Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Mar;61(3):1124–1132. doi: 10.1128/aem.61.3.1124-1132.1995

Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry

S V Avery, J L Harwood, D Lloyd
PMCID: PMC1388394  PMID: 16534962

Abstract

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.

Full Text

The Full Text of this article is available as a PDF (358.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alexander M. Why microbial predators and parasites do not eliminate their prey and hosts. Annu Rev Microbiol. 1981;35:113–133. doi: 10.1146/annurev.mi.35.100181.000553. [DOI] [PubMed] [Google Scholar]
  2. Allen P. G., Dawidowicz E. A. Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast. J Cell Physiol. 1990 Dec;145(3):508–513. doi: 10.1002/jcp.1041450317. [DOI] [PubMed] [Google Scholar]
  3. Allen P. G., Dawidowicz E. A. Phagocytosis in Acanthamoeba: II. Soluble and insoluble mannose-rich ligands stimulate phosphoinositide metabolism. J Cell Physiol. 1990 Dec;145(3):514–521. doi: 10.1002/jcp.1041450318. [DOI] [PubMed] [Google Scholar]
  4. Bloem J., Ellenbroek F. M., Bär-Gilissen M. J., Cappenberg T. E. Protozoan grazing and bacterial production in stratified lake vechten estimated with fluorescently labeled bacteria and by thymidine incorporation. Appl Environ Microbiol. 1989 Jul;55(7):1787–1795. doi: 10.1128/aem.55.7.1787-1795.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bottone E. J., Madayag R. M., Qureshi M. N. Acanthamoeba keratitis: synergy between amebic and bacterial cocontaminants in contact lens care systems as a prelude to infection. J Clin Microbiol. 1992 Sep;30(9):2447–2450. doi: 10.1128/jcm.30.9.2447-2450.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bottone E. J., Perez A. A., Gordon R. E., Qureshi M. N. Differential binding capacity and internalisation of bacterial substrates as factors in growth rate of Acanthamoeba spp. J Med Microbiol. 1994 Feb;40(2):148–154. doi: 10.1099/00222615-40-2-148. [DOI] [PubMed] [Google Scholar]
  7. Bowers B. Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii. Exp Cell Res. 1977 Dec;110(2):409–417. doi: 10.1016/0014-4827(77)90307-x. [DOI] [PubMed] [Google Scholar]
  8. Bowers B., Olszewski T. E. Acanthamoeba discriminates internally between digestible and indigestible particles. J Cell Biol. 1983 Aug;97(2):317–322. doi: 10.1083/jcb.97.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Byers T. J., Kim B. G., King L. E., Hugo E. R. Molecular aspects of the cell cycle and encystment of Acanthamoeba. Rev Infect Dis. 1991 Mar-Apr;13 (Suppl 5):S373–S384. doi: 10.1093/clind/13.supplement_5.s373. [DOI] [PubMed] [Google Scholar]
  10. Cucci T. L., Shumway S. E., Brown W. S., Newell C. R. Using phytoplankton and flow cytometry to analyze grazing by marine organisms. Cytometry. 1989 Sep;10(5):659–669. doi: 10.1002/cyto.990100523. [DOI] [PubMed] [Google Scholar]
  11. Davies P., Fox R. I., Polyzonis M., Allison A. C., Haswell A. D. The inhibition of phagocytosis and facilitation of exocytosis in rabbit polymorphonuclear leukocytes by cytochalasin B. Lab Invest. 1973 Jan;28(1):16–22. [PubMed] [Google Scholar]
  12. Drozanski W., Chmielewski T. Electron microscopic studies of Acanthamoeba castellanii infected with obligate intracellular bacterial parasite. Acta Microbiol Pol. 1979;28(2):123–133. [PubMed] [Google Scholar]
  13. Drozański W. Activity and distribution of bacteriolytic N-acetyl-muramidase during growth of Acanthamoeba castellanii in axenic culture. Acta Microbiol Pol. 1978;27(3):243–256. [PubMed] [Google Scholar]
  14. Fritsche T. R., Gautom R. K., Seyedirashti S., Bergeron D. L., Lindquist T. D. Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J Clin Microbiol. 1993 May;31(5):1122–1126. doi: 10.1128/jcm.31.5.1122-1126.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hed J., Hallden G., Johansson S. G., Larsson P. The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods. 1987 Jul 16;101(1):119–125. doi: 10.1016/0022-1759(87)90224-9. [DOI] [PubMed] [Google Scholar]
  16. Hohman T. C., Bowers B. Hydrolase compartmentalization limits rate of digestion in Acanthamoeba. J Eukaryot Microbiol. 1993 Sep-Oct;40(5):589–593. doi: 10.1111/j.1550-7408.1993.tb06112.x. [DOI] [PubMed] [Google Scholar]
  17. Hook G. R., Odeyale C. O. Confocal scanning fluorescence microscopy: a new method for phagocytosis research. J Leukoc Biol. 1989 Apr;45(4):277–282. doi: 10.1002/jlb.45.4.277. [DOI] [PubMed] [Google Scholar]
  18. King C. H., Shotts E. B., Jr, Wooley R. E., Porter K. G. Survival of coliforms and bacterial pathogens within protozoa during chlorination. Appl Environ Microbiol. 1988 Dec;54(12):3023–3033. doi: 10.1128/aem.54.12.3023-3033.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Korn E. D., Wright P. L. Macromolecular composition of an amoeba plasma membrane. J Biol Chem. 1973 Jan 25;248(2):439–447. [PubMed] [Google Scholar]
  20. Lloyd D. Biochemistry of the cell cycle. Biochem J. 1987 Mar 1;242(2):313–321. doi: 10.1042/bj2420313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moffat J. F., Tompkins L. S. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infect Immun. 1992 Jan;60(1):296–301. doi: 10.1128/iai.60.1.296-301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rabinovitch M., De Stefano M. J. Phagocytosis of erythrocytes by Acanthamoeba sp. Exp Cell Res. 1971 Feb;64(2):275–284. doi: 10.1016/0014-4827(71)90077-2. [DOI] [PubMed] [Google Scholar]
  23. Ryter A., Bowers B. Localization of acid phosphatase in Acanthamoeba castellanii with light and electron microscopy during growth and after phagocytosis. J Ultrastruct Res. 1976 Dec;57(3):309–321. doi: 10.1016/s0022-5320(76)80119-0. [DOI] [PubMed] [Google Scholar]
  24. Sahlin S., Hed J., Rundquist I. Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J Immunol Methods. 1983 May 27;60(1-2):115–124. doi: 10.1016/0022-1759(83)90340-x. [DOI] [PubMed] [Google Scholar]
  25. Sherr B. F., Sherr E. B., Rassoulzadegan F. Rates of digestion of bacteria by marine phagotrophic protozoa: temperature dependence. Appl Environ Microbiol. 1988 May;54(5):1091–1095. doi: 10.1128/aem.54.5.1091-1095.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  27. Steinkamp J. A., Wilson J. S., Saunders G. C., Stewart C. C. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science. 1982 Jan 1;215(4528):64–66. doi: 10.1126/science.7053559. [DOI] [PubMed] [Google Scholar]
  28. Ulsamer A. G., Wright P. L., Wetzel M. G., Korn E. D. Plasma and phagosome membranes of Acanthamoeba castellanii. J Cell Biol. 1971 Oct;51(1):193–215. doi: 10.1083/jcb.51.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wan C. P., Park C. S., Lau B. H. A rapid and simple microfluorometric phagocytosis assay. J Immunol Methods. 1993 Jun 4;162(1):1–7. doi: 10.1016/0022-1759(93)90400-2. [DOI] [PubMed] [Google Scholar]
  30. Weekers P. H., Bodelier P. L., Wijen J. P., Vogels G. D. Effects of Grazing by the Free-Living Soil Amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis on Various Bacteria. Appl Environ Microbiol. 1993 Jul;59(7):2317–2319. doi: 10.1128/aem.59.7.2317-2319.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weisman R. A., Korn E. D. Phagocytosis of latex beads by Acanthamoeba. I. Biochemical properties. Biochemistry. 1967 Feb;6(2):485–497. doi: 10.1021/bi00854a017. [DOI] [PubMed] [Google Scholar]
  32. Wetzel M. G., Korn E. D. Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes. J Cell Biol. 1969 Oct;43(1):90–104. doi: 10.1083/jcb.43.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES