Coulter counter is a commercially available device for determining the size distribution of electrically nonconducting particles suspended in a conducting medium.

1. Coulter Counter
Physiology Seminar
Mrinmoy Pal

2. Graham, Marshall Don. "The Coulter principle: foundation of an industry." Journal of the Association for Laboratory
Automation 8.6 (2003): 72-81.
Wallace H. Coulter Joseph R. Coulter
Wallace H Coulter discovered the Coulter Principle in the late 1940s (though a
patent was not awarded until October 20, 1953). Coulter was influenced by
the atomic bombs dropped on Hiroshima and Nagasaki. These events
motivated Coulter to simplify and improve blood cell analysis so that large
populations could be screened rapidly, as would be necessary in the event of a
nuclear war. Remembering his visits to hospitals, where he observed lab
workers hunched over microscopes manually counting blood cells smeared
on glass, Wallace focused the first application on counting red blood cells.
1935, he joined General Electric X-Ray as a sales and service engineer, electronics companies

3. Coulter Counter
Coulter counter is commercially available device for
determining the size distribution of electrically
nonconducting particles suspended in a conducting medium.
Two electrodes passing constant current are placed on
either side of a small hole or aperture through which the
suspension is sucked. Because of the smallness of the
aperture the major resistance in the circuit is at the aperture
and when a nonconducting particle passes through , the
resistance is changed giving rise to electrical pulse.
. The number of pulses is equal to the number of cells
counted and the strength of the signal (pulse height) is
directly proportional to the cell volume.
The electrical response of the instrument is essentially
independent of the shape of particles with the same volume,
an exception to this may occur with some extreme shapes.
Color or refractive index of the particles does not affect the
results.
Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer." Proc Natl Electron Conf. Vol. 12. 1956.

4. Graham, Marshall Don. "The Coulter principle: foundation of an industry." Journal of the Association for Laboratory
Automation 8.6 (2003): 72-81.
Working Principle

5. Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer." Proc Natl Electron Conf. Vol. 12. 1956.
The pulses produced at the orifice are amplified and displayed on the oscilloscope screen and appear as
vertical lines or spikes. The height of an individual pulse spike from the baseline is a measure of relative size
of the cell. The threshold control dial enables the user to set a cut off. Only if the pulse exceeds the threshold,
it gets counted. In addition to a display of relative cell size the oscilloscope also indicates the threshold control by
brightening that portion above the threshold level .
Electronic System & Output

6. The Model A Coulter Counter
The round black object at the upper right
of the stand is the stirrer motor used to
keep heavy particles in suspension. The
console contains, from left to right, the
mechanical totalizer used for slowly
accumulating high-value digits, the three
decade counters used for the rapidly
accumulating low-value digits, and the
oscilloscope display tube. Controls for the
single threshold and the aperture current
appear below the display.
Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer." Proc Natl Electron Conf. Vol. 12. 1956.
Major Drawbacks
 Single threshold: Multiple sample runs at successively increasing thresholds were required
for generating a cumulative size distribution.
 The voltage source: Accurate counting and sizing was hampered by instrument’s sensitivity
to aperture dimension and temperature-induced resistivity change of the electrolyte.
Commercial Models

7. The Model B Coulter Counter FN Model
Commercial Models
Improvements:
Dual-threshold current-sensitive amplifier
Multi-channel Pulse Height Analyzers
Graham, Marshall Don. "The Coulter principle: foundation of an industry." Journal of the Association for Laboratory
Automation 8.6 (2003): 72-81.

8. Experimental Considerations
• Coincidence:
Anomalous electrical pulses can be generated when multiple particles
enter the aperture simultaneously. This situation is known as
coincidence. This occurs because there is no way to ensure that a single large
pulse is the result of a single large particle or multiple small particles
entering the aperture at once. To prevent this situation, samples must be fairly
dilute.
Primary Coincidence Secondary Coincidence

9. Experimental Considerations
• Porous particles
The Coulter Principle measures the volume of an object, since the
disturbance in the electric field is proportional to the volume of
electrolyte displaced from the aperture. This leads to some confusion
amongst those who are used to optical measurements from microscopes or
other systems that only view two dimensions and also show the boundaries of
an object. For porous particles the volume of pores that are parallel to the
electric field lines is not measured, while the volume of pores perpendicular
to these lines does contribute to the measured volume. Thus, Particles with
interconnected pores produce a size related to their solid volume as shown
below.
Envelope
volume
Solid
volume
Porous
particle

10. Experimental Considerations
• Particle path
The shape of the generated electrical pulse varies with the particle path
through the aperture. The use of the Edit facility (signal processing
algorithm) eliminates the distorted pulses, so that the recovered size
distribution is close to the correct one. Using a longer tunnel aperture
may enhance the accuracy of the results for samples with a narrow size
distribution. This behaviour of long tunnel apertures is mostly due to the
greater approach to parabolic flow inside the bore. Still better engineering
technique was required to correct for artifacts resulting from particle path.

11. The method consists of feeding the suspension from a 30 μ diameter hole positioned 2 to 4 mm from
the aperture of the Coulter device. Both tubes are immersed in a bath of particle-free electrolyte.
When the electrolyte is sucked through the sensing aperture, the stream tube containing the
suspension is accelerated and thinned so that the particles all travel essentially the same
streamline, and consequently have uniformity of approach and passage through the sensing
aperture.
Spielman, Lloyd, and Simon L. Goren. "Improving resolution in Coulter counting by hydrodynamic focusing." Journal of Colloid
and Interface Science 26.2 (1968): 175-182.
Hydrodynamic Focusing

12. Sweet, R.G. 1965. High frequency recording with electrostatically deflected ink jets. Rev. Sci. Instr. 36: 131–136.
Richard Sweet
RG Sweet’s Ink Jet ‘‘Oscillograph’’ which was the basis for the oscillation
for separating cells into single droplets in Fulwyler’s cell sorter.
Cell Sorter

13. Fulwyler, Mack J. "Electronic separation of biological cells by volume." Science 150.3698 (1965): 910-911.
Mack Fulwyler
Mack Fulwyler used Wallace Coulter’s Coulter principle as the basis for
identifying different red blood cells in his cell sorter invention.
Cell Sorter

14. Cell counting and sizing: A cell suspension enters the droplet
generator (C) by way of a tube (D) and emerges as a high-
velocity fluid jet (E). Cell volume is sensed as the cell passes
through a Coulter aperture within the droplet generator (C). An
electric pulse proportional to cell volume is obtained at J.
Droplet formation: A piezoelectric crystal (A), driven at a
frequency of 72,000 cy/sec, produces vibrations which pass
down the rod (B) into the liquid within the droplet generator.
The shape of the rod serves to amplify the magnitude of the
vibrations within the liquid. The velocity fluctuations of the
emerging liquid produce bunching of the liquid column.
Cell Sorting: Now the isolated cell droplet arrives at the
separation point (I). The size of the charging pulse needed to
deflect droplets into the proper vessel is electronically
determined from the cell-volume pulse and the droplet is
charged electronically at the charging collar F. The charged
droplets are then deflected (H) on entering the electrostatic
field between the deflection plates (G). Finally, a series of
collection vessels (L) receive the deflected droplets.
Fulwyler, Mack J. "Electronic separation of biological cells by volume." Science 150.3698 (1965): 910-911.
Cell Sorter

15. FACS: Fluorescence-activated cell sorting
Becton Dickinson (1974) Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf
The Flow system (fluidics) The Optical system (light sensing)
Once the sheath fluid is running at laminar flow, the cells are
injected into the center of the stream, at a slightly higher
pressure. The principles of hydrodynamic focusing cause the
cells to align in a single file.
Advancement in technologies such as fluorescence tagging and light scattering
The intensity of fs signal has been attributed to cell size and
the intensity of ss signal is proportional to the amount of
cytosolic structure in the cell.

16. FACS: Fluorescence-activated cell sorting
Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf
Application:
Immunophenotyping, DNA Ploidy and S-phase Fraction Analysis
The Electronic system (signal processing)

17. Lab-on-a-chip Coulter Counter
Mei, Zhe, et al. "Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter." 2012 Annual International
Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2012.
A lab-on-a-chip Coulter counter allows cancer patients going through chemotherapy and patients with
compromised immune systems to take less than 10uL of blood to measure lymphocyte concentration.
The test can be self-administered or performed at the point-of-care clinics to reduce costs and risks of
hospital infection. Instead of electroplated or deposited metal electrodes, off-the-shelf gold pins were used as
electrodes to simplify fabrication process and to achieve superior uniformity in E-field distribution for
improved signal quality.

18. A life tree showing the impact of Wallace Coulter’s inventions and discoveries up to the date of his death in 1998. On the
right are some of the 85 patents issued to Wallace Coulter; technologies that made major contributions to the field are
shown on the left side of the tree. In the centre are some of the most important technologies with which Wallace Coulter
was directly involved or, as in the case of monoclonal antibodies, in which he immediately saw the opportunity and
built significant businesses and technologies that capitalized on these inventions
Robinson, J. Paul. "Wallace H. Coulter: decades of invention and discovery." Cytometry Part A 83.5 (2013): 424-438.