U.S. patent application number 09/824160 was filed with the patent office on 2001-09-27 for method and devices for partitioning biological sample liquids into microvolumes.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Berg, James G., Calhoun, Clyde D., Debe, Mark K., Halverson, Kurt J., Krejcarek, Gary E., Qiu, Jean, Wei, Ai-Ping, Wickert, Peter D., Williams, Michael G..
Application Number | 20010024805 09/824160 |
Document ID | / |
Family ID | 27126024 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010024805 |
Kind Code |
A1 |
Williams, Michael G. ; et
al. |
September 27, 2001 |
Method and devices for partitioning biological sample liquids into
microvolumes
Abstract
A method for partitioning an aqueous biological liquid sample
into discrete microvolumes for detection and enumeration of
microorganisms is described. The method involves distributing
microvolumes of a sample to a plurality of hydrophilic
liquid-retaining zones of a culture device, where each
liquid-retaining zone is surrounded by a portion of a hydrophobic
"land" area. Also disclosed are devices for carrying out these
methods.
Inventors: |
Williams, Michael G.;
(Vadnais Heights, MN) ; Halverson, Kurt J.; (Lake
Elmo, MN) ; Krejcarek, Gary E.; (White Bear Lake,
MN) ; Wei, Ai-Ping; (Woodbury, MN) ; Berg,
James G.; (Lino Lakes, MN) ; Wickert, Peter D.;
(St. Paul, MN) ; Calhoun, Clyde D.; (Stillwater,
MN) ; Debe, Mark K.; (Stillwater, MN) ; Qiu,
Jean; (Andover, MA) |
Correspondence
Address: |
Attention: Christopher D. Gram
Office of Intellectual Property Counsel
3M Innovative Properties Company
P.O. Box 33427
St. Paul
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
27126024 |
Appl. No.: |
09/824160 |
Filed: |
April 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09824160 |
Apr 2, 2001 |
|
|
|
08997337 |
Dec 23, 1997 |
|
|
|
08997337 |
Dec 23, 1997 |
|
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08838397 |
Apr 9, 1997 |
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Current U.S.
Class: |
435/29 ;
435/305.1 |
Current CPC
Class: |
C12M 41/36 20130101;
C12M 23/20 20130101; C12M 23/12 20130101; C12M 23/44 20130101; C12M
25/04 20130101; B01L 3/5085 20130101 |
Class at
Publication: |
435/29 ;
435/305.1 |
International
Class: |
C12Q 001/02; C12M
001/22 |
Claims
What is claimed is:
1. A culture device for detection or enumeration of microorganisms,
said device comprising a substrate having liquid retaining discs
wherein said substrate is hydrophobic relative to said liquid
retaining discs and wherein the discs have media for growth of
microorganisms and said discs have a microvolume capacity of liquid
retention.
2. The culture device of claim 1 wherein said discs are constructed
at least in part of a material selected from the group consisting
of cellulosics, polyolefins, polyamides and polyesters.
3. The culture device of claim 2 wherein the discs are constructed
at least in part of alpha cellulose.
4. The culture device of claim 2 wherein the discs are constructed
at least in part of rayon.
5. The culture device of claim 2 wherein the discs are constructed
at least in part nylon.
6. The culture device of claim 2 wherein the discs are constructed
at least in part of polylactic acid.
7. The culture device of claim 1 wherein said device has about 10
to about 10,000 discs.
8. The culture device of claim 1, wherein said device has about 400
to about 600 discs.
9. The culture device of claim 1, wherein each said disc has a
liquid retention capacity of about 1 to about 2 microliters.
10. The culture device of claim 1, having an indicator substance on
said discs.
11. The culture device of claim 1 wherein the discs are adhered to
the substrate with adhesive, and wherein said adhesive has an
indicator for detecting microorganisms.
12. The culture device of claim 10, wherein said indicator
substance is selected from the group consisting of a chromogenic
indicator, a fluorescent indicator, a luminescent indicator and an
electrochemical indicator.
13. The culture device of claim 1, wherein said culture device
comprises a plurality of sets of hydrophilic liquid-retaining
discs, each said set having discs of uniform size, said sets
varying in liquid retention capacity, and said device having at
least two sets of discs.
14. A method for partitioning an aqueous liquid sample into
discrete microvolumes, comprising: a) providing a device for
culturing a microorganism, said device having an assay surface,
said assay surface comprising hydrophilic liquid-retaining discs
and a hydrophobic land area between said discs, each said disc
having a microvolume capacity of liquid retention and having media
for growing microorganisms; and b) contacting said liquid sample
with said assay surface such that said liquid sample is partitioned
into said hydrophilic liquid-retaining discs.
15. The method of claim 14, wherein the discs are constructed of a
material selected from the group consisting of cellulosics,
polyolefins, polyamides and polyesters.
16. The method of claim 15, wherein the discs are coated with a
growth medium.
17. The method of claim 14, wherein said discs have at least one
indicator substance coated thereon.
18. The method of claim 17, wherein said indicator substance is
selected from the group consisting of a chromogenic indicator, a
fluorescent indicator, a luminescent indicator and an
electrochemical indicator.
19. The method of claim 14, wherein said discs are of uniform size
and each said disc has a liquid retention capacity of about 0.01 to
about 25 microliters.
20. The method of claim 14 wherein each said disc has a liquid
retention capacity of about 1 to about 2 microliters.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 08/997,337, filed Dec. 23, 1997, which is a
continuation-in-part of U.S. patent application Ser. No.
08/838,397, filed Apr. 9, 1997.
FIELD
[0002] This invention relates to methods and devices for
partitioning biological samples into microvolume aliquots, based on
the tendency for aqueous liquids to be retained within hydrophilic
zones of the devices while being substantially excluded from
hydrophobic areas of the devices, and detecting and enumerating
microorganisms present within the samples.
BACKGROUND
[0003] The detection and enumeration of microorganisms is practiced
in numerous settings, including the food-processing industry
(testing for the contamination of food by microorganisms such as E.
coli and S. aureus), the health care industry (testing of patient
samples and other clinical samples for infection or contamination),
environmental testing industry, the pharmaceutical industry, and
the cosmetic industry.
[0004] Growth-based detection and enumeration of microorganisms is
commonly practiced using either liquid nutrient media (most
probable number analysis (MPN)) or semi-solid nutrient media (agar
petri dishes). Enumeration using the liquid MPN method is typically
achieved by placing serial 10-fold dilutions of a sample of
interest in replicate sets of tubes containing selective media and
chemical indicators. The tubes are incubated at elevated
temperature (24-48 hours) followed by examination for growth of
organisms. A statistical formula, based on the volume of sample
tested and the number of positive and negative tubes for each set,
is used to estimate the number of organisms present in the initial
sample.
[0005] This method of performing MPN analysis has several
disadvantages. It is labor intensive because of the multiple
diluting and pipetting steps necessary to perform the analysis. In
addition, in practice it is only practical to use replicate sets of
about three to five tubes for each dilution. As a result, the 95%
confidence limits for an MPN estimate for microbial concentration
are extremely wide. For example, a three tube MPN estimate of 20
has 95% confidence limits ranging from 7 to 89. Furthermore,
results typically are not obtainable in less than twenty-four
hours.
[0006] In contrast to the method described above, a direct count of
viable microorganisms in a sample can be achieved by spreading the
sample over a defined area using nutrient media containing a
gelling agent. The gelling agent (agar) prevents diffusion of the
organisms during incubation (24-48 hours), producing a colony in
the area where the original organism was deposited. There is,
however, a limit to the number of colonies that can fit on a given
area of nutrient media before fusion with neighboring colonies
makes counting difficult. This usually necessitates performing
several dilutions for each sample. In addition, the classes of
chemical indicator molecules that can be used for identifying
individual types of microorganisms present within a mixed
population are limited to those that produce a product that is
insoluble in the gelled media. Furthermore, rapid detection, i.e.,
in less than twenty-four hours, and enumeration is not feasible
using this method.
[0007] In addition to these disadvantages, both the currently used
MPN analysis and gel-based systems require a relatively long
incubation time before a positive result can be detected.
SUMMARY
[0008] The invention is based on the discovery that biological
liquid samples can be partitioned into discrete microvolumes with
only minimal manipulation on the part of an operator. The method of
partitioning employs devices that have hydrophilic liquid-retaining
zones surrounded by hydrophobic "land" areas. The methods and
devices provide a system for the detection and enumeration of
microorganisms and other biological materials that solves the
problems associated with currently used systems. The system is a
liquid-based system, allowing efficient and effective partitioning
of the sample into discrete microvolumes for testing, and allows
for rapid detection and enumeration.
[0009] In the case of MPN analysis for the detection and
enumeration of microorganisms, the approaches described herein
allow for the use of water-soluble indicator species, and reduce or
eliminate the need for the several dilutions typically required in
current MPN analysis.
[0010] In general, the invention features a method for partitioning
an aqueous liquid sample, into discrete microvolumes,
comprising
[0011] a) providing a device for culturing a microorganism, said
device having an assay surface, the assay surface comprising
hydrophilic liquid-retaining zones and a hydrophobic land area
between the zones, each zone having a microvolume capacity of
liquid retention; and
[0012] b) contacting the liquid sample with the assay surface such
that the liquid sample is partitioned into the hydrophilic
liquid-retaining zones.
[0013] The zones may comprise a coating or deposition of assay
reagent, such as a nutrient medium or indicator substance.
Appropriate indicator substances include without limitation
chromogenic indicators, fluorescent indicators, luminescent
indicators and electrochemical indicators.
[0014] The zones may be of uniform size, with each zone having a
liquid retention capacity of about 0.01 to about 25 microliters,
more preferably about 1 to about 2 microliters.
[0015] The culture device can have, for example, about 10 to about
10,000 hydrophilic liquid-retaining zones, more preferably about
400 to about 600 hydrophilic liquid-retaining zones.
[0016] The hydrophilic liquid-retaining zones may comprise
microvolume wells surrounded by a hydrophobic land area.
Alternatively, the culture device may have a land area comprising a
treated nanostructured film. In further alternative embodiments,
the hydrophilic liquid-retaining zones may comprise hydrophilic
fiber material projecting from the assay surface. The fiber
material can be constructed of hydrophilic absorbent discs or of
hydrophilic nonwoven fiber loop material. Hydrophilic absorbent
discs may have media provided thereon to facilitate growth of
microorganisms. The media may be selective for one or more types of
microorganisms. The discs are biocompatible with the microorganisms
such that the materials do not substantially interfere with the
growth or detection of the microorganisms.
[0017] In an alternative embodiment, the culture device may
comprise a plurality of sets of hydrophilic liquid-retaining zones,
each of the sets having zones of uniform size, the sets varying in
liquid retention capacity, and the device having at least two sets
of zones.
[0018] In another aspect, the invention features a culture device
for detection or enumeration of microorganisms, the device
comprising an assay surface, the assay surface comprising
hydrophilic liquid-retaining zones and a hydrophobic land area
between the zones, each zone having a microvolume capacity of
liquid retention, and at least some of the zones comprising an
assay reagent.
[0019] As used herein, the term "microorganism" includes all
microscopic living organisms and cells, including without
limitation bacteria, mycoplasmas, rickettsias, spirochetes, yeasts,
molds, protozoans, as well as microscopic forms of eukaryotic
cells, for example single cells (cultured or derived directly from
a tissue or organ) or small clumps of cells. Microorganisms are
detected and/or enumerated not only when whole cells are detected
directly, but also when such cells are detected indirectly, such as
through detection or quantitation of cell fragments, cell-derived
biological molecules, or cell by-products.
[0020] As used herein, "microvolume" refers to a volume of less
than about 25 microliters, and includes volumes in the
sub-microliter range.
[0021] The terms "hydrophobic" and "hydrophilic" are herein given
the meanings commonly understood in the art. Thus, a "hydrophobic"
material has relatively little or no affinity for water or aqueous
media, while a "hydrophilic" material has relatively strong
affinity for water or aqueous media. The relative hydrophobicities
and hydrophilicities of the devices described herein are such as to
ensure partitioning of liquid samples substantially into the
described hydrophilic liquid-retaining zones upon application of
the sample. The required levels of hydrophobicity and
hydrophilicity may vary depending on the nature of the sample, but
may be readily adjusted based on simple empirical observations of
the liquid sample when applied to the devices.
[0022] The term "electrochemical" means a chemical indicator that
changes the resistance of conductance of the sample upon reaction
with the microorganism.
[0023] Other advantages of the invention will be apparent from the
following detailed description and the figures.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 is a perspective view of one embodiment of an assay
device.
[0025] FIG. 2 is a perspective view of an assay device having sets
of hydrophilic liquid-retaining zones varying in microvolume
capacity of liquid retention.
[0026] FIG. 3 is a schematic representation of an assay device
including a hydrophobic nanostructured film.
[0027] FIG. 4 is a schematic representation of an assay device in
which the hydrophilic liquid-retaining zones are constructed of
paper discs.
[0028] FIG. 5 is a perspective view of an assay device having two
sets of different volume discs and a coversheet.
[0029] FIG. 6 is a perspective view of an assay device having discs
within wells.
[0030] FIG. 7a is a perspective view of an assay device in which
the hydrophilic liquid-retaining zones are constructed of nonwoven
fiber loop material.
[0031] FIG. 7b is an expanded top view of the device depicted in
FIG. 7a.
[0032] FIG. 8a is a photograph of a top view of an assay device in
which the assay surface is hydrophilic.
[0033] FIG. 8b is a photograph of a top view of an assay device
with hydrophilic liquid-retaining zones and hydrophobic land
areas.
DETAILED DESCRIPTION
[0034] This invention relates to methods and devices for
partitioning of biological samples into microvolume liquid sample
aliquots for signal-based detection and enumeration of
microorganisms in liquid samples.
[0035] Among the problems encountered in the art relating to the
testing of liquid samples for microorganisms are relatively lengthy
incubation times, the need to undertake multiple pipetting
operations for aliquots being tested, and the need for a relatively
large volume of sample for testing.
[0036] The present invention provides a solution to these and other
problems associated with such testing. Methods and devices are
provided for partitioning a liquid sample into microvolume
compartments of a test device, with only minimal manipulation of
the liquid sample required of the laboratory technician or other
operator. In one embodiment, the invention provides absorbent disc
materials that are absorbent yet are biocompatible. These materials
are also compatible with fluorescent indicator systems. The
materials lend themselves easily to the manufacturing process.
[0037] The present inventors have discovered that the use of
microvolumes in signal-based detection of microorganisms in liquid
samples results in remarkably shorter incubation times required to
produce a detectable signal. Because shorter incubation times are
highly desirable in this field, this feature of the invention
provides a distinct advantage.
[0038] In addition to achieving shorter incubation times, the use
of microvolumes in the testing of liquid samples may allow for the
use of substantially smaller test samples. Very small volume test
samples are sometimes necessary due to very small volume sample
sources. Small volume liquid test samples are also sometimes
desirable, for example to ease handling or transport of the sample
to a testing facility.
[0039] The present inventors have developed a number of novel
devices for partitioning of biological liquid samples into discrete
microvolumes within hydrophilic liquid-retaining zones (also
referred to herein as "liquid-retaining zones" or "zones").
Non-limiting examples of these devices include: micro-embossed or
pressed films having a plurality of microcompartments, for example
microvolume wells, functioning as liquid-retaining zones, with the
area between the wells ("land area") being hydrophobic and the
wells being hydrophilic; nanostructured hydrophobic films in which
discrete liquid-retaining zones of the film are hydrophilic and are
adapted to retain microvolumes of a liquid sample for testing; and
devices having hydrophilic liquid-retaining zones and hydrophobic
land areas, where a given hydrophilic zone is fabricated from
hydrophilic fiber material and projects upward or downward from the
plane of the surrounding land area. One particularly useful example
of a device of the present invention is a device having hydrophilic
liquid-retaining zones and hydrophobic land areas, where a given
hydrophilic zone is fabricated from hydrophilic fiber material in
the form of a disc that projects upward from the plane of the
surrounding land area.
[0040] Advantageously, the above-summarized devices allow for the
testing of liquid samples using microvolume aliquots in a single
device, eliminating the need for separate vessels in such testing.
A test sample may be distributed among hundreds or even thousands
of discrete liquid-retaining zones, i.e., hydrophilic wells or
discs, substantially increasing the number of data points in a test
of the liquid sample.
[0041] A particularly useful application of these methods and
devices is in the growth-based detection and enumeration of
microorganisms in liquid test samples. Such growth-based detection
and enumeration is very important in the testing of food,
environmental, clinical, pharmaceutical, cosmetic, and other
samples for contamination by microorganisms. The methods and
devices of this invention allow for the efficient, accurate,
convenient, and cost-effective testing of such samples. A preferred
use of the methods and devices of this invention in such
microbiological testing is in MPN. In traditional MPN, a sample of
interest is serially diluted (10 fold) and pipetted in equal
amounts into replicate sets of tubes containing selective growth
media and chemical indicators. The tubes are incubated at elevated
temperature for about 24-48 hours followed by examination for
growth of organisms. A statistical formula, based on the number of
positive and negative tubes for each set, is used to estimate the
number of organisms present (per volume) in the initial sample As
currently used, this traditional method has several disadvantages.
It is labor intensive because of the multiple diluting and
pipetting steps required to perform the analysis. As a practical
matter, only replicate sets of about three to five tubes for each
dilution are commonly used. As a result, the 95% confidence limits
for an MPN estimate of microbial concentration using this method
are extremely wide. For example, a nine tube (3 ten-fold dilutions)
MPN estimate of 20 has 95% confidence limits ranging from 7 to
89.
[0042] The use of the methods and devices of the present invention
in MPN analysis overcomes several of the above-noted disadvantages.
The amount of labor is greatly reduced because no pipetting into
individual tubes is necessary, and very little or no agitation or
other manipulations are required. Instead, the liquid sample is
distributed to microvolume liquid-retaining zones by simply
contacting the liquid sample with the device. In addition, fewer
sample dilutions are necessary when large numbers of
liquid-retaining zones are present in the device. The relatively
large number of liquid-retaining zones also provides a more
accurate estimate of microbial concentration. This is because the
correspondingly larger number of data points provides a
correspondingly narrower confidence limit interval.
[0043] Accordingly, the present invention provides a method for
detecting and enumerating a microorganism in a liquid test sample.
The method involves distributing microvolumes of the test sample to
a plurality of hydrophilic liquid-retaining zones of an assay
device. The assay device may be any device that includes an assay
surface having a plurality of hydrophilic liquid-retaining zones,
where each zone has a microvolume capacity of liquid retention. The
device also includes a land area between the zones that is
hydrophobic and remains substantially free of liquid after the
biological sample has become distributed into the liquid-retaining
zones. Non-limiting examples of such assay devices include those
described herein.
[0044] The liquid-retaining zones in the assay device preferably
are of uniform size and each zone has a liquid-retention capacity
of about 0.01 to about 25 microliters of the liquid sample.
Preferably, each zone has a liquid retention capacity of about 0.1
to about 10 microliters, and more preferably about 1 to about 2
microliters. The assay device preferably contains between 1 and
about 100,000 liquid-retaining zones, more preferably about 10 to
about 10,000 zones, even more preferably about 200 to about 5,000
zones and most preferably about 400 to about 600 zones.
[0045] The use of a device having about 400 to about 600
hydrophilic liquid-retaining zones is particularly useful in the
context of testing a liquid sample for microorganism concentration
using MPN. Certain regulatory requirements may dictate that a
testing method must be able to detect one microorganism in a
one-to-five-milliliter sample. Such a sample size is standard in
the food processing industry for microbiological testing. Thus, for
example, an assay device having 500 hydrophilic liquid-retaining
zones, where each zone has a liquid capacity of about 2
microliters, would be very useful for testing a 1-ml sample. A
liquid-retention zone having a capacity of 2 microliters allows for
rapid development of a detectable signal in accordance with the
invention, and the use of about 400 to about 600 zones provides a
sufficiently large number of data points to substantially improve
the confidence interval for an MPN calculation. In addition, it is
feasible to perform a manual count of liquid-retaining zones
testing positive for the microorganism. Use of devices having
substantially more than 400 liquid-retaining zones may require, as
a practical matter, instrument-assisted or automated counting.
[0046] The liquid test sample may be any sample of interest, from
any source. The sample may be distributed to the plurality of
liquid-retaining zones directly, or the sample may be diluted
before distribution to the zones. The determination as to whether
sample dilution is necessary will depend on a variety of factors
such as sample source and age, and such determination is a routine
matter to those of skill in the art.
[0047] The liquid test sample may include selective nutrient growth
media for the microorganism of interest, and/or an indicator
substance that produces a signal in the presence of the growing
microorganism. Optionally, the nutrient medium may include a
gelling agent that assists in "encapsulating" the growing
microorganisms. Such gelling agents are known to those of skill in
the art, and include any water-absorbing material that becomes a
gel upon addition of an aqueous liquid.
[0048] Alternatively, one or both of the selective nutrient growth
media and the indicator substance may be present as a coating or
other deposition within a liquid-retaining zone, in amounts
sufficient to achieve desired concentrations when a microvolume of
the liquid test sample is distributed into the zone. Such a coating
may be achieved, for example, by placing or distributing a solution
of the nutrient medium (with or without gelling agent) and/or
indicator substance into the liquid-retaining zone and drying the
solution to produce a coating or deposition of the nutrient medium
and/or indicator substance in the zone. For devices in which the
liquid-retaining zones include discs, components of the media may
be present in the adhesive or other substance that binds the discs
(if applicable) to the substrate. The media ultimately diffuses
into the disc material.
[0049] A wide variety of selective growth media for a wide variety
of microorganisms of interest is known, as is a wide variety of
indicator substances for a wide variety of microorganisms, and any
of these media or indicator substances is suitable for use in the
method of the invention. An advantage of the present invention is
that soluble indicators can be used, since diffusion is prevented
by confinement of the aqueous biological sample liquid in the
hydrophilic liquid-retaining zones.
[0050] Various methods may be employed to distribute a liquid test
sample to the liquid-retaining zones. More than one method may be
applicable to a particular device, although the preferred method
may depend to some extent on the configuration of a particular
assay device. For example, for film devices containing hydrophilic
microvolume wells or for devices in which the zones comprise
hydrophilic discs projecting from the plane of the assay surface,
the sample may be poured or pipetted over the device and the sample
spread to the liquid-retaining zones by tilting or rocking the
device. The hydrophilic/hydrophobic interaction acts to retain the
sample on the discs and substantially excludes the sample from the
substrate.
[0051] Alternatively, the assay surface of the device can be
immersed in the sample as described in Example 4. Upon removal of
the assay surface from the liquid sample, liquid is retained in the
hydrophilic liquid-retaining zones and is likewise substantially
excluded from the hydrophobic land area.
[0052] After the sample is distributed to the hydrophilic
liquid-retaining zones of the assay device, various assays may be
carried out depending on desired uses. For microbial detection or
enumeration, the assay device may be incubated for a time
sufficient to permit at least one cell division cycle of the
microorganism. For these purposes, the device is generally
incubated at about 25.degree. C. to about 45.degree. C., more
preferably at about 30.degree. C. to about 37.degree. C. The
incubation time for bacterial detection will vary. The detection
time for most bacteria will range from about 20 minutes to about 24
hours in order to produce detectable growth as demonstrated by the
indicator substance in the incubated liquid test sample. Detection
time may vary depending on the growth rate and the number of
microorganisms present in the sample. Taking into account these
considerations, detection time for purposes of enumeration may be
as little as about 10 hours. This relatively short incubation time
represents a distinct advantage over detection methods currently
used, which typically require incubation times of about 24 hours or
more.
[0053] Following incubation of the assay device, the presence or
absence of the microorganism in the liquid-retaining zones (and
thus in the liquid test sample) is detected. The mode of detection
depends on the type of indicator substance used in the method. Any
indicator substance that is capable of providing a detectable
signal may be used. Such indicators include but are not limited to
fluorescent, chromogenic, luminescent, and electrochemical
indicators. The presence or absence of a microorganism in a zone
can be visually detected, with the naked eye or microscopically, if
a chromogenic or luminescent indicator is used. When the
liquid-retaining zones include discs, the indicator may be coated
or otherwise incorporated into the discs. The indicators may also
be included in the adhesive or other substance that binds the discs
(if applicable) to the substrate. In this instance, the indicator
ultimately diffuses into the disc material. If a fluorescent
indicator substance is used, equipment and methods for detecting a
fluorescent signal may be employed for detection. There are
numerous indicator substances and signal detection systems,
including systems for detecting electrochemical changes, known in
the art for detecting microorganisms. Any such substance or system
may be used in accordance with the present invention.
[0054] Fluorescent indicators are useful in the method of the
present invention because they may be detected at relatively low
concentrations. Suitable indicators include 4-methylumbelliferyl
phosphate and 4-methylumbelliferyl-.beta.-D-glucopyranoside,
L-phenylalanine-7-amido-4-- methylcoumarin. Others may include
4-methylumbelliferyl acetate and 4-methylumbelliferyl sulfate.
[0055] The detection of microorganisms in the liquid sample may
further involve the enumeration of a microorganism count in the
liquid test sample. In a preferred embodiment, the enumeration is
performed using MPN. Once the number of liquid-retaining zones
containing the microorganism of interest is determined, an MPN
calculation can be made using known MPN techniques. If desired, the
number of microorganisms in an individual zone can then be
determined using known techniques, for example, signal intensity
compared to a known standard, or by plating the contents of the
zone. Advantageously, the large number of liquid-retaining zones
used in the method of the invention allows for narrower intervals
for the 95% confidence limits in an MPN analysis of a liquid test
sample.
[0056] Because of the large number of liquid-retaining zones that
may be manufactured in a single device, it is possible to use a
single device in the detection and enumeration of multiple
microorganisms of interest, while retaining the advantages of the
invention. For example, a single liquid test sample can be tested
for the presence or concentration of E. coli and S. aureus. One
portion of an assay device can contain hydrophilic liquid-retaining
zones for the detection and enumeration of one of these
microorganisms, while a second set of zones can be directed to
detection and enumeration of another microorganism of interest.
This is accomplished, for example, by including
microorganism-specific nutrients and/or indicator substances in the
respective sets of liquid-retaining zones. Alternatively, all
liquid-retaining zones can contain assay reagents designed for the
simultaneous detection of multiple microorganisms. For example, E.
coli can be detected with a fluorescent indicator substance while,
at the same time, other coliforms are detected with a chromogenic
indicator substance.
[0057] When the liquid-retaining zone include discs, subsequent
tests may be conducted. For example, the discs can be removed from
the device and transferred into a test tube in order to
differentiate the specific microorganisms growing thereon.
[0058] In another embodiment, the distribution step can involve
distributing aliquots of the liquid test sample to a plurality of
hydrophilic liquid-retaining zones of an assay device, wherein the
assay device includes a plurality of sets of zones. Each set has
zones of uniform size, and the device has at least two sets of
zones. For example, the assay device can include a plurality of
lanes, with the hydrophilic liquid-retaining zones in a particular
lane having the same liquid-retention capacities. Alternatively,
the assay device may have a plurality of hydrophilic discs, as
described more fully below. Disc volumes may be constant within a
set, but may vary between sets. Whether the device has a plurality
of lanes or a plurality of disc sets, the liquid test sample may be
distributed into different test volume sizes within a single assay
device. In MPN, this feature provides a significant advantage in
that, for a highly concentrated sample, an appropriate volume size
may be selected and MPN analysis performed using a single
distribution step in a single device without the need for serial
dilutions.
[0059] As stated above, the methods of this invention may be
practiced using any assay device containing hydrophilic
liquid-retaining zones and a hydrophobic land area, depending on
the particular embodiment being practiced. The present inventors
have developed several novel devices suitable for use in the
methods of this invention. The following are non-limiting examples
of such devices.
[0060] Referring to FIG. 1, a device 10 comprises a substrate 12
having a plurality of hydrophilic liquid-retaining zones in the
form of hydrophilic microvolume wells 14. The substrate 12 can be
fabricated from any material in which microvolume wells can be
fashioned and in which the microvolume wells retain their
respective shapes throughout the useful life span of the device 10.
Substrate 12 can be fabricated, for example, from polymeric films
or other appropriate materials. Appropriate polymers include
without limitation polyethylene, polypropylene, polyimides,
fluoropolymers, polycarbonates, polyurethanes, and polystyrenes.
Should a particular polymer not be sufficiently hydrophilic, it can
be treated to impart hydrophilicity. For example, a surfactant can
be included in the film to impart hydrophilicity. Those skilled in
the art will recognize other means to impart surface
hydrophilicity. Microvolume wells 14 can be formed by any process
appropriate to the substrate 12 material. Such processes include
without limitation thermal embossing, cast embossing, laser
drilling, etching with reactive materials, or lamination of a sheet
of patterned material containing a plurality of small openings onto
a support film. Polyethylene or polypropylene films can be, for
example, pressed embossed or extrusion embossed, and can include
various pigments and surfactants.
[0061] Referring again to FIG. 1, the area 13 between microvolume
wells 14 ("land area") is fabricated to be hydrophobic. This serves
to prevent aqueous liquid from bridging between the microvolume
wells 14, thereby preventing cross-contamination. The land area 13
can be rendered hydrophobic in various ways. For example, the land
area on an extrusion embossed polyethylene film, that had been
rendered hydrophilic by incorporation of a surfactant, can be
rendered hydrophobic by transferring a thin layer of acrylated
silicone or other hydrophobic material to the land area. Those
skilled in the art will recognize other means to impart surface
hydrophobicity.
[0062] The device 10 can include any desired number of microvolume
wells. Additionally, the device 10 can include relatively large
reservoirs or other compartments adapted to hold larger volumes of
liquid for maintenance of an appropriate humidity level within the
device. Although the number of microvolume wells can be relatively
small (e.g., 2-50) for certain applications such as preliminary
screening, the small sizes of the microvolume wells allow
relatively large numbers of wells to be fabricated on a single
device 10. Preferably, the device has about 10 to about 10,000
liquid-retaining zones, even more preferably about 200 to about
5,000 zones, and most preferably about 400 to about 600 zones. The
device 10 can have a population of uniformly sized microvolume
wells 14 or wells of differing sizes. For example, a device 16 as
depicted in FIG. 2 can have sets (e.g., rows) of microvolume wells
in which volumes are constant within a set, but vary between sets.
As depicted in FIG. 2, the volumes can vary incrementally over an
array of sets of wells, with the smaller wells 18 holding
sub-microliter volumes and the larger wells 20 holding microliter
volumes. It is even possible for the largest wells in a device such
as depicted in FIG. 2 to include wells 22 that would not be
classified as "microvolume" wells. Such wells might have a
liquid-retention capacity, for example, of substantially more than
25 microliters.
[0063] In an alternative embodiment, the substrate 12 can be coated
with a hydrophobic nanostructured film. For example, polyimide or
fluoropolymer webs can be vapor coated with organic pigments, lead,
gold and other materials to create specific nanostructured films,
then made hydrophobic by coating with an organized molecular
assembly, such as octadecyl mercaptan or a fluorocarbon-hydrocarbon
thiol, as described in Patent Application WO 96/34697. Relatively
hydrophilic microvolume wells and other liquid-retaining zones may
be fashioned by removing the hydrophobic nanostructured elements
from selected areas of the substrate 12. This can be accomplished
in various ways, including without limitation
encapsulation/delamination and laser ablation as described in
Example 3, below.
[0064] A representative hydrophobic nanostructured film device 24
is depicted schematically in FIG. 3. Such devices can be loaded
with sample simply by dipping in an aqueous sample solution. To
this end, the device 24 can include a handle 26. Handle 26 allows
an operator to place the device 24 in a liquid sample to any
desired depth up to and including total immersion of the device 24
in the liquid sample, while avoiding contact of the operator's
fingers with the sample. Upon removal of device 24 from the sample,
liquid sample remains attached to the device only at the locations
of the hydrophilic liquid-retaining zones 28. Incubation and
detection are then performed as described above.
[0065] Assay devices also can be manufactured with hydrophilic
liquid-retaining zones constructed of hydrophilic absorbent
materials arrayed on a hydrophobic surface. For example, the zones
may have a plurality of absorbent discs having circular, oval,
square, polygonal or other appropriate shapes. Discs may be
constructed from a variety of materials, including cellulosics,
polyolefins, polyesters, and polyamides. Suitable cellulosics
include paper, wood pulp and rayon and may include chemically
modified cellulosics, such as cellulose esters. Suitable
polyolefins include hydrophilic polyethylene or hydrophilic
polypropylene fibers. Suitable polyamides include nylon. Suitable
polyesters include polyactic acid.
[0066] In the device 36 illustrated in FIG. 4, for example, discs
of cotton linter binderless paper 30 can be laminated to a
silicone-coated film 32 to form hydrophilic liquid-retaining areas
34 that project from the plane of the surrounding hydrophobic
surface 36. When present, discs 30 may be attached to the substrate
32 by various means known in the art, including without limitation,
by using adhesives. Preferred adhesives include water-insoluble
isooctyl acrylate adhesives as disclosed in U.S. Pat. No.
5,409,838. The area between the discs 38 is fabricated to be
hydrophobic. This serves to prevent aqueous liquid from bridging
the between discs 30, thereby preventing cross-contamination. The
area between the discs 38 may be rendered hydrophobic in any manner
described above with respect to devices having microvolume
wells.
[0067] The device can include any desired number of discs 30.
Additionally, the device can include relatively large reservoirs or
other compartments adapted to hold larger volumes of liquid for
maintenance of any appropriate humidity level within the device. As
described above with respect to devices comprising microvolume
wells, the devices may have a relatively small number (e.g., 2-50)
of discs for certain applications. Alternatively, the small sizes
of the discs allow relatively large numbers of discs to be affixed
to a single device. A single device may have as many as about
10,000 discs.
[0068] As shown in FIG. 5, the device can have a population of
uniformly sized discs or the discs may be of differing sizes. For
example, a device 40 may have sets (e.g., rows) of discs in which
volumes are constant within a set, but vary between sets. For
example, a certain embodiment can have 100 discs in which 50 discs
have a volume of 2 microliters 10 and 50 discs have a volume of 20
microliters. Other embodiments may have volumes that vary
incrementally over an array of sets of discs, with smaller discs 46
holding sub-microliter volumes and larger discs 44 holding
microliter volumes. The largest discs 42 may even have
liquid-retention capacities exceeding 25 microliters.
[0069] The materials of embodiments of the present invention that
include discs are biocompatible and may be used with fluorescent
indicators. The materials do not exhibit significant inherent
fluorescence that would interfere with the use of indicators. In
addition, the discs do not exhibit significant absorption at the
emission wavelength of the indicators. Also, the film substrate
should not exhibit fluorescent or light-absorbing properties that
would interfere with any fluorescent indicator system that is
used.
[0070] Optionally, the device may include a coversheet 48 to
protect the discs from contamination or desiccation once the sample
has been added to the device. The coversheet 48 may further be
sealed to the device along its edges with a pressure sensitive
adhesive.
[0071] In an alternative embodiment, as depicted in FIG. 6, the
device 50 may include discs 56 contained in microwells 54 that have
been made in the substrate 52 of the device. As with other
embodiments, the numbers and size of the discs may be varied.
[0072] Alternatively, the hydrophilic liquid-retaining zones may be
constructed of nonwoven fiber loop material that likewise protrudes
(projects) from the plane of the surrounding hydrophobic land area.
For example, as illustrated in FIGS. 7a and 7b, the assay device 60
may comprise a sheet of hydrophobic polypropylene film 62
containing arrays of protrusions 64 fabricated from
surfactant-containing polypropylene nonwoven fiber loop
material.
[0073] Assay reagents can be coated or otherwise deposited within
the liquid-retaining zones of the assay devices. Such assay
reagents can include without limitation nutrients for growth of
microorganisms; gelling agents; indicator substances such as
chromogenic indicators, fluorescent indicators, luminescent
indicators, and electrochemical indicators. The assay reagents can
be immobilized in the liquid-retaining zones by any of numerous
methods for immobilizing assay reagents on solid substrates known
to those of skill in the art. Such methods include for example
drying down assay reagent-containing liquids in the zones, as well
as other methods for noncovalently attaching biomolecules and other
assay reagents to a solid substrate. Alternatively, various methods
may be employed to covalently attach assay reagents to the
substrate 12 material within the wells 14 by methods well known to
those of skill in the art.
[0074] As discussed above, the presence of hydrophilic
liquid-retaining zones with microvolume liquid-retention capacity
in an assay device allows for separation of a liquid test sample
into a relatively large number of test volumes. The ability to
separate a liquid sample into microvolume aliquots and to perform
MPN or other assays without cross-contamination between aliquots is
an advantage of the present method and devices.
[0075] All references and publications cited herein are expressly
incorporated herein by reference into this disclosure. Particular
embodiments of this invention will be discussed in detail and
reference has been made to possible variations within the scope of
this invention. There are a variety of alternative techniques and
procedures available to those of skill in the art that would
similarly permit one to successfully practice the intended
invention.
EXAMPLES
[0076] The following examples are offered to aid in understanding
of the present invention and are not to be construed as limiting
the scope thereof. Unless otherwise indicated, all parts and
percentages are by weight.
Example 1
Embossed Film Culture Devices
[0077] Embossed film culture devices containing a plurality of
microcompartments and capable of being used for the detection of
microorganisms in a liquid test sample were constructed as
described in this example.
[0078] The hydrophilic liquid-retaining zones can be formed in a
substrate by a number of processes, examples of which are thermal
embossing, cast embossing, laser drilling, and by etching the
surface with a reactive material. Detailed descriptions of how to
make recesses or microvolume wells in polymeric films are provided
in U.S. Pat. Nos. 5,192,548; 5,219,462; 5,344,681; and 5,437,754.
The following descriptions are representative of specific embossed
film culture devices used in the subsequent examples.
[0079] A. Pressed Embossed Films Containing a Plurality of
Microvolume Wells
[0080] Polyethylene (Eastman Chemical Company Resin #18BOA)
containing 10% by weight TiO.sub.2 (50% TiO.sub.2/50% Polyethylene
Pigment Concentrate) and 0.5% by weight Triton X-35 Surfactant
(Sigma Chemical Company) or polypropylene was extrusion cast into a
film (4-mil thickness). The film was cut into sheets and stacked
(.about.20 sheets) onto photolithographically etched magnesium
alloy tooling as described in U.S. Pat. No. 5,219,462, designed to
form a plurality of microvolume wells. The etched magnesium tooling
contained protuberances arranged in the patterns described in
subsequent examples. The stacked polyethylene sheets were embossed
on a heated hydraulic press (132.degree. C., 1.4 N/m.sup.2, 120
second dwell) as described in U.S. Pat. No. 5,219,462. The samples
were allowed to cool, at which time the tooling was removed to
provide a single layer film containing the "negative" image of the
tooling.
[0081] B. Extrusion Embossed Films Containing a Plurality of
Microvolume Wells
[0082] Photolithographically etched magnesium master tooling was
attached to a steel roll using pressure-sensitive transfer
adhesive. The polyethylene, pigment, and surfactant composition
described in Example 1A was blended together and extrusion cast
onto the roll as described in U.S. Pat. No. 5,192,548. Embossed
films lacking the Triton X-35 surfactant were also prepared in this
manner.
[0083] C. Extrusion Embossed Films with Hydrophobic "Land" Area
[0084] Extrusion embossed polyethylene films containing Triton X-35
Surfactant were prepared according to Example 1B. The area between
microvolume wells ("land" area) was rendered hydrophobic by
transferring a thin layer of acrylated silicone (Goldschmidt FC
711) containing 4.8% of a cross linking agent (Darocur 1173) with a
roll-to-roll coating apparatus (Straub Design Co.). The hydrophobic
coating was cured by exposing the film to ultraviolet radiation
under nitrogen atmosphere using a Fusion Systems UV lamp with an H
bulb providing a dosage of 85 millijoules/cm.sup.2.
Example 2
Method of Inoculation
(Method Utilizing Plurality of Microvolume Wells)
[0085] A. Inoculation with Indicator Solution
[0086] An aqueous solution containing phenol red indicator (to
provide contrast) was applied by pipette onto silicone-treated and
silicone-untreated polyethylene embossed films (Examples 1C and 1B,
respectively) containing a plurality of microvolume wells (about
1.3 .mu.l/well). The microvolume wells were arranged in a hexagonal
array (.about.19 wells/cm.sup.2) and each well was in the shape of
an inverted truncated cone, having a diameter of approximately 1.9
mm at the surface and 1.0 mm at its depth, which was about 1.1 mm.
The microvolume wells were filled as described in U.S. Pat. No.
5,219,462 by drawing the diluted sample solution down the film with
the edge of a razor blade. The samples treated with the hydrophobic
silicone coating were shown to partition liquid into individual
microvolume wells without fluid bridging between the wells, whereas
bridging of liquid was observed on the untreated films.
[0087] B. Inoculation with Microorganism-Containing Samples
[0088] The method of inoculating embossed film culture devices
containing a plurality of microvolume wells with
bacteria-containing media was demonstrated in this example. The
inoculated devices were utilized to detect and enumerate E. coli
bacteria.
[0089] An overnight broth culture of E. coli ATCC 51813
(.about.10.sup.9 CFU/ml in Tryptic Soy Broth (TSB) media) was
serially diluted into Violet Red Bile (VRB) media (7.0 g/l Bacto
peptone, 3.0 g/l yeast extract, and 1.5 g/l bile salts) containing
4-methylumbelliferyl-.beta.-D-glucuronide (0.5 mg/ml) (MUG,
Biosynth International, Naperville, L). The dilution was prepared
to the approximate bacterial concentration of 100 CFU/ml. The
diluted sample (0.5 ml) was applied by pipette onto
silicone-treated and silicone-untreated polyethylene embossed films
(406 microvolume wells) as described in Example 2A. The inoculated
embossed films 73 were placed inside petri dishes, and incubated
for 12 hours at 37.degree. C. Twenty-eight microvolume wells 74
exhibited sharp, discrete fluorescent spots on the silicone-treated
film 76 (FIG. 8b). In contrast, significant well-to-well
cross-contamination was observed on the untreated film (FIG. 8a).
For the silicone-treated film, 28 positive wells corresponds to a
most probable number (MPN) of 58 CFU/ml, as calculated using the
formula MPN=N 1n (N/N-X) where N is the total number of filled
wells and X is the total number of wells showing a positive
reaction.
[0090] The results of this example show that microorganisms can be
readily detected and enumerated using an embossed film culture
device having a plurality of microvolume wells and that
well-to-well cross-contamination can be eliminated by coating a
hydrophobic substance on the land area between wells.
Example 3
Nanostructured Film Culture Devices
[0091] Nanostructured film culture devices containing a plurality
of hydrophilic microvolume liquid-retaining zones arrayed on a
substrate coated with a hydrophobic nanostructured film were
constructed as described in this example.
[0092] A. Nanostructured Film
[0093] Processes for generating nanostructured surfaces are
disclosed in U.S. Pat. Nos. 4,812,352 and 5,039,561. Briefly, the
organic pigment C.I. Pigment Red 149 (American Hoechst-Celanese,
Somerset, N.J.) was vacuum vapor deposited to a thickness of 250 nm
onto a 0.0125-nm thick, 30.times.30 cm sheet of polyimide web,
which had previously been metal vapor coated with 700 .ANG. of
lead. The sample was annealed in a vacuum oven at 264.degree. C.
for greater than 30 minutes, which was sufficient to convert the PR
149 pigment to a dense distribution of discrete crystalline
whiskers oriented perpendicular to the web substrate. The whiskers
were vapor coated with a mass equivalent thickness of 2500 .ANG. of
gold, which resulted in a conformational coating of gold particles,
.about.2 .mu.m tall and .about.0.15 mm in diameter, with an areal
number density of 5 per (.mu.m).sup.2, as determined by SEM.
[0094] Alternatively, the polyimide was replaced with a transparent
fluorenone polyester (FPE, 3M Co.) and vapor coated with 50 .ANG.
of gold, which prevented surface charging during deposition of the
PR 149, yet remained essentially transparent.
[0095] B. Hydrophobic Nanostructured Film
[0096] The nanostructured film was then made hydrophobic by
immersing in a 0.1 mM solution of
C.sub.8F.sub.17(CH.sub.2).sub.11SH in ethanol for 4 hours, followed
by rinsing with pure ethanol and air drying. The resulting highly
hydrophobic surface was measured to have identical advancing and
receding contact angles of 178.degree. for water. This process is
described in
[0097] C. Nanostructured Film Culture Devices
[0098] Nanostructured film culture devices were constructed by
using an encapsulation/delamination of nanostructured films process
described in U.S. Pat. No. 5,336,558. Briefly, pieces of the
nanostructured hydrophobic film were cut into 1.5.times.2.0 cm
strips. A 0.25-mm thick perforated steel sheet, having a square
array of 1.5-mm diameter holes spaced .about.4 mm apart, was laid
over the nanostructured side of the strips. A fast curing vinyl
polysiloxane encapsulate (3M EXPRESS dental impression material, 3M
Co.) was applied liberally over the steel plate to cause the
material to penetrate through the holes and encapsulate the
nanostructured whiskers. After several minutes, the impression
material was set and the steel sheet was removed, thereby removing
the nanostructured elements cleanly from the polyimide web only at
the location of the array of holes. The exposed metal-coated
polyimide substrate in the areas under the holes was relatively
hydrophilic compared to the remainder of the surface. This was
demonstrated by dipping the strips into an aqueous solution and
observing that small droplets remained attached only at the array
of exposed spot or zone areas.
[0099] Alternatively and preferably, laser ablation was utilized
for removing the nanostructured elements from the polyimide web to
provide the desired array of relatively hydrophilic
liquid-retaining zones. The strips of nanostructured hydrophobic
film were ablated with a Nd-YAG laser with a collimated beam 1 mm
in diameter and operated in a Q-switched mode with approximately 2
mjoule, 60 nanosecond pulses. Single pulses were used to ablate
rows of 1-mm diameter zones on 4- and 5-cm center-to-center
spacing. Larger zones, .about.1.6.times.1.6 mm square, were
produced by overlapping a 3.times.3 matrix of nine 1-mm diameter
zones. The resulting nanostructured film culture device with 40
(4.times.10) zones was submersed in water for 1 minute initially to
make the ablated zone areas hydrophilic. Upon withdrawing the
plate, each of the 40 zones had an .about.1 -mm diameter,
hemispherical droplet attached to it.
Example 4
Method of Inoculation
(Method Utilizing
Nanostructured Film Culture Devices)
[0100] A. Inoculation with Aqueous Liquid Sample
[0101] To inoculate and measure the amount of liquid selectively
retrieved by the nanostructured film culture devices (Example 3C),
a plate with 12 hydrophilic liquid-retaining zones, ranging in size
from 1 to 2.5 mm in diameter (average 2 mm), was dipped into pure
water and the amount of water extracted onto the zones was measured
gravimetrically. The plate was first dipped at a slow withdrawal
rate of .about.3 seconds/cm. After withdrawal, the back of the
plate was touched against tissue paper to remove any water droplets
clinging to the back of the polyimide plate, and the plate was then
placed on a mass balance (0.1 mg minimum sensitivity) and the mass
recorded 15 seconds later. This was repeated 15 times. The mean and
standard deviation of the mass of the 12 water zones was 3.7.+-.0.2
mg, giving an average zone volume of 0.310 .mu.l.+-.5%. The
procedure was then repeated a fast withdrawal rate with the plate
pulled from the water in a time estimated to be 0.1 second. At this
rate, the amount of liquid that remained on the hydrophilic zones
was larger, because the liquid did not have time to "stretch" and
dynamically equilibrate. The mean and standard deviation of the 15
trials was 6.0.+-.0.5 mg, giving an average zone volume of 0.500
.mu.l.+-.12%.
[0102] B. Inoculation with S. Aureus-Containing Samples
[0103] The method of inoculating nanostructured film culture
devices containing a plurality of microvolume liquid-retaining
zones with bacteria-containing media was demonstrated in this
example. The inoculated devices were utilized to detect and
enumerate S. aureus (Example 4B) and E. coli (Example 4C)
bacteria.
[0104] A mixture (5 .mu.l) of molten (.about.60.degree. C.)
bacteriological growth media BHI (Brain Heart Infusion, Becton
Dickinson and Co.) and agar (1.2% weight/volume) was spotted onto
the hydrophilic zones of the nanostructured film culture devices
prepared as described in Example 3C. The agar "spots" were allowed
to cool and solidify at room temperature. One plate was dipped
briefly into a growing culture of Staphylococcus aureus
(.about.10.sup.8 cells/ml) in BHI broth medium. Other plates were
dipped similarly into 1:10 and 1:1000 dilutions of the S. aureus
culture, representing 10.sup.7 and 10.sup.5 cells/ml, respectively.
The plates were placed into plastic petri dishes containing
water-saturated filter paper to maintain humidity, and incubated at
37.degree. C. for 4 hours. The plates were then dipped into a
solution containing 900 .mu.l of HEPES Buffer (Sigma Chemical Co.,
pH 8.0); 120 Pl of fluorescent indicator solution (1.0 mg/ml
Boc-Val-Pro-Arg-AMC HCl (NovaBiochem, San Diego, Calif.) in 72 mM
triethanolamine, 144 mM NaCl, pH 8.4); and, 30 .mu.l human
prothrombin (Sigma Chemical Co., 50 mg/ml in 5 mM Tris buffer, 50
mM NaCl, pH 8.0). The plates were incubated for one additional hour
under the same conditions described above and then examined under
UV light (.about.366 nm, Mineralite, UVP, Inc., San Gabriel,
Calif.). The zones containing agar media, bacterial suspension, and
indicator solution all showed visible, intense bluish fluorescence
as compared to no visible fluorescence in the control samples,
which were prepared without any added bacteria. No
cross-contamination between zones was observed.
[0105] C. Inoculation with E. coli-Containing Samples
[0106] Agar medium was prepared by combining the following
ingredients: pancreatic digest of gelatin (10 g, Peptone G,
Acumedia Manufacturers, Inc., Baltimore, Md.); Bacto Bile Salts
Number 3 (2.5 g, Difco Labs, Detroit, Mich.); Agar (6 g, Difco
Labs); and deionized water (500 ml). The mixture was stirred and
heated to 100.degree. C. until the agar melted, autoclaved at
121.degree. C. for 15 minutes to sterilize, and then cooled to room
temperature to solidify. An IPTG stock solution was prepared from
filter-sterilized (0.2 mm) isopropyl-.beta.-D-galactoside (IPTG,
CalBiochem Corp., La Jolla, Calif.) in deionized water (200 mg/ml)
and stored at -20.degree. C. until use. A MU-Gal stock solution was
prepared from 4-methylumbelliferyl-.beta.-D-galactoside (MU-Gal) in
N,N-dimethylformamide (10 mg/ml) and stored at 4.degree. C. until
use. Immediately before use the agar medium was melted at
100.degree. C. and 25 ml was transferred to a sterile 50-ml tube.
The IPTG stock solution (12.5 ml) and the MU-Gal stock solution
(150 mil) were then mixed into the cooled (-60.degree. C.) agar
suspension. The mixture was immediately transferred (4-.mu.l
aliquots) to the nanostructured film culture device zones as
described in Example 4B. After cooling to room temperature, the
plates were dipped into a mid-exponential growing culture of E.
coli ATCC 51813 (.about.10.sup.8 cells/ml in LB medium 3) and
incubated in individual humidified petri dishes at 37.degree. C.
After 4 hours of incubation, the plates were checked for
fluorescence with a Mineralite UV lamp. The inoculated zones
exhibited slightly more fluorescence than that observed in the
uninoculated zones. The plates were then incubated for an
additional 16 hours and rechecked. The inoculated zones showed
significantly more blue fluorescence than the uninoculated zones.
The plate prepared with clear-film substrate (Example 3A utilizing
FPE) was particularly convenient to measure because it could be
illuminated from one side and viewed or photographed from the other
side. No cross-contamination between zones was observed.
Example 5
Absorbent Disc Culture Devices
[0107] Absorbent disc culture devices containing a plurality of
hydrophilic absorbent discs arrayed on a hydrophobic surface and
capable of being used for the detection and enumeration of
microorganisms in a liquid test sample were constructed as
described in this example.
[0108] A. Culture Devices Constructed with Absorbent Paper
Discs
[0109] A sheet of absorbent material (Schleicher & Schuell
Grade 903 Paper; absorbs about 4.5 g of water/100 cm.sup.2) was
laminated to a Rexam silicone-coated film (Grade #15819 D 2MIL CL
PET MM34P/000 having a clear 2-mil thick polyester film as a
substrate, Rexam Release, Oak Brook, Ill.) with an acrylate
pressure sensitive adhesive (PSA) containing the chromogenic
indicator 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) (Amresco,
Solon, Ohio). The material was saturated with tryptic soy broth
(TSB) growth nutrient containing 0.5% of the fluorescent indicators
4-methylumbelliferyl phosphate (100 .mu.g/ml, Sigma, St. Louis,
Mo.) and 4-methylumbellifery-.alpha.-D-glucoside (50 .mu.g/ml,
Sigma), wiped with a wire-wound rod, and dried at 110.degree. C.
for 10 minutes. Circular discs approximately 0.635 cm in diameter
were punched out of the laminate and the silicone-coated film
backing removed. The discs with PSA were then adhered to another
sheet of Rexam silicone-coated film so that the discs were
patterned in equally spaced parallel rows. The film and discs that
the discs were patterned in equally spaced parallel rows. The film
and discs assemblies were gamma irradiated to a level of 8.9 kGy,
cut to size, and then taped into a petri dish such that each dish
contained a piece of film with 20 discs. Based on gravimetric
measurements, each disc in the resulting culture devices had a
capacity to retain about 40 .mu.l of liquid.
[0110] B. Culture Devices Constructed with Various Polymeric
Absorbent Disc Materials
[0111] Silicone-coated polyester release liner (as described in
Example 5A) and biaxially-oriented polypropylene (BOPP) film
(1.6-mil thickness, 3M Co., St. Paul, Minn.) were cut into
7.6-cm.times.10.2-cm rectangular pieces. Pieces of each material
were joined at one end with SCOTCH.TM. brand double-coated adhesive
tape (No. 665, 3M Co.) with the silicone-coated side of the release
liner oriented toward the BOPP film. The release liner functioned
as the base of the culture device and the BOPP film functioned as
the top film.
[0112] Sheets of the following polymeric absorbent materials were
laminated onto separate layers of an acrylate adhesive (No. Y966,
3M Co.): Product No.10201-9 cellulose (Dexter, Windsor Locks,
Conn.), Grade 903 cotton lint paper (Schleichter & Schuell,
Keene, N.H.), Product No. P-110 Supersorbent polyolefin (3M Co.),
Product No. 9208283 polyester (Veratec, Walpole, Mass.), Spunbond
Nylon (4 ounces per square yard) polyamide (Cerex Advanced Fabrics,
Cantonment, Fla.), and polylactic acid polyester [absorbent
nonwoven meltblown web prepared from polylactic acid pellets
(HEPLON.TM., Chronopol, Inc., Golden, Col.) as described in U.S.
Pat. No. 5,230,701]. Circular discs (approximately 0.64-cm
diameter) were punched out of the resulting laminates and adhered
to the silicone-coated side of the polyester release liner. Each
culture device contained 12 discs equally spaced in a 3.times.4
array of parallel rows. After construction was completed, the
culture devices were gamma irradiated to a level of 8 kGy. Each
disc had the capacity to retain about 10 .mu.ls.
Example 6
Method of Inoculation
(Method Utilizing Absorbent Disc Culture Devices)
[0113] The method of inoculating absorbent disc culture devices
containing a plurality of microvolume liquid-retaining zones
(absorbent discs) with bacteria-containing media was demonstrated
in this example. The inoculated devices were utilized to detect and
enumerate E. coli bacteria.
[0114] A. Microbial Assay Using Culture Devices Constructed with
Absorbent Paper Discs (from Example 5A)
[0115] A culture of E. coli ATCC 51813 was diluted to produce
suspensions containing about 10 CFU/ml and 1 CFU/ml. Samples (1 to
2 ml) of the suspensions were applied by pipette to the absorbent
disc culture devices described in Example 5A. Excess liquid sample
was poured off, thereby leaving about 0.8 ml retained on the device
(20 discs, about 40 .mu.l of liquid per disc). The inoculated
devices were incubated at 35.degree. C. for 23 hours and inspected
under ultraviolet light. The number of discs exhibiting
fluorescence was counted for each device and most probable number
(MPN) values calculated using the formula described in Example 2B.
The MPN per milliliter was calculated by dividing the value
obtained by the total volume of the sample (0.8 ml). Results are
provided in Table 1 and are compared with counts obtained from
standard testing with Coliform Count PETRIFILM.TM. Plates (3M Co.).
The fluorescent discs often showed the red TTC color, usually as
discrete spots within the discs. No cross-contamination between
absorbent discs was observed.
1TABLE 1 Enumeration of Microorganisms (E. coli) Bacterial
Suspension Positive Discs Coliform Count (- CFU/ml) (Out of 20) MPN
(CFU/ml) PETRIFILM .TM. 10 17 47 22 10 19 74 24 1 2 2.6 5 1 3 4.1
4
[0116] The results of this example show that absorbent disc culture
devices having a plurality of absorbent discs arrayed on a
hydrophobic film can be easily inoculated with bacteria-containing
liquid samples and that the inoculated devices can be utilized for
the detection and enumeration of E. coli, with the values obtained
being comparable with those obtained from commercial Coliform Count
PETRIFILM.TM. Plates.
[0117] B. Microbial Assay Using Culture Devices Constructed with
Various Polymeric Absorbent Disc Materials (from Example 5B)
[0118] Cultures of different bacterial strains (Table 2) were grown
overnight at 35.degree. C. in 5 ml of TBS media. A 0.01-ml volume
of each culture was diluted into 99 ml of sterile Butterfield's
diluent (Fisher Scientific, Pittsburgh, Pa.), to obtain initial
10.sup.-4 dilutions of the original bacterial suspensions. Three
subsequent 10-fold dilutions (10.sup.-5, 10.sup.-6, and 10.sup.-7)
of the bacterial suspensions were made in Standard Methods Broth
containing the following ingredients: Pancreatic Digest of Casein
(10.0 g/l, Difco Labs), Yeast Extract (5.0 g/l, Difco Labs),
Glucose (2.0 g/l, Becton Dickinson and Co., Cockeysville, Md.), and
the fluorescent indicator 4-methylumbelliferylpho- sphate (0.05
g/l, Biosynth International). With the top covers of the culture
devices (from Example 5B) raised, three 0.01-ml aliquots of the
10.sup.-5, 10.sup.-6, and 10.sup.-7 dilutions were transferred by
pipette onto nine individual discs on each of the devices. An
equivalent volume of sterile medium was transferred to the
remaining three discs on each device to serve as sterility
controls. The top covers of the inoculated culture devices were
closed, and the devices placed into GLAD-LOCK.RTM. ZIPPER.TM.
storage bags (First Brands Corp., Danbury, Conn.), each containing
a moistened paper towel. The bags were placed in a 35.degree. C.
incubator for 24 hours, after which the culture devices were
examined under a long-wave ultraviolet light source. Positive
growth and detection was evidenced by a bluish fluorescence.
Results are provided in Table 2.
2TABLE 2 Growth and Detection of Bacteria on Various Disc Materials
No. of Positive Discs (at designated Disc dilutions) Bacterial
Strain Material 10.sup.-5 10.sup.-6 10.sup.-7 Control Escherichia
coli P18 Cellulose 3 3 3 0 (Clinical isolate; (Dexter) obtained
Paper 3 3 3 0 from Centers for Disease (S & S) Control and
Prevention, Polyolefin 3 3 3 0 Atlanta, GA) (3M) Polyester 0 0 0 0
(Veratec) Polyamide 3 3 3 0 (Cerex) Bacillus sp. L11 Cellulose 3 3
0 0 (Food isolate) (Dexter) Paper 3 2 0 0 (S & S) Polyolefin 0
0 0 0 (3M) Polyester 0 0 0 0 (Veratec) Polyamide 1 0 0 0 (Cerex)
Polylactic 2 0 1 0 Acid Polyester (Chronopol) Streptococcus faecium
Cellulose 3 1 0 0 P92 (Dexter) (Clinical isolate; Paper 3 0 0 0
obtained (S & S) from Centers for Disease Polyolefin 0 0 0 0
Control and Prevention) (3M) Polyester 0 0 0 0 (Veratec) Polyamide
3 0 0 0 (Cerex) Polylactic 3 1 0 0 Acid Polyester (Chronopol)
Hafnia alvei 3026 Cellulose 3 3 1 0 (Obtained from the (Dexter)
University of Paper 3 2 0 0 Minnesota) (S & S) Polyolefin 1 0 0
0 (3M) Polyester 0 0 0 0 (Veratec) Polyamide 3 1 0 0 (Cerex)
Polylactic 3 3 0 0 Acid Polyester (Chronopol)
[0119] The results of this example show that culture discs
constructed with an array of discs made from different absorbent
materials can be utilized for the detection of various bacterial
strains. Especially effective in this example were absorbent discs
made from cellulosic, polyamide, and polyolefin materials.
Example 7
Method of Inoculation
(Method Utilizing Hydrophilic Fiber Culture Devices)
[0120] The method of constructing and inoculating hydrophilic fiber
culture devices containing a plurality of microvolume
liquid-retaining zones (nonwoven fiber loops) with indicator
solution and with bacteria-containing media were demonstrated in
this example. The inoculated devices were utilized to detect and
enumerate E. coli bacteria.
[0121] A. Device Construction
[0122] A sheet of hydrophobic polypropylene film containing an
array of relatively hydrophilic surfactant-containing polypropylene
nonwoven fiber loop protrusions was prepared as described in U.S.
Pat. No. 5,256,231. The sheet was cut to size and taped to the
bottom of a petri dish to form a culture device. Each device
contained film having about 200 fiber loop protrusions patterned
hexagonally in equally spaced parallel rows. Each hemispherical
protrusion was hexagonal at its base (side length about 3 mm,
height about 2 mm) and had a capacity to retain about 15 .mu.l of
liquid.
[0123] B. Inoculation with Indicator Solution
[0124] A sample (1 ml) of phosphate buffer ("Butterfield", Fisher
Scientific) containing phenol red indicator (to provide contrast)
was applied by pipette onto the film in the center of the device.
The liquid was observed to wick into the hydrophilic fiber loop
protrusions radially from the point of inoculation. The liquid was
observed to quickly partition into the loop protrusions while
"draining" from the hydrophobic polypropylene land areas. About 65
of the 200 protrusions were filled. No bridging of the colored
liquid across the land areas between loop protrusions was
observed.
[0125] C. Inoculation with Microorganism-Containing Sample
[0126] An overnight culture of E. coli (ATCC 51813, .about.10.sup.9
CFU/ml in TSB media) was serially diluted into VRB Media (7.0 g/l
Bacto peptone, 3.0 g/l yeast extract, 1.5 g/l bile salts)
containing 4-methylumbelliferone-.beta.-D-glucuronide (0.5 mg/ml).
A dilution of 10.sup.-8 was prepared corresponding to a bacterial
concentration of about 10 CFU/ml. A sample (1 ml) was pipetted onto
the film in the center of a hydrophilic fiber culture device
(Example 7A) as described in Example 7B. After inoculation, the
petri dish was covered and sealed using electrical tape to prevent
evaporation. The device was then inverted and incubated at
37.degree. C. for 19 hours. After incubation, the number of
protrusions exhibiting fluorescence under 365 nm irradiation were
counted. Five separate, discrete protrusions were observed to have
significant fluorescence. No fluorescence was observed between
protrusions, thereby indicating no cross-contamination. The MPN
value was calculated to be 5 CFU/ml, using the formula described in
Example 2B.
[0127] The results of this example show that hydrophilic fiber
culture devices having a plurality of hydrophilic fiber zones
arrayed on a hydrophobic film can be easily inoculated with
bacteria-containing liquid samples and that the inoculated devices
can be utilized for the detection and enumeration of E. coli.
[0128] Various modifications and alterations of this invention will
be apparent to those skilled in the art without departing from the
scope and spirit of this invention, and it should be understood
that this invention is not limited to the illustrative embodiments
set forth herein.
* * * * *