147
meets pre-set criteria is charged, the rest are not. The droplet is
then passed through deflection plates and sorted right (+) or left
(-) depending upon the charge, or dropped into a center reject vial.
While analysis can be accomplished with distilled water as the sheath
fluid, the sheath fluid must be saline (at least 0.5%) to maintain
charge for sorting.
We customarily use filtered seawater (0.4 llm
Nuc1epore membrane) or appropriate culture medium for marine phytoplankton.
However, the saline sheath would be expected to cause
osmotic stress for freshwater aquatic cells.
Even under conditions
optimal for marine phytoplankton, the sheath fluid does substantially
dilute the final recovered cells.
In 14c experiments, this has been
estimated (by tracers) to be between 50-100 times dilution (P.J.1eB.
Williams, pers. corom.).
The instrument, designed with the objective of sort purity, has
resulted in computer controlled anti-coincidence feedback loops which
result in highly pure sorts to the right and to the left. The reject
container, however, is in no way a "pure" sample.
Contents will
include 1) particles failing to meet sort criteria, 2) particles
which meet sort criteria but are rej ected due to coincidence, 3)
debris, and 4) excess sheath fluid.
If the objective is to recover
most of the cells, the reject container must be resorted repeatedly,
which further dilutes the cells with sheath fluid.
Due to computer speed limitations, effective sorting cannot be
accomplished as rapidly as analysis.
In general, while analysis can
cope with up to 0.4 m1 per minute, only 1 m1 per hour can be sorted.
Yield is dependent on particle concentration per m1 and initial
percentage of desired particles in the mixture/natural population.
For example, one has to sort ten times as long to collect x number of
cells if the desired cells are 2% of the population as compared with
20% of the population.
For microscopic verification of sorted cells
and electron microscope work, sufficient cell numbers are readily
achieved.
On the other hand, for subsequent physiological experiments or biochemical measurements,
sort times are not always
practical within a time frame permissible to insure that little or no
change has occurred during the sort time itself.
We predict and urge a general thrust to develop micro-methods
for elemental analysis, tracer incorporation, etc. which
resu1 t in the capability to deal with small numbers of cells
10 4 cells, irrespective of the fluid volume from which recovered).
Such development would permit maximum utility of the flow cytometer/
sorter and the necessary coupling with more traditional methods which
meets pre-set criteria is charged, the rest are not. The droplet is
then passed through deflection plates and sorted right (+) or left
(-) depending upon the charge, or dropped into a center reject vial.
While analysis can be accomplished with distilled water as the sheath
fluid, the sheath fluid must be saline (at least 0.5%) to maintain
charge for sorting.
We customarily use filtered seawater (0.4 llm
Nuc1epore membrane) or appropriate culture medium for marine phytoplankton.
However, the saline sheath would be expected to cause
osmotic stress for freshwater aquatic cells.
Even under conditions
optimal for marine phytoplankton, the sheath fluid does substantially
dilute the final recovered cells.
In 14c experiments, this has been
estimated (by tracers) to be between 50-100 times dilution (P.J.1eB.
Williams, pers. corom.).
The instrument, designed with the objective of sort purity, has
resulted in computer controlled anti-coincidence feedback loops which
result in highly pure sorts to the right and to the left. The reject
container, however, is in no way a "pure" sample.
Contents will
include 1) particles failing to meet sort criteria, 2) particles
which meet sort criteria but are rej ected due to coincidence, 3)
debris, and 4) excess sheath fluid.
If the objective is to recover
most of the cells, the reject container must be resorted repeatedly,
which further dilutes the cells with sheath fluid.
Due to computer speed limitations, effective sorting cannot be
accomplished as rapidly as analysis.
In general, while analysis can
cope with up to 0.4 m1 per minute, only 1 m1 per hour can be sorted.
Yield is dependent on particle concentration per m1 and initial
percentage of desired particles in the mixture/natural population.
For example, one has to sort ten times as long to collect x number of
cells if the desired cells are 2% of the population as compared with
20% of the population.
For microscopic verification of sorted cells
and electron microscope work, sufficient cell numbers are readily
achieved.
On the other hand, for subsequent physiological experiments or biochemical measurements,
sort times are not always
practical within a time frame permissible to insure that little or no
change has occurred during the sort time itself.
We predict and urge a general thrust to develop micro-methods
for elemental analysis, tracer incorporation, etc. which
resu1 t in the capability to deal with small numbers of cells
10 4 cells, irrespective of the fluid volume from which recovered).
Such development would permit maximum utility of the flow cytometer/
sorter and the necessary coupling with more traditional methods which