Parasitology Research

, Volume 108, Issue 2, pp 425–430 | Cite as

Comparison of protein-free defined media, and effect of l-cysteine and ascorbic acid supplementation on viability of axenic Entamoeba histolytica

  • Weng Kin Wong
  • Zi Ning Tan
  • Boon Huat Lim
  • Zeehaida Mohamed
  • Alfonso Olivos-Garcia
  • Rahmah NoordinEmail author
Original Paper


Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory–secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank’s balanced salt solution (HBSS). Concurrently, the effect of adding l-cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P > 0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only onefold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.


Entamoeba Histolytica Trypan Blue Exclusion Method Amoebic Liver Abscess Amoeboid Movement Parasite Viability 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This study was supported by Universiti Sains Malaysia Research University grant, no. 1001/PPSK/813009, FRGS grant, no. 203/CIPPM/6711122, and USM-RU-PRGS no. 1001/INFORMM/8032030. The first and second authors received financial support from USM Fellowship program.


  1. Akgun Y, Tacyildiz IH, Celik Y (1999) Amebic liver abscess: changing trends over 20 years. World J Surg 23:102–106CrossRefPubMedGoogle Scholar
  2. Band RN, Cirrito H (1979) Growth response of axenic Entamoeba histolytica to hydrogen, carbon dioxide, and oxygen. J Protozool 26:282–286PubMedGoogle Scholar
  3. Debnath A, Akbar MA, Mazumder A, Kumar S, Das P (2005) Entamoeba histolytica: characterization of human collagen type I and Ca2+ activated differentially expressed genes. Exp Parasitol 110:214–219CrossRefPubMedGoogle Scholar
  4. Diamond L (1961) Axenic cultivation of Entamoeba histolytica. Science 134: 336–337Google Scholar
  5. Dutta GP (1981) Experimental and clinical studies on amoebiasis. McGraw-Hill, New DelhiGoogle Scholar
  6. Gillin FD, Diamond LS (1980a) Attachment and short-term maintenance of motility and viability of Entamoeba histolytica in a defined medium. J Protozool 27:220–225PubMedGoogle Scholar
  7. Gillin FD, Diamond LS (1980b) Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid. J Protozool 27:474–478PubMedGoogle Scholar
  8. Gitler C, Calef E, Rosenberg I (1984) Cytopathogenicity of Entamoeba histolytica. Philos Trans R Soc Lond B Biol Sci 307:73–85CrossRefPubMedGoogle Scholar
  9. Guerrero-Manriquez GG, Sanchez-Ibarra F, Avila EE (1998) Inhibition of Entamoeba histolytica proteolytic activity by human salivary IgA antibodies. APMIS 106:1088–1094CrossRefPubMedGoogle Scholar
  10. Gupta S, Naik S, Naik SR (1999) Vaccine potential of 56–66 kDa protease secreted by Entamoeba histolytica. Indian J Med Res 109:141–146PubMedGoogle Scholar
  11. Guy RA, Bertrand S, Faubert GM (1991) Modification of RPMI 1640 for use in vitro immunological studies of host-parasite interactions in giardiasis. J Clin Microbiol 29:627–629PubMedGoogle Scholar
  12. Jimenez JC, Fontaine J, Grzych JM, Dei-Cas E, Capron M (2004) Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis. Clin Diagn Lab Immunol 11:152–160PubMedGoogle Scholar
  13. Martinez-Palomo A (1982) The biology of Entamoeba histolytica. Research Studies Press, ChichesterGoogle Scholar
  14. Moncada D, Keller K, Chadee K (2005) Entamoeba histolytica-secreted products degrade colonic mucin oligosaccharides. Infect Immun 73:3790–3793CrossRefPubMedGoogle Scholar
  15. Pal S, Sengupta K, Manna B, Sarkar S, Bhattacharya S, Das P (1996) Comparative evaluation of somatic & excretory-secretory antigens of Entamoeba histolytica in serodiagnosis of human amoebiasis by ELISA. Indian J Med Res 104:152–156PubMedGoogle Scholar
  16. Petri WA Jr, Singh U (1999) Diagnosis and management of amebiasis. Clin Infect Dis 29:1117–1125CrossRefPubMedGoogle Scholar
  17. Que X, Reed SL (2000) Cysteine proteinases and the pathogenesis of amebiasis. Clin Microbiol Rev 13:196–206CrossRefPubMedGoogle Scholar
  18. Reed SL, Keene WE, McKerrow JH (1989) Thiol proteinase expression and pathogenicity of Entamoeba histolytica. J Clin Microbiol 27:2772–2777PubMedGoogle Scholar
  19. Sen A, Chatterjee NS, Akbar MA, Nandi N, Das P (2007) The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress. Eukaryot Cell 6:664–673CrossRefPubMedGoogle Scholar
  20. Sengupta S, Akbar A, Mukhopadhyay P, Ganguly S, Sen P, Das P (2000) Role of excretory-secretory products of Entamoeba histolytica in human amebiasis. Arch Med Res 31(4 Suppl):S226–S228CrossRefPubMedGoogle Scholar
  21. Tekwani BL, Mehlotra RK (1999) Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia. Microbes Infect 1:385–394CrossRefPubMedGoogle Scholar
  22. Wells CD, Arguedas M (2004) Amebic liver abscess. S Med J 97:673–682CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Weng Kin Wong
    • 1
  • Zi Ning Tan
    • 3
  • Boon Huat Lim
    • 3
  • Zeehaida Mohamed
    • 4
  • Alfonso Olivos-Garcia
    • 5
  • Rahmah Noordin
    • 2
    Email author
  1. 1.Institute for Research in Molecular Medicine (INFORMM)Universiti Sains MalaysiaKubang KerianMalaysia
  2. 2.Institute for Research in Molecular Medicine (INFORMM)Universiti Sains MalaysiaPenangMalaysia
  3. 3.School of Health SciencesUniversiti Sains MalaysiaKubang KerianMalaysia
  4. 4.Department of Medical Microbiology and Parasitology, School of Medical SciencesUniversiti Sains MalaysiaKubang KerianMalaysia
  5. 5.Departamento de Medicina Experimental, Facultad de MedicinaUniversidad Nacional Autónoma de MéxicoMéxicoMexico

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