Bogdan I. Gerashchenko

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria

BACKGROUNDnThe stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena.nnnMETHODSnFlow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host.nnnRESULTSnNeither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae.nnnCONCLUSIONSnFlow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements

BACKGROUNDnThe ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle.nnnMETHODSnAsynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks.nnnRESULTSnCell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively.nnnCONCLUSIONSnGrowth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.

Life Cycle Analysis of Unicellular Algae

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.

Flow Cytometry‐Based Quantification of Cell Proliferation in the Mixed Cell Co‐Culture

In cell communities, among the crucial signals that govern cell function are those generated locally by surrounding cells. A co‐culture of mixed homotypic or heterotypic cells, which is often used in various fields of experimental biology and medicine, can be applied for elucidation of the role of cell proximity in modulating proliferative responses. Quick and reliable quantification of the changes in proliferation of each of the mixed cell populations as a result of their co‐culture is of importance. For this purpose, flow cytometry together with fluorescent tracers that do not affect cell proliferation can be used. Curr. Protoc. Cytom. 63:9.40.1‐9.40.10.