium II: TPS-1 without modification, using a 20 years old batch of the original trypticase. Medium III: TPS-1 in which trypticase had been replaced by Bacto-Casitone@ (Difco, 025901). Medium IV: TPS-1 with casein hydrolysate (Sigma, type III, C-1026) replacing trypticase. Medium V: PEHPS medium (SAID-FERNANDEZ et al., 1988)
(1991)
85, 746-747
for the axenic culture of Entamoeba
F. Guhl, A. Aguirre and F. Devia
y Parasitologia,
1$6i,
AND HYGIENE
Universidad Colombia
de 10sAndes, Laboratorio
de Microbiologia
without casein peptone and with fresh liver and pancreas extracts of swine and ox replacing [email protected] VI: medium V plus 1 g of casein hydrolysate per 100 ml of medium. All media contained 5% or 10% of locally obtained adult bovine serum (ABS). Growth of the amoebas was monitored according to the methods recommended by L&E&REVILLA & RODRIGUEZ BAEZ (1981). Subcultures were initiated with an inoculum of 0.1 ml of each medium containing 1250 to 1500 trophozoites. The recovery and growth rates in the 6 media were recorded after cryopreservation of 150 000 trophozoites per vial. The procedures of cryopreservation and thawing were those described by FARRI et al. (1983). The cultures were placed at 36°C immediately after thawing and first examined 24 h later, becausethis had been found to increase the recovery rate. Counts of trophozoites were made at 24 h intervals until a monolayer was observed, when the content of the tube was subcultured. To calculate the numbers of trophozoites, the tubes were chilled for 25 min in ice-water and then centrifuged. The sediment was diluted in 10 ml of saline glucose solution at pH 7.2 and counts were made in a Neubauer cell counter. For less accurate daily routine estimates of the number of trophozoites, counts were made per microscope field directly under an inverted microscope. (x 160). Results A complete monolayer on the flat side of the tubes corresponded to about 310-440 trophozoites per microscope field., approximately 3.5 x IO5trophozoites per ml of medmm. Figs 1 and 2 represent the average values of 10 observations at different time intervals of the numbers of amoebae. Growth in media I, II, IV, V and VI was satisfactory and rather similar when 10% ABS was B
A
P
Fig. 1. Entamoeba (X) and VI (0).
A
histo&ica: average numbers of trophozoites per microscope field (X 160) in six different media: I (O), II (+), A, media containing 10% adult bovine serum; B, media containing 5% adult bovine serum.
III (*), IV (Cl), V
747 II
Fig. 2. Entamoeba histolytica: averagenumbers of trophozoites per microscopefield (X 160)in medium IV plus 10% adult bovine serum after two (*), eight (+) and sixteen (0) months cryopreservation.
used, although growth in medium V was slower. Medium III with 10% ABS showed a small increase of trophozoites over the first 72 h, but afterwards the cells died off slowly. Continuous growth of amoebae in media III, V and VI was not possible when 5% ABS was used. Trophozoites have now been grown for more than one year using medium IV with both 5% and 10% ABS. Recovery of trophozoites after maintenance for 2, 8 or 16 months in liauid nitrogen was alwavs nossible when medium IV with 10% ABS was used. Cultures preserved for 2 months produced a monolayer much faster than those frozen for 8 or 16 months (Fig. 2). .
1
Although amoebae could be grown in medium VI with 10% ABS. extraction of the organs needed for its preparation is’ cumbersome and acquisition of pig pancreas is difficult. However, the experiment demonstrated the stimulating effect of casein hydrolysate, if the results are compared with growth in medium V. Medium IV had the great advantage that, with 5% ABS, the amoebae grew well but more slowly and therefore needed less frequent subcultures. The requirement for labour and expensive serum can thus be-reduced by half. When rapid production of large numbers of trophozoites is required, 10% ABS can be added to the medium. The layer of trophozoites
cultured in medium IV was collected after 48 h growth since the rapid multiplication did not allow them to be maintained for another day without rounding up or sloughing off. This explains why the total vield per field averaged 320 instead of 420 (Fig. 1A). -We did not succeedin maintaining trophozoiie cultures in media I or IV when imnorted ABS (Flow Laboratories) was used at 5%. In-contrast to media III, V and VI, medium IV with 10% ABS was suitable for recovery of cultures which had been preserved in liquid nitrogen. Since both the casein digest peptone (BBL, 97023) and the amoeba growth-supporting type of tjpticase (BBL. 11921) are no longer nroduced. we recommend the use of casein hydroly&e (Sigma, C-1026). Acknowledgements
We thank Dr D. C. Warhurst(London Schoolof Hvaiene
and Tropical Medicine) for the trophozoite culture and his continuous encouragement. Part of the investigation was supported by CEC grant TSD 00711597. References
Diamond, L. S. (1961). Axenic cultivation of Entamoeba histolytica. Science, 134, 336337. Diamond, L. S. (1968). Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica like amoebae. Journal of Parasitology? 54, 1047-1056. Diamond, L. S. (1980). Axenic cultivatton of Entamoeba histolytica: progress and problems. Archives de Investigacidnes Mkdicas (Mhico), 11, supplement 47-54. Farri, T. A., Warhurst, D. C. & Marshall, T. F. de C. (1983). The use of infectivity titrations for measurement of the viability of Entamoeba histolytica after cryopreservation. Transactions of the Royal Society of Tropical Medicine
and Hygiene,
Hygiene,
82, 249-253.
Lopez-Revilla,
77, 259-266.
R. & Rodriguez-Baez, J. (1981). Manual para el Cultivo Axkico de Entamoeba histolytica. MCxice: Ciencia y Desarrollo, CONACYT,pp. 21-27. Said-Fernandez, S., Vargas, V. J., Castro, G. J., Mata, C. B. D., Navarro, M. L., Lozano, G. G. & Martinez, R. H. (1988). PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens. Transactions of the Royal Society of Tropical Medicine and Received 18 February March 1991
1991; accepted for publication
12
TRANSACTIONS OFTHEROYALSOCIETY OFTROEWAL
A modification histolytica C. J. Marinkelle,
of Diamond’s
MEDICINE
medium
Apartado
Akreo
4976,
Bogotci,
D. E.,
Abstract The use of casein hydrolysate in Diamond’s axenic culture medium TPS-1 in replacement of trypticase allowed good growth of the trophozoites of Entamoeba histolytica. This modified medium also supported growth of trophozoites preserved for 16 months in liquid nitrogen. Considerable labour and cost of serum can be saved by using 5% instead of 10% bovine serum in combination with this modified medium. , Introduction Until 1980, trypticase (BBL) was used in most parts of the world m media for axenic growth of Entamoeba histolytica. Recently this substance has not supported good growth and most investigators have replaced it with casein digest peptone (BBL). However, production of casein digest peptone was unfortunately stopped in 1987. The objective of the present investigation was, therefore, to find a medium which allowed good growth of E. histolytica and also permitted recovery of growth after long-term preservation in liquid nitrogen. Materials and Methods E. histolytica strain HM-1 (IMSS) was grown and subcultured in Nunc 12 ml flat-bottomed screwcanned tubes bv standard nrocedures (DIAMOND. 1968).
-
Growth in the following 6 different media was CornDared. Medium I: modified TPS-1 (DIAMOND. 198d), in which trypticase (BBL, 11921) Gas replaced by the now-discontinued casein digest peptone (BBL, 97023). Medium II: TPS-1 without modification, using a 20 years old batch of the original trypticase. Medium III: TPS-1 in which trypticase had been replaced by Bacto-Casitone@ (Difco, 025901). Medium IV: TPS-1 with casein hydrolysate (Sigma, type III, C-1026) replacing trypticase. Medium V: PEHPS medium (SAID-FERNANDEZ et al., 1988)
(1991)
85, 746-747
for the axenic culture of Entamoeba
F. Guhl, A. Aguirre and F. Devia
y Parasitologia,
1$6i,
AND HYGIENE
Universidad Colombia
de 10sAndes, Laboratorio
de Microbiologia
without casein peptone and with fresh liver and pancreas extracts of swine and ox replacing [email protected] VI: medium V plus 1 g of casein hydrolysate per 100 ml of medium. All media contained 5% or 10% of locally obtained adult bovine serum (ABS). Growth of the amoebas was monitored according to the methods recommended by L&E&REVILLA & RODRIGUEZ BAEZ (1981). Subcultures were initiated with an inoculum of 0.1 ml of each medium containing 1250 to 1500 trophozoites. The recovery and growth rates in the 6 media were recorded after cryopreservation of 150 000 trophozoites per vial. The procedures of cryopreservation and thawing were those described by FARRI et al. (1983). The cultures were placed at 36°C immediately after thawing and first examined 24 h later, becausethis had been found to increase the recovery rate. Counts of trophozoites were made at 24 h intervals until a monolayer was observed, when the content of the tube was subcultured. To calculate the numbers of trophozoites, the tubes were chilled for 25 min in ice-water and then centrifuged. The sediment was diluted in 10 ml of saline glucose solution at pH 7.2 and counts were made in a Neubauer cell counter. For less accurate daily routine estimates of the number of trophozoites, counts were made per microscope field directly under an inverted microscope. (x 160). Results A complete monolayer on the flat side of the tubes corresponded to about 310-440 trophozoites per microscope field., approximately 3.5 x IO5trophozoites per ml of medmm. Figs 1 and 2 represent the average values of 10 observations at different time intervals of the numbers of amoebae. Growth in media I, II, IV, V and VI was satisfactory and rather similar when 10% ABS was B
A
P
Fig. 1. Entamoeba (X) and VI (0).
A
histo&ica: average numbers of trophozoites per microscope field (X 160) in six different media: I (O), II (+), A, media containing 10% adult bovine serum; B, media containing 5% adult bovine serum.
III (*), IV (Cl), V
747 II
Fig. 2. Entamoeba histolytica: averagenumbers of trophozoites per microscopefield (X 160)in medium IV plus 10% adult bovine serum after two (*), eight (+) and sixteen (0) months cryopreservation.
used, although growth in medium V was slower. Medium III with 10% ABS showed a small increase of trophozoites over the first 72 h, but afterwards the cells died off slowly. Continuous growth of amoebae in media III, V and VI was not possible when 5% ABS was used. Trophozoites have now been grown for more than one year using medium IV with both 5% and 10% ABS. Recovery of trophozoites after maintenance for 2, 8 or 16 months in liauid nitrogen was alwavs nossible when medium IV with 10% ABS was used. Cultures preserved for 2 months produced a monolayer much faster than those frozen for 8 or 16 months (Fig. 2). .
1
Although amoebae could be grown in medium VI with 10% ABS. extraction of the organs needed for its preparation is’ cumbersome and acquisition of pig pancreas is difficult. However, the experiment demonstrated the stimulating effect of casein hydrolysate, if the results are compared with growth in medium V. Medium IV had the great advantage that, with 5% ABS, the amoebae grew well but more slowly and therefore needed less frequent subcultures. The requirement for labour and expensive serum can thus be-reduced by half. When rapid production of large numbers of trophozoites is required, 10% ABS can be added to the medium. The layer of trophozoites
cultured in medium IV was collected after 48 h growth since the rapid multiplication did not allow them to be maintained for another day without rounding up or sloughing off. This explains why the total vield per field averaged 320 instead of 420 (Fig. 1A). -We did not succeedin maintaining trophozoiie cultures in media I or IV when imnorted ABS (Flow Laboratories) was used at 5%. In-contrast to media III, V and VI, medium IV with 10% ABS was suitable for recovery of cultures which had been preserved in liquid nitrogen. Since both the casein digest peptone (BBL, 97023) and the amoeba growth-supporting type of tjpticase (BBL. 11921) are no longer nroduced. we recommend the use of casein hydroly&e (Sigma, C-1026). Acknowledgements
We thank Dr D. C. Warhurst(London Schoolof Hvaiene
and Tropical Medicine) for the trophozoite culture and his continuous encouragement. Part of the investigation was supported by CEC grant TSD 00711597. References
Diamond, L. S. (1961). Axenic cultivation of Entamoeba histolytica. Science, 134, 336337. Diamond, L. S. (1968). Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica like amoebae. Journal of Parasitology? 54, 1047-1056. Diamond, L. S. (1980). Axenic cultivatton of Entamoeba histolytica: progress and problems. Archives de Investigacidnes Mkdicas (Mhico), 11, supplement 47-54. Farri, T. A., Warhurst, D. C. & Marshall, T. F. de C. (1983). The use of infectivity titrations for measurement of the viability of Entamoeba histolytica after cryopreservation. Transactions of the Royal Society of Tropical Medicine
and Hygiene,
Hygiene,
82, 249-253.
Lopez-Revilla,
77, 259-266.
R. & Rodriguez-Baez, J. (1981). Manual para el Cultivo Axkico de Entamoeba histolytica. MCxice: Ciencia y Desarrollo, CONACYT,pp. 21-27. Said-Fernandez, S., Vargas, V. J., Castro, G. J., Mata, C. B. D., Navarro, M. L., Lozano, G. G. & Martinez, R. H. (1988). PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens. Transactions of the Royal Society of Tropical Medicine and Received 18 February March 1991
1991; accepted for publication
12