Bacteria sorting ?

Alice L. Givan Alice.L.Givan at dartmouth.edu
Tue Mar 5 10:12:53 EST 2002


Andy,
Here is my take on this.

"We are using a 50 µm nozzle at 30psi (BD vantage) and trying to single cell
sort bacteria onto  96 well plate. The first question is how large are the droplets
(volume) ? "

Using an approximation that the diameter of the drop is the same as the diameter of
the nozzle,  then the volume of your drops is going to be  Volume = 4/3 *(3.14) r^3  --
where the radius, r, is equal to 25 µm (half of the nozzle diameter)  or 25 x 10^-4 cm.
So, Volume is 6.5 x 10^-8 ml for each drop.    The trouble with this approximation is
that the drop diameter is often a bit less than the diameter of the nozzle (as the
stream contracts a bit as it leaves the nozzle).  Someone else may be able to help
you to get this number more accurately, if necessary.

"Can you determine the dilution factor of your samples (bacteria) ?"

If you are sorting in "one drop envelopes," then each bacterium is diluted into the
volume above (one drop).  Therefore,  a million cells will be in 0.06 ml.  If you
are sorting 2 drop envelopes,  then each bacterium is in twice that volume (and a
million cells will be in 0.12 ml).  However,  we usually sort onto a volume (cushion)
of medium --- so the concentration of bacteria in the sort tube would actually be much
lower if you do this.


"We are sorting bacteria and triggering on the fluorescence signal to reduce
background noise.  The questions is : at what rate of sample can we assume
that the instrument is detecting a single cell in a droplet ?"

It all depends on how fast the cytometer drop drive is going (drop drive frequency
or ddf).  If  the drop drive is going at, say, 40,000 drops per second,  then you could
have your cells going at 40,000 cells per second and you will have ON AVERAGE one cell
in each drop.  The problem with this is that there is a Poisson distribution of cells
flowing (some will be closer to other cells in the flow stream and some will be far
apart from other cells).  So that even if you have,  ON AVERAGE,  one cell in each drop,
a significant number of drops will have no cells and a significant number of drops will
have two or more cells.  To get away from this problem (and without going through the
math of the Gaussian distribution -- Jim Watson's book (Introduction to Flow Cytometry --
Cambridge University Press) has good discussions on this),  people usually have their
cells flowing at something like one cell in every 5-10 drops.  That means that you
should aim to have your cells flowing at one fifth or one tenth the drop drive frequency
if you want to make sure that you almost  never have more than one cell in a drop.
However, you should also realize that most cytometers have "abort" conditions ---
if you have more than one cell in a drop,  the cell(s) will probably not be sorted
(the sort will be aborted).  So having more than one cell in a drop means that you
will lose cells,  but probably will not lose purity (cytometers do different things
with these abort conditions -- and there are often different ways you can set the
cytometer up to provide different sort decisions when multiple cells occur in a drop).

"Looking at the numbers it would appear that non-fluorescent bacteria must
be in the same droplets, but I may be assuming something incorrectly."

Hmmm -- I am not sure about this.  Are you sure your cytometer is sorting correctly
(does it sort fluorescent beads from non-fluorescent beads?).  If it is sorting beads
well,  then it could be that the so-called non-fluorescent bacteria that you are
seeing are simply instrument noise.  How small are the bacteria that you are sorting?
If you run a sample of medium without any bacteria through the cytometer,  do you see
lots of events at a high rate (falling in the same place as your "bacteria" fall on
the scatter and fluorescence plots)?

It will be interesting to see what other suggestions people come up with.

Alice

Alice L. Givan
Englert Cell Analysis Laboratory
of the Norris Cotton Cancer Center
Dartmouth Medical School
Lebanon, New Hampshire NH 03756
tel 603-650-7661
fax 603-650-6130
givan at dartmouth.edu




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