U.S. patent number 3,586,859 [Application Number 05/011,628] was granted by the patent office on 1971-06-22 for fluorescent cell viability counter.
Invention is credited to Morris A. Benjaminson, Irwin J. Katz, Nicholas M. Satriano.
United States Patent |
3,586,859 |
Katz , et al. |
June 22, 1971 |
FLUORESCENT CELL VIABILITY COUNTER
Abstract
Method and apparatus for automatically ascertaining the number
of dead cells in a batch. All of the cells in the batch are stained
with a specific fluorescent dye in a reservoir, then the cells are
passed through a capillary tube by means of a peristaltic pump
under a microscope while illuminated by an ultraviolet light. The
microscope is coupled to a photomultiplier which in turn has its
output applied to an electronic counter with a readout. After the
dead cells are counted, the batch is immersed in a heating bath in
which all the viable cells are killed and another count is obtained
by repassing the batch beneath the microscope. The difference in
the counts is the number of original viable cells while the first
count is the original number of dead cells.
Inventors: |
Katz; Irwin J. (Brooklyn,
NY), Satriano; Nicholas M. (Rego Park, NY), Benjaminson;
Morris A. (New York, NY) |
Family
ID: |
21751284 |
Appl.
No.: |
05/011,628 |
Filed: |
February 16, 1970 |
Current U.S.
Class: |
250/459.1;
435/39; 435/808; 436/63; 436/172 |
Current CPC
Class: |
G06M
1/101 (20130101); C12M 41/36 (20130101); G01N
2015/1486 (20130101); Y10S 435/808 (20130101) |
Current International
Class: |
C12M
1/34 (20060101); G01N 15/14 (20060101); G06M
1/10 (20060101); G06M 1/00 (20060101); G01n
021/22 (); G01n 021/26 () |
Field of
Search: |
;250/71,71.5,83.3UV
;356/36,39 ;424/7 ;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Borchelt; Archie R.
Claims
We claim:
1. An apparatus for determining the number of viable and nonviable
cells in a 5mg./ml. saline solution of 4 acetamido 440
isothiocyanostilbene 2,2 disulfonic acid Disodium which
comprises:
an elongated restricted transparent passageway having an open end
and connected at its other end to a circulating means for flowing a
liquid through said passageway, the opposite end of said
circulating means being connected to means for killing said viable
cells;
coupling means connecting said killing means to said open end of
said passageway;
a fluorescent microscope having an objective disposed on one side
of said passageway;
a source of ultraviolet light disposed on the opposite side of said
passageway from said objective for directing ultraviolet light
through said passageway into said objective;
light amplifying and detecting means connected to said microscope
for detecting light pulses therefrom and converting them to
electric signals and, computer means connected to said detecting
means for counting said light pulses; and
whereby when the solution is circulated twice through said
apparatus a count of the number of nonviable and the total number
of cells will be obtained.
2. The apparatus according to claim 1 wherein said restricted
passageway is a capillary tube.
3. The apparatus according to claim 2 wherein said circulating
means is a peristaltic pump.
4. The apparatus according to claim 3 wherein said means for
killing is a heating module through which the cells are passed.
5. The apparatus according to claim 4 wherein said amplifying and
detecting means is a photomultiplier circuit and tube.
6. The apparatus according to claim 5 further including a specimen
reservoir disposed in line with and proximate said open end of said
passageway.
7. A method for ascertaining the number of viable and nonviable
cells within a specimen which comprises the steps of:
preparing a solution of said specimen cells with a saline solution
of a nontoxic fluorescent dye;
passing said prepared solution through a restricted passage;
exposing said prepared solution in said passage to an ultraviolet
light;
detecting and counting those cells which fluoresce;
killing any viable cells in said prepared solution; and
repassing said prepared solution after killing said viable cells
through said passage and detecting and counting the fluorescent
cells.
8. The method according to claim 7 wherein said dye is 4 acetamido
4' isothiocyanostilbene 2, 2 disulfonic acid Disodium.
9. The method according to claim 8 wherein said prepared solution
consists of said specimen cells added to 5 mg. of said dye for
every ml. of saline solution.
Description
STATEMENT OF GOVERNMENT INTEREST
The invention described herein may be manufactured and used by or
for the Government of the United States of America for governmental
purposes without the payment of any royalties thereon or
therefor.
BACKGROUND OF THE INVENTION
1.
Field of the Invention
The present invention relates to a system for counting and
discriminating between living and dead cells and more particularly
pertains to staining all the cells with a specific fluorescent dye
and automatically counting the dead cells, and thereafter killing
the viable cells and then counting all the cells, providing a rapid
count of each.
2.
Description of the Prior Art
In the field of counting and discriminating dead and viable cells,
it has been the general practice to employ a standard technique of
visually and manually counting the cells exposed to trypan blue.
This method is both tedious and extremely time consuming. A more
recent method contemplates the use of an automatic cell
spectrophotometer on cells which have been stained with trypan
blue. This instrument is based upon the principle of light
absorption and scattering which requires highly complex
instrumentation. The technique itself is less sensitive than other
techniques. Such prior art devices have been unsatisfactory in that
errors are introduced by undissolved dye particulates, fluids in
which the cells are suspended and in counting small size cells.
Erythrocytes must be eliminated since they refract light and do not
take up trypan blue, thereby resulting in a false count of the
living cells.
SUMMARY OF THE INVENTION
The general purpose of this invention is to provide a method and
apparatus that has all the advantages of similarly employed prior
art devices and has none of the above described disadvantages. To
attain this, the present invention provides a unique technique in
which all the cells are stained with 4 acetamido 4'
isothiocyanostilbene 2,2 disulfonic acid Disodium (SITS) a
fluorescent dye and then passed through a capillary, illuminated by
an ultraviolet light, beneath a microscope. The microscope is
connected to a photomultiplier which provides a count only for
those cells which fluoresce and applies to to a counter. All the
cells are then killed by heat and recounted, thus giving, first, a
count of the nonviable cells and, thereafter, a total count of all
the cells, whereby an accurate rapid count of both viable and
nonviable cells is provided.
An object of the present invention is to provide a simple,
accurate, reliable inexpensive and automatic device for
ascertaining a count of both dead and living cells in a
specimen.
Another object is to provide a method and technique which is
direct, simple and automatic for counting both viable and nonviable
cells.
Other objects and many of the attendant advantages of this
invention will be readily appreciated as the same becomes better
understood by reference to the following detailed description when
considered in connection with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE is a block diagram of an embodiment made in accordance
with the principle of this invention and illustrates the method
employed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In the illustrated embodiment, a small specimen reservoir 10 is
provided at one end of the flow tube 11, which is disposed directly
above a larger reservoir 12 whose inner wall 13 converges
downwardly toward a small opening 14 at the base and is connected
to a transparent capillary tube 15. The capillary tube 15, which is
transparent to fluorescent light, and the reservoir 12 are
supported by a clear plastic slide 16. In order to maintain balance
and prevent distortion, a solid plastic block 17 is seated above
the capillary tube. The inner diameter of tube 15 is selected for
the particular size of the cells to be counted. It serves to
restrict the multiple simultaneous passage of the cells
therethrough in order to permit observation of the cells
individually. The capillary tube extends directly between the
objective 18 of a standard fluorescent microscope 19 and the light
condenser 20. The eyepiece end 21 of the microscope is connected to
a photomultiplier tube 22 in any of the standard methods so that
the multiplier tube directly senses the ultraviolet light from
source 20' impinging on the eyepiece 21 and provides an output
count for each light burst detected at the microscope. The electric
pulse from the multiplier is applied to an electronic counter and
computer 23 which also includes an output indication as by a
printout. This type of counter/computer is well known in the art
and is commercially available.
The capillary 15 extends beyond the plastic block 17 and is
connected to a circulating means or a peristaltic pump 24 via a
flexible coupling 25 while a second flexible coupling 26 connects
the opposite end of the pump to a valve diverter 27. The flexible
couplings decouple the pump movement and vibrations from the flow
system. This form of pump prevents damage or destruction of the
cells while supplying the necessary motive force to circulate the
cells. The valve 27 either directs the flow into the waste
receptacle 28 or through the tube 29 to a heating module 30. Within
the module is a solution 31 which is heated by element 32 connected
to a source of electrical power 33 and the module may include a
thermostat 34. The flow tube 35 is coiled within the heating module
to permit the heating action to act on the flowing cells. After the
heating module, the cells are directed back to the reservoir along
flow tube 11.
Having fully described the apparatus employed, the process or
technique will now be detailed. It has been discovered that 4
acetamido 4' isothiocyanostilbene 2, 2 disulfonic acid Disodium
(SITS), a fluorescent dye, has the ability to distinguish between
living and nonliving cells. Viable cells take up the dye and retain
it in their vesicles and do not fluoresce brightly if at all, while
nonviable cells fluoresce very brightly, since the dye escapes from
their vesicles. This dye is not pH dependent, has an emission
spectrum of 415--420 millimicrons and an activation spectrum of
345--360 millimicrons. Five milligrams of the dye are added to each
milliliter of a buffered physiological saline solution to form a
5mg./ml. solution which is added to the unknown cell suspension. In
the above concentration the dye (SITS) is nontoxic.
The cell suspension in the dye solution is placed in the specimen
reservoir 10, the ultraviolet source 20' activated and the pump 24
started. When the cell suspension solution reaches flow through the
capillary tube 15 and passes beneath the objective 18 of the
ultraviolet microscope 19, the photomultiplier 22 will respond only
to those cells with a high fluorescent output and provide a count
at computer 23 only of the dead or nonviable cells per unit
volume.
After all the dead cells are counted, the solution proceeds into
the heating module 30, where all the viable cells are killed,
resulting in their releasing the SITS from their vesicles and
providing bright fluorescent staining. The solution then is
recirculated to the specimen reservoir 10 and held until all the
initial or original contents therein have been depleted.
With all the cells now dead, the solution is again recirculated and
counted as previously described. This count is of the total number
of cells initially in solution. The computer 23 is also programmed
to subtract the number of nonviable cells counted in the first
passage from the total number of cells counted in the second
passage so as to print out the following information:
1. number of viable cells
2. number of nonviable cells
3. total number of cells Bypass or diverting valve 27 is opened to
the waste receptacle 28 and the specimen solution discarded by
being pumped therein. The system is then flushed by adding washing
fluid to the specimen reservoir and circulating as necessary and is
thereafter discarded into the waste receptacle.
The method of this invention comprises the following steps:
a. applying the specimen cells to be counted to a saline solution
of a fluorescent dye SITS;
b. passing the cell solution through a restricted passage;
c. exposing said cell solution in said passage to an ultraviolet
light;
d. detecting and counting the fluorescent light pulses thereof;
e. killing any viable cells in said solution; and then
f. detecting and recounting said fluorescent pulses.
The above described invention is not limited as to the cell type
which may be counted or examined and the dye is specific for
biological material, and the problem of false counts is eliminated
by the excellent solubility of the dye. It should be understood, of
course, that the foregoing disclosure relates to only a preferred
embodiment of the invention and that numerous modifications or
alterations may be made therein without departing from the spirit
and the scope of the invention as set forth in the appended
claims.
* * * * *