Authors: H. James Tripp
The Guava Easy-Cyte flow cytometer accepts 96 well plates containing stained cells in liquid media and reports the cells/ml for each well in Excel format (csv file). Marine microbes stained with SYBR-Grn I in natural seawater can be counted within approximately 5% error at densities of 1.0E4 to 5.5E5 cells per ml. With exceptional care, cell cultures can be reliably detected but not accurately counted down to 2E3 cells per ml. At densities above 5.5E5, cells must be diluted for accurate counting. There are a few critical adaptations to the manufacturer’s operating instructions that must be made relating to use of seawater media for extremely small SAR11 cells. Neutral pH detergent must be used for cleaning or salts will precipitate and foul the fluid pump of the cytometer. Stain concentration must be higher than recommended and made fresh daily, voltage must be higher than recommended, and rinse tubes must be changed daily. Also, freeze/thaw cycles of SYBR-Grn I stock should be kept to a minimum, so it is wise to aloquot stocks upon purchase and receipt.
Step 7 is critical. All aspects of the procedure come together here. Properly stained cells can only be accurately counted with the correct settings in a clean machine.
See Figure 1.
Figure 1: Histogram with gating and threshold set properly for properly stained cells in a clean machine.
The large red peak repesents the green light signal from cells. The pink line segment flanking the peak is the gating band for cells. A small amount of background noise appears to the left of the red peak. The vertical dotted line represents the location of the threshold.
SAR11 marine bacteria require exogenous reduced sulphur for growth, H. James Tripp, Joshua B. Kitner, Michael S. Schwalbach, John W. H. Dacey, Larry J. Wilhelm, and Stephen J. Giovannoni, Nature 452 (7188) 741 - 744 10/04/2008 doi:10.1038/nature06776
H. James Tripp, Oregon State University
Correspondence to: H. James Tripp ([email protected])
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.29. Originally published online 18 March 2008.