February, 1994

ABSTRACT

Flow cytometry has become well established for the study of animal cells but its use in microbiology has been more limited. However, during the last fifteen years instrumentation capable of detecting and measuring microbes has begun to emerge and now the application of flow cytometry in microbiology is increasing.

The introductory chapter discusses the historical development of the flow cytometer. Flow cytometric measurement of light scattering and fluorescence and their applications are described together with a range of data analysis techniques.

Chapter 2 discusses the use of forward light scatter measurements for determination of cell size and presents a method for obtaining absolute cell size by providing a calibration for the difference in light scattering behaviour of latex beads and of microbial cells.

A common application of flow cytometry is the measurement of DNA content, and Chapter 3 discusses the selection of a stain for this purpose. It was found that staining with a combination of mithramycin and ethidium bromide gave optimal separation from autofluorescence, together with rapid and stable staining.

In microbiology it is often desirable to determine the number of viable cells in a sample. This is usually achieved by plate counts, a method that is slow and can lead to an underestimation of the true viable count. Rhodamine 123 is accumulated by living cells and Chapter 4 describes its use for the rapid assessment of viability.

Since flow cytometry yields data on individual cells, rather than on cell populations, large volumes of data are rapidly produced. Consequently an efficient method of handling the data is required. Chapter 5 describes a program that I have written to accomplish this task.

Finally, Chapter 6 describes the application of flow cytometry for monitoring both batch and turbidostat-type fermentations, and illustrates the heterogeneity of populations present in such systems.

ACKNOWLEDGEMENTS

I am grateful to the Science and Engineering Research Council (U.K.) for financial support, without which my undertaking of this project would not have been possible.

I would also like to convey my thanks to the members of staff in the Institute of Biological Sciences at Aberystwyth for their encouragement and intellectual support. My thanks also go to those people with whom I have collaborated during the course of this project, namely Chris Davey and Arseny Kaprelyants. I also gratefully acknowledge the help of the many people whose useful discussions and varied expertise have helped me with my work, in particular Gary Salter, Andy Woodward, Pedro Mendes, Herbert Sauro and Edward Tidswell.

I would especially like to thank my supervisor Professor Douglas Kell for his help with my project and for providing an intellectually stimulating environment in which to pursue my research. My thanks also go to Professor J. Gareth Morris for making the department's resources available to me, and for useful discussions.

In particular I would like to thank my husband Chris Davey for his support and encouragement throughout this project.

Chapter 1 An Introduction to Flow Cytometry

Chapter 2 On the Determination of the Size of Microbial Cells using Flow Cytometry

Chapter 3 Fluorescence and Flow Cytometry: Selecting a Stain for Microbial DNA Analysis

Chapter 4 Flow Cytometric Analysis, using Rhodamine 123, of Micrococcus luteus at Low Growth Rate in Chemostat Culture

Chapter 5 SKATGRAF: A Stand-Alone Program for the Calibration and Plotting of Flow Cytometric Data

Chapter 6 The Exploitation of Dielectric Spectroscopy for the Measurement of Biomass at High Volume Fractions and for the Monitoring and Control of Continuous Cultures

References