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Description  |
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FIELD OF THE INVENTION
The invention generally relates to a small, closed, easy-to-use device for
performing chemical, biochemical, immunological, biomedical, or
microbiological tests, and in particular to a device for the
multiparameter testing and/or identification of bacteria.
BACKGROUND
Modern diagnostic medicine depends on the routine testing of biological
samples from sources such as blood, serum, spinal fluid, urine, tissue
specimens, etc. In addition, many other industries and research facilities
run both chemical and biological tests in large numbers. In order to
perform the running of large numbers of tests efficiently, accurately, and
safely, the "hardware" used in the performance of the tests can be of
critical importance.
Microtiter plates or "microplates" were introduced in the 1960's to
facilitate laboratory testing in situations where a large number of tests
were run simultaneously. The most typical microplates contain ninety-six
(96) molded plastic wells (in an 8.times.12 array) with a typical sample
volume capacity of about 200 microliters. A wide variety of mechanical
fluid handling devices are now available so that specimens, chemical
solutions and other liquids can be transferred into the wells. Usually a
row of eight (8) or twelve (12) wells are filled simultaneously, but some
handling devices can simultaneously add sample to all of the wells.
The design of the microplate is less than optimal in several ways. First,
the microplate wells are open wells. Most microplates have loose fitting
lids, but these do not seal the top of the well. As a consequence, liquid
can spill out of the well or aerosols can form during filling. This can
ruin the test and may also create a hazard if the testing involves
infectious material. Moreover, liquid can evaporate from the wells. This
can also ruin the test or limit the duration of the test. Thus, it is
preferable to have testing hardware which can be easily sealed.
Second, although filling devices are available to fill more than one well
at a time, these devices are costly and still time consuming to use. It
would, therefore, be preferable for the testing device to be easy and fast
to fill without expensive equipment.
Third, the volume of the well is relatively large. Often the sample is in
short supply or the testing reagents are costly. It would, therefore, be
preferable to have wells with a smaller capacity.
Finally, microplates are relatively large and heavy. They take up a great
deal of space in the laboratory refrigerators and incubators, and they are
costly to ship in large quantities.
An improvement over the microplate format is disclosed in U.S. Pat. No.
4,038,151 to Fadler et al. This device has an enclosed format (minimizing
spills and aerosols) and is relatively smaller and lighter in weight. The
well volume is reduced and thus requires a smaller sample size.
Nonetheless, this design has distinct disadvantages as well. In order to
fill the Fadler test device with liquid, the air in the wells must leave
so that the liquid can enter. The Fadler et al. device accomplishes this
with a very slow, elaborate, and expensive procedure in which the device
is placed in a vacuum chamber and the air is removed as the vacuum is
created. When the vacuum is released, the liquid flows into the device.
Since, even with the elaborate vacuum system, the air removal may not be
complete, this design provides a small well connected to each main well as
an appendage for the purpose of holding residual air. These appendage
wells, take up space that might otherwise be used to accommodate more test
wells.
An alternative to this design is disclosed in U.S. Pat. No. 4,806,316 to
Johnson et al. This device allows air to escape from the wells via escape
channels which connect back to the air space in the reservoir from which
the sample originates. This additional channelling is necessary so that
the liquid can flow to the wells. Not only does this additional
channelling take up space in the device making it larger or reducing the
number of test wells that will fit on it, but, more importantly, this
design requires the use of a reservoir with a special cap having two vent
pipes, which is an expensive component when, for reasons of potential
contamination, such reservoir and cap must be single-use and disposable.
What is needed is a device that is simpler, faster and more economical to
fill. Such a device should not require the use of filling procedures
having additional risk nor which require expensive and cumbersome
equipment, but it should be able to accommodate the simultaneous filling
of a large number of test wells.
SUMMARY OF THE INVENTION
The invention generally relates to the chemical, biochemical,
immunological, biomedical, or microbiological testing of a sample in a
small, closed and easy-to-fill device, and in particular to the
multiparameter testing of microorganisms for the purpose of identification
or for other purposes. Depending on the type of test being run, such
device might contain one or more dry test chemicals, or it may, initially,
be empty. The device requires only a small volume of sample (e.g., one
milliliter).
The invention represents a much improved and fundamentally different
design. The invention contemplates permitting air in the device to escape
during filling by taking advantage of the physical properties of some
newly available materials that act as vents. These materials have the
useful property that they allow gas to pass readily, and yet they provide
a strong barrier to aqueous liquids. In this manner, discrimination is
made between liquids and gasses without the need for a valve-like
mechanism.
The barrier to the passage of liquids can be achieved in several ways. One
way is for the liquid-contacting surfaces of the material, particularly
internally within its pores, to be hydrophobic so as to repel the liquid
and thereby making it difficult for the liquid to pass through the
material. Another way for the material to prevent the passage of gas would
be for it to swell and close off the pores when contacted by liquid; and
yet still another way would be for it to form a film or gel which would
effectively plug or close the pores upon exposure to liquid. By
incorporating one of such venting materials into the design, a simple
means is provided for permitting air to escape from the testing device
during filling, while preventing the escape of liquid. Such a fluid flow
discriminator operates automatically, without any moving parts.
In one embodiment, the present invention contemplates a device comprising:
a) a housing; b) a testing region contained within said housing; c) a
liquid receiving means on an external surface of said housing; d) a liquid
flow-directing means providing liquid communication between said testing
region and said liquid receiving means; and e) a gas-venting, liquid
barrier in fluidic communication with said testing region.
It is contemplated that a test sample, such as a suspension of
microorganisms in water or saline solution, would be taken up in liquid
form by a pipette or other suitable means and delivered, under positive
pressure, to the wells within the housing by way of the port and the
connecting channels. It is further contemplated that as the liquid fills
the connecting channels, the air within the device would exit through the
gas-venting, liquid barrier ahead of the advancing liquid.
After venting is complete, a non-venting material may be affixed to cover
the exposed outside surface of the barrier thereby sealing; it against the
evaporation of the liquid through the vent material. Such non-venting
material can be one of any number of tapes each differing in its ability
to permit the molecular diffusion of oxygen so as to allow the desired
amount of oxygen to enter or leave the well as may be needed for
maintaining the appropriate environment for the microorganisms or test
chemistry within.
Following the filling, a non-venting tape can also be applied to cover over
the filling port. In this manner, the present invention provides a device
wherein the microorganisms are completely contained so that little hazard
exists of having spills or aerosols and the device is convenient and easy
to handle. In a preferred embodiment, the non-venting tape is a plastic
film, e.g., Mylar.
It is not intended that the present invention be limited to particular
materials. In one embodiment, the housing is made of hard plastic such a
polystyrene. In a preferred embodiment, the gas-venting, liquid barrier is
a membrane or plug made of a porous, hydrophobic material that is capable
of allowing air to pass readily during delivery of the liquid under
positive pressure, yet which will block the flow of liquid under the
positive pressure normally applied in the practice of the invention.
In one embodiment, the gas-venting, liquid barrier is made of material
selected from the group comprising polycarbonate, polypropylene, and
polysulfone. In another embodiment, the gas-venting, liquid barrier is
made of material selected from the group comprising polyvanilidine
chloride (PVDC) and polyvanilidine difluoride (PVDF). In still another
embodiment, the gas-venting, liquid barrier is made of expanded, sintered
polytetrafluoroethylene (PTFE).
As a membrane, the pore size for these materials is generally greater than
or equal to 0.1 microns, and preferably greater than 3.0 microns. The
positive pressure normally applied in the practice of the invention will
be greater than 1 pound per square inch (psi), and preferably greater than
5 psi. The gas-venting, liquid barrier in one embodiment blocks the
passage of liquid at pressures less than 75 pounds per square inch.
It is also not intended that the invention be limited to any particular
configuration of wells within the housing, or to any one configuration of
channels within the housing. In one embodiment, the channels in
communication with the wells are of decreasing cross-section.
It is also not intended that the invention be limited to the type of
sample. The present invention can be employed with success with all types
of liquid samples, including suspensions of biological material. It is
also not intended that the invention be limited by the type of suspended
microorganism. The device of the present invention is useful for
identification of a great number of microorganism species and subspecies.
In a preferred embodiment, the device can be held conveniently in a user's
hand. Due to its small size, the device conserves media and test
chemicals. It is also preferred that the background color of the device be
uniform (e.g., white polystyrene or clear polystyrene). In this manner,
the results of the test can be read as a visual change either by eye or,
alternatively, by a simple or automated instrument. In the case of an
opaque background the test would be read by reflected light, and in the
case of a transparent background the test could be read by transmitted
light. In any case, one of each well's surfaces must be transparent to
permit it to be read.
While the actual placement of the barrier may have an impact on the nature
of the background (i.e., opaque or transparent), it is not intended that
the barrier be positioned in only one manner. The invention contemplates
an embodiment wherein the barrier is adapted to completely enclose one of
the sides of the testing wells. However, the invention contemplates an
embodiment wherein the barrier is adapted to be only a portion of one of
the sides of the well. In still another embodiment, it is contemplated
that the barrier is internal to the wells. In yet another embodiment, it
is contemplated that the barrier is separated from the well by a conduit.
The conduit itself can be configured in a number of ways (e.g., tube,
passageway, etc.).
The present invention also contemplates a method for testing chemical or
biological samples. In one embodiment, the invention comprises: (a)
providing a sample in liquid form; (b) providing a sample delivering
means; (c) providing a device comprising: i) a housing, ii) a testing
region contained within said housing, iii) a liquid receiving means on an
external surface of said housing, iv) a liquid flow-directing means
providing liquid communication between said testing region and said liquid
receiving means, and v) a gas-venting, liquid barrier in fluidic
communication with said testing region; and (d) delivering, internal to
said housing, said sample via said sample delivering means to said testing
region such that said liquid enters said device at said liquid receiving
means under positive pressure and the air in said device is vented through
said gas-venting liquid barrier.
In one embodiment, the method further comprises, prior to step (d),
prefilling said test wells with a test formula. For example, the test
formula may comprise basal medium, one or more carbon sources, and an
indicator, such as a redox indicator.
The device can be configured such that the sample can be delivered without
the need for cumbersome equipment. In such an embodiment, the liquid
receiving means may comprise one or more liquid entry ports on an external
surface of said housing and the sample delivering means may comprise a
pipette adapted to fit the liquid entry port. For better fit, the port can
be configured with internal grooves and the pipette can be molded with
corresponding threads (or the pipette can be molded with internal grooves
and the port can be configured with corresponding threads) so that a
substantially liquid-tight seal is formed.
DESCRIPTION OF THE FIGURES
FIG. 1 is an exploded perspective view of one embodiment of the device of
the present invention.
FIG. 2 is a top plan view of the device shown in FIG. 1.
FIG. 3 is a cross-sectional view of the device shown in FIG. 2 along the
lines of 3--3.
FIG. 4 is a bottom plan view of the device shown in FIG. 1.
FIG. 5 is a top plan view of a second embodiment of the device of the
present invention.
FIG. 6 is a cross-sectional view of the device shown in FIG. 5 along the
lines of 6--6.
FIG. 7 is a top plan view of a third embodiment of the device of the
present invention.
FIG. 8 is a top plan view of a fourth embodiment of the device of the
present invention.
FIG. 9 is an enlarged bottom plan view of FIG. 8.
FIG. 10 is one embodiment of an automated analyzer for reading the
embodiment of the device shown in FIGS. 8 and 9.
FIG. 11 is a cross-section of the analyzer of FIG. 10.
FIG. 12 is a perspective view of a fifth embodiment of the device of the
present invention.
FIG. 13 is a top plan view of a sixth embodiment of the device of the
present invention.
FIG. 14 is a cross-sectional view of the embodiment shown in FIG. 13 along
lines 14--14.
FIG. 15 is a perspective view of the embodiment shown in FIGS. 13 and 14.
DESCRIPTION OF THE INVENTION
The invention generally relates to a device for the single or
multiparameter testing, of chemical, biochemical, immunological,
biomedical, or microbiological samples in liquid or liquid suspension form
in a small, closed, easy-to-fill device, and is particularly suitable for
multiparameter testing and identification of microorganisms. To this end,
the present invention contemplates a device comprising: a) a housing; b) a
testing region contained within said housing; c) a liquid receiving means
on an external surface of said housing; d) a liquid flow-directing means
providing liquid communication between said testing region and said liquid
receiving means; and e) a gas-venting, liquid barrier in fluidic
communication with said testing region.
After the device has been filled, a non-venting, sealing tape can be
applied to the device to cover the gas-venting, liquid barrier to reduce
the evaporation of the liquid from the device; the tape can permit the
molecular diffusion of oxygen into or out of the device to maintain the
desired chemical or biochemical environment within the device for
successful performance of the test. Where the liquid receiving means
comprises liquid entry ports, a similar closing tape can be applied to
close the port or ports to prevent spilling and evaporation of the liquid
therefrom.
One of the principal objects of the present invention is to provide, unlike
other approaches, a method and device for carrying out chemical,
biochemical, immunological and microbiological tests which greatly
simplifies the removal of air to allow filling. A further object is to
provide a closed, spill-proof device which is small and compact and easy
to read either by eye or by automated instrumentation wherein the visible
result of the test within the device can be detected
spectrophotometrically by passing light through the sample, or the visible
result can be detected by imaging such as with a video camera. The
results, however read, can be inputted into a computer wherein an
algorithm then determines the best match of the inputted pattern to the
patterns of known species in a data base.
The small size and spill-proof configuration of the present device permits
many such devices to be held in a cartridge, similar to those that hold 35
mm photographic slides for viewing in a Kodak.TM. Carousel slide projector
or a Kodak.TM. slide-to-video converter. Such cartridge and reading
mechanism can be housed within a temperature controlled enclosure wherein
the devices can be incubated and read automatically. The visual result
that is detected by eye or by instrument can be any optically perceptible
change such as a change in turbidity, a change in color, or the emission
of light, such as by chemiluminescence, bioluminescence, or by Stokes
shift. Color indicators may be, but are not limited to, redox indicators
(e.g., tetrazolium), pH indicators, or various dyes and the like. Various
dyes are described in U.S. Pat. Nos. 4,129,483, 4,235,964 and 5,134,063 to
Barry R. Bochner, hereby incorporated by reference. See also B. R.
Bochner, Nature 339:157 (1989); Bochner, B. R. ASM News 55:536 (1990); B.
R. Bochner, Amer. Clin. Lab. April:14 (1991). A generalized indicator
useful for practice of the present invention is also described by Bochner
and Savageau. See B. Bochner and M. Savageau, Appl. Environ. Microbiol.
33:434 (1977).
Testing based on the redox technology is extremely easy and convenient to
perform. A cell suspension is prepared and introduced into the testing
wells of the device. Each well is prefilled with a different substrate.
In a preferred embodiment, all wells are prefilled with test formula
comprising a basal medium that provides nutrients for the microorganisms,
and a color-change indicator, and each well is prefilled with a different
carbon compound or "substrate," against which the microorganism is tested.
"Basal medium," as used herein, refers to a medium which provides
nutrients for the microorganisms, but does not contain sufficient
concentrations of carbon compounds to trigger a color response from the
indicator. "Carbon compound," "carbon source" and "substrate" are
equivalent terms, and are used interchangeably herein to refer to a carbon
chemical in sufficient concentration as to trigger a color response from
the indicator when it is utilized (metabolized) by a microorganism.
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