Life Cycle Analysis of Unicellular Algae

Bogdan I. Gerashchenko1, Toshiyuki Takahashi2, Toshikazu Kosaka3, Hiroshi Hosoya3

1 R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv, Ukraine, 2 Miyakonojo National College of Technology, Miyakonojo, Japan, 3 Hiroshima University, Hiroshima, Japan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.19
DOI:  10.1002/0471142956.cy1119s52
Online Posting Date:  April, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Unicellular green alga is a very convenient object for flow cytometric characterization. Flow cytometry has been proposed as a quick and reliable tool for studying life cycle and growth of unicellular algae. Cell size of vegetating algae can be monitored in association with their DNA and endogenous chlorophyll content. Cells of interest (e.g., group of cells of a certain stage of the life cycle) in an asynchronously proliferating cell population can be sorted out for further microscopical or molecular biology studies. This methodological approach can be helpful for researchers who are interested in algal proliferation. Curr. Protoc. Cytom. 52:11.19.1-11.19.6. © 2010 by John Wiley & Sons, Inc.

Keywords: unicellular algae; Chlorella; light scatter; DNA content; chlorophyll content; life cycle; flow cytometry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

 Basic Protocol
 Materials
  • Unicellular algae (e.g., Chlorella sp.)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Ethanol (³99.5% purity)
  • DNA staining solution (see recipe)
  • Formaldehyde (methanol free)
  • DAPI
  • Culture cabinet (thermostated at 24°C) with a shaker inside generating 180 reciprocal movements per min and with a light source producing the light of ~2000 lux
  • 5-ml centrifuge tubes
  • Hemacytometer
  • Centrifuge for concentration of algal suspensions and for washing steps
  • Refrigerator for incubation at 4°C
  • 37°C incubator
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer/cell sorter equipped with an argon-ion laser (488 nm)
  • Software for analyzing data obtained by flow cytometry (e.g., CELLQuest software from Becton-Dickinson Immunocytometry Systems)
  • Microscopes (bright field and fluorescence)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 11.19.1 Analysis of asynchronous culture of exsymbiotic algae (SA-1) in stationary growth phase using two-parameter FSC-H vs. SSC-H (A), FSC-H vs. FL2-H (DNA-bound PI fluorescence) (B), and FSC-H vs. FL3-H (chlorophyll fluorescence) (C) plots. The algae analyzed with FSC-H vs. FL3-H (chlorophyll fluorescence) plot (C) were fixed with formaldehyde (sample of aliquot #2), while the algae analyzed with FSC-H vs. SSC-H (A), and FSC-H vs. FL2-H (DNA-bound PI fluorescence) (B) plots were fixed with ethanol (sample of aliquot #1). DNA content histogram (D) shows the presence of two minor hyperhaploid peaks 2C and 4C that correspond to ripening/ripened algae. Photomicrographs (E) represent Nomarski differential interference contrast (a, c) and fluorescence microscopy (b, d) images of algae with two (a, b) and four (c, d) nuclei identified in the fraction of sorted cells [regions R1 and R2 in the FSC-H vs. FL2-H plot (B)]. Scale bar = 8 µm. The life cycle of algae is schematically illustrated in FSC-H vs. SSC-H (A) and FSC-H vs. FL3-H (chlorophyll fluorescence) (C) plots. Abbreviations: Dv, stage of “division” or “autospore liberation;” Gr, stage of “growth;” Rp, stage of “ripening.” Modified from Gerashchenko et al., 2001.
    View Image
  •  FigureFigure 11.19.2 Analysis of asynchronous culture of Chlorella vulgaris (c-27) of the late logarithmic growth phase using two-parameter FSC-H vs. SSC-H (A) and FSC-H vs. FL3-H plots (chlorophyll fluorescence) (B) plots. The algae analyzed with these plots were not fixed with any fixatives. The percentages of cells that constitute each of the optically compartmentalized cell subpopulations are shown in the plots. The life cycle of algae is schematically illustrated in contour FSC-H vs. SSC-H (C) and FSC-H vs. FL3-H (chlorophyll fluorescence) plots (D). Abbreviations: Dv, stage of “division” or “autospore liberation;” Gr, stage of “growth;” Rp, stage of “ripening.” Modified from Gerashchenko et al., 2002.
    View Image

Videos

Literature Cited

Literature Cited
    Gerashchenko, B.I., Nishihara, N., Ohara, T., Tosuji, H., Kosaka, T., and Hosoya, H. 2000. Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria. Cytometry 41:209-215.
    Gerashchenko, B.I., Kosaka, T., and Hosoya, H. 2001. Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements. Cytometry 44:257-263.
    Gerashchenko, B.I., Kosaka, T., and Hosoya, H. 2002. Optical compartmentation of vegetating algae species as a basis for their growth-specific characterization. Cytometry 48:153-158.
    Kadono, T., Kawano, T., Hosoya, H., and Kosaka, T. 2004. Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium. Protoplasma 223:133-141.
    McAuley, P.J. and Muscatine, L. 1986. The cell cycle of symbiotic Chlorella. IV. DNA content of algae slowly increases during host starvation of green hydra. J. Cell Sci. 85:73-84.
    Nishihara, N., Horiike, S., Takahashi, T., Kosaka, T., Shigenaka, Y., and Hosoya, H. 1998. Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria. Protoplasma 203:91-99.
    Takahashi, T., Shirai, Y., Kosaka, T., and Hosoya, H. 2007. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia. PLoS ONE 12:e1352.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library