Rapid, Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer, and a Comparison to Other Methods.
In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument's automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
Cory J
CJ
Krediet
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
Jan C
JC
DeNofrio
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
Carlo
C
Caruso
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
Matthew S
MS
Burriesci
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
Kristen
K
Cella
Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.
John R
JR
Pringle
D000818:Animals; D003063:Cnidaria / Q000166:cytology; D004141:Dinoflagellida / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D013559:Symbiosis / Q000502:physiology