U.S. patent application number 15/945228 was filed with the patent office on 2018-08-09 for microorganism concentration process and device.
The applicant listed for this patent is 3M INNOVATIVE PROPERTIES COMPANY. Invention is credited to Manjiri T. Kshirsagar, Andrew W. Rabins.
Application Number | 20180224360 15/945228 |
Document ID | / |
Family ID | 42828630 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180224360 |
Kind Code |
A1 |
Kshirsagar; Manjiri T. ; et
al. |
August 9, 2018 |
MICROORGANISM CONCENTRATION PROCESS AND DEVICE
Abstract
A process for capturing or concentrating microorganisms for
detection or assay comprises (a) providing a concentration device
comprising a sintered porous polymer matrix comprising at least one
concentration agent that comprises diatomaceous earth bearing, on
at least a portion of its surface, a surface treatment comprising a
surface modifier comprising ferric oxide, titanium dioxide,
fine-nanoscale gold or platinum, or a combination thereof; (b)
providing a sample comprising at least one microorganism strain;
and (c) contacting the concentration device with the sample such
that at least a portion of the at least one microorganism strain is
bound to or captured by the concentration device.
Inventors: |
Kshirsagar; Manjiri T.;
(Woodbury, MN) ; Rabins; Andrew W.; (St. Paul,
MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
3M INNOVATIVE PROPERTIES COMPANY |
St. Paul |
MN |
US |
|
|
Family ID: |
42828630 |
Appl. No.: |
15/945228 |
Filed: |
April 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13257406 |
Sep 19, 2011 |
9964474 |
|
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PCT/US10/28111 |
Mar 22, 2010 |
|
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15945228 |
|
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61166262 |
Apr 3, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 1/40 20130101; G01N
1/44 20130101 |
International
Class: |
G01N 1/40 20060101
G01N001/40 |
Claims
1-18. (canceled)
19. A concentration device comprising a sintered porous polymer
matrix comprising at least one concentration agent that comprises
diatomaceous earth bearing, on at least a portion of its surface, a
surface treatment comprising a surface modifier comprising ferric
oxide, titanium dioxide, fine-nanoscale gold or platinum, or a
combination thereof.
20. A process for preparing a concentration device comprising (a)
providing a mixture comprising (1) at least one particulate,
sinterable polymer and (2) at least one particulate concentration
agent that comprises diatomaceous earth bearing, on at least a
portion of its surface, a surface treatment comprising a surface
modifier comprising ferric oxide, titanium dioxide, fine-nanoscale
gold or platinum, or a combination thereof; and (b) heating the
mixture to a temperature sufficient to sinter the polymer, so as to
form a sintered porous polymer matrix comprising the particulate
concentration agent.
21. The concentration device of claim 19, wherein each of the at
least one concentration agent has a negative zeta potential at a pH
of 7.
22. The concentration device of claim 19, wherein the sintered
porous polymer matrix comprises a tortuous path.
23. The concentration device of claim 19, wherein the sintered
porous polymer matrix comprises at least one thermoplastic
polymer.
24. The concentration device of claim 23, wherein the thermoplastic
polymer is selected from olefin homopolymers, olefin copolymers,
copolymers of olefins and other vinyl monomers, and combinations
thereof.
25. The concentration device of claim 24, wherein the thermoplastic
polymer is selected from olefin homopolymers and combinations
thereof.
26. The concentration device of claim 24, wherein the olefin
homopolymer is polyethylene.
27. The concentration device of claim 19, wherein the surface
modifier is selected from the group consisting of fine-nanoscale
gold, fine-nanoscale platinum, fine-nanoscale gold in combination
with at least one metal oxide, titanium dioxide, titanium dioxide
in combination with at least one other metal oxide that is not
titanium dioxide, ferric oxide, and ferric oxide in combination
with at least one other metal oxide that is not ferric oxide.
28. The concentration device of claim 27, wherein the at least one
metal oxide is selected from the group consisting of ferric oxide,
titanium dioxide, zinc oxide, aluminum oxide, and combinations
thereof.
29. The concentration device of claim 27, wherein the surface
modifier is selected from the group consisting of fine-nanoscale
gold, fine-nanoscale platinum, fine-nanoscale gold in combination
with at least ferric oxide or titanium dioxide, titanium dioxide,
titanium dioxide in combination with at least ferric oxide, and
ferric oxide.
30. The concentration device of claim 29, wherein the surface
modifier is selected from the group consisting of fine-nanoscale
gold, fine-nanoscale platinum, fine-nanoscale gold in combination
with ferric oxide or titanium dioxide, titanium dioxide, and
titanium dioxide in combination with ferric oxide.
31. The concentration device of claim 30, wherein the surface
modifier is selected from the group consisting of fine-nanoscale
gold, fine-nanoscale gold in combination with ferric oxide or
titanium dioxide, and titanium dioxide in combination with ferric
oxide.
32. The concentration device of claim 31, wherein the surface
modifier is selected from the group consisting of fine-nanoscale
gold, and fine-nanoscale gold in combination with ferric oxide or
titanium dioxide.
33. The concentration device of claim 29, wherein the at least one
concentration agent is embedded in or on the surface of the porous
polymer matrix.
34. The concentration device of claim 19, wherein the at least one
concentration agent comprises diatomaceous earth bearing, on at
least a portion of its surface, fine-nanoscale gold or
fine-nanoscale platinum deposited on the diatomaceous earth using
physical vapor deposition.
Description
STATEMENT OF PRIORITY
[0001] This application is a divisional application of U.S.
application Ser. No. 13/257,406, filed Sep. 19, 2011, which is a
national stage filing under 35 U.S.C. 371 of PCT/US2010/028111,
filed Mar. 22, 2010, which claims the benefit of U.S. Application
No. 61/166,262, filed Apr. 3, 2009, the disclosure of which is
incorporated by reference in its/their entirety herein.
FIELD
[0002] This invention relates to processes for capturing or
concentrating microorganisms such that they remain viable for
detection or assay. In other aspects, this invention also relates
to concentration devices (and diagnostic kits comprising the
devices) for use in carrying out such processes and to methods for
device preparation.
BACKGROUND
[0003] Food-borne illnesses and hospital-acquired infections
resulting from microorganism contamination are a concern in
numerous locations all over the world. Thus, it is often desirable
or necessary to assay for the presence of bacteria or other
microorganisms in various clinical, food, environmental, or other
samples, in order to determine the identity and/or the quantity of
the microorganisms present.
[0004] Bacterial DNA or bacterial RNA, for example, can be assayed
to assess the presence or absence of a particular bacterial species
even in the presence of other bacterial species. The ability to
detect the presence of a particular bacterium, however, depends, at
least in part, on the concentration of the bacterium in the sample
being analyzed. Bacterial samples can be plated or cultured to
increase the numbers of the bacteria in the sample to ensure an
adequate level for detection, but the culturing step often requires
substantial time and therefore can significantly delay the
assessment results.
[0005] Concentration of the bacteria in the sample can shorten the
culturing time or even eliminate the need for a culturing step.
Thus, methods have been developed to isolate (and thereby
concentrate) particular bacterial strains by using antibodies
specific to the strain (for example, in the form of antibody-coated
magnetic or non-magnetic particles). Such methods, however, have
tended to be expensive and still somewhat slower than desired for
at least some diagnostic applications.
[0006] Concentration methods that are not strain-specific have also
been used (for example, to obtain a more general assessment of the
microorganisms present in a sample). After concentration of a mixed
population of microorganisms, the presence of particular strains
can be determined, if desired, by using strain-specific probes.
[0007] Non-specific concentration or capture of microorganisms has
been achieved through methods based upon carbohydrate and lectin
protein interactions. Chitosan-coated supports have been used as
non-specific capture devices, and substances (for example,
carbohydrates, vitamins, iron-chelating compounds, and
siderophores) that serve as nutrients for microorganisms have also
been described as being useful as ligands to provide non-specific
capture of microorganisms.
[0008] Various inorganic materials (for example, hydroxyapatite and
metal hydroxides) have been used to non-specifically bind and
concentrate bacteria. Physical concentration methods (for example,
filtration, chromatography, centrifugation, and gravitational
settling) have also been utilized for non-specific capture, with
and/or without the use of inorganic binding agents. Such
non-specific concentration methods have varied in speed (at least
some food testing procedures still requiring at least overnight
incubation as a primary cultural enrichment step), cost (at least
some requiring expensive equipment, materials, and/or trained
technicians), sample requirements (for example, sample nature
and/or volume limitations), space requirements, ease of use (at
least some requiring complicated multi-step processes), suitability
for on-site use, and/or effectiveness.
SUMMARY
[0009] Thus, we recognize that there is an urgent need for
processes for rapidly detecting pathogenic microorganisms. Such
processes will preferably be not only rapid but also low in cost,
simple (involving no complex equipment or procedures), and/or
effective under a variety of conditions (for example, with varying
types of sample matrices and/or pathogenic microorganisms, varying
microorganism loads, and varying sample volumes).
[0010] Briefly, in one aspect, this invention provides a process
for non-specifically concentrating the strains of microorganisms
(for example, strains of bacteria, fungi, yeasts, protozoans,
viruses (including both non-enveloped and enveloped viruses), and
bacterial endospores) present in a sample, such that the
microorganisms remain viable for the purpose of detection or assay
of one or more of the strains. The process comprises (a) providing
a concentration device comprising a sintered porous polymer matrix
comprising at least one concentration agent that comprises
diatomaceous earth bearing, on at least a portion of its surface, a
surface treatment comprising a surface modifier comprising ferric
oxide, titanium dioxide, fine-nanoscale gold or platinum, or a
combination thereof; (b) providing a sample (preferably, in the
form of a fluid) comprising at least one microorganism strain; and
(c) contacting the concentration device with the sample
(preferably, by passing the sample through the concentration
device) such that at least a portion of the at least one
microorganism strain is bound to or captured by the concentration
device.
[0011] Preferably, the process further comprises detecting the
presence of at least one bound microorganism strain (for example,
by culture-based, microscopy/imaging, genetic, luminescence-based,
or immunologic detection methods). The process can optionally
further comprise separating the concentration device from the
sample and/or culturally enriching at least one bound microorganism
strain (for example, by incubating the separated concentration
device in a general or microorganism-specific culture medium,
depending upon whether general or selective microorganism
enrichment is desired) and/or isolating or separating captured
microorganisms (or one or more components thereof) from the
concentration device after sample contacting (for example, by
passing an elution agent or a lysis agent through the concentration
device).
[0012] The process of the invention does not target a specific
microorganism strain. Rather, it has been discovered that a
concentration device comprising certain relatively inexpensive,
inorganic materials in a sintered porous polymer matrix can be
surprisingly effective in capturing a variety of microorganisms
(and surprisingly effective in isolating or separating the captured
microorganisms via elution, relative to corresponding devices
without the inorganic material). Such devices can be used to
concentrate the microorganism strains present in a sample (for
example, a food sample) in a non-strain-specific manner, so that
one or more of the microorganism strains (preferably, one or more
strains of bacteria) can be more easily and rapidly assayed.
[0013] The process of the invention is relatively simple and low in
cost (requiring no complex equipment or expensive strain-specific
materials) and can be relatively fast (preferred embodiments
capturing at least about 70 percent (more preferably, at least
about 80 percent; most preferably, at least about 90 percent) of
the microorganisms present in a relatively homogeneous fluid sample
in less than about 30 minutes, relative to a corresponding control
sample having no contact with the concentration device). In
addition, the process can be effective with a variety of
microoganisms (including pathogens such as both gram positive and
gram negative bacteria) and with a variety of samples (different
sample matrices and, unlike at least some prior art methods, even
samples having low microorganism content and/or large volumes).
Thus, at least some embodiments of the process of the invention can
meet the above-cited urgent need for low-cost, simple processes for
rapidly detecting pathogenic microorganisms under a variety of
conditions.
[0014] The process of the invention can be especially advantageous
for concentrating the microorganisms in food samples (for example,
particulate-containing food samples, especially those comprising
relatively coarse particulates), as the concentration device used
in the process can exhibit at least somewhat greater resistance to
clogging than at least some filtration devices such as absolute
micron filters. This can facilitate more complete sample processing
(which is essential to eliminating false negative assays in food
testing) and the handling of relatively large volume samples (for
example, under field conditions).
[0015] In another aspect, the invention also provides a
concentration device comprising a sintered porous polymer matrix
comprising at least one concentration agent that comprises
diatomaceous earth bearing, on at least a portion of its surface, a
surface treatment comprising a surface modifier comprising ferric
oxide, titanium dioxide, fine-nanoscale gold or platinum, or a
combination thereof. The invention also provides a diagnostic kit
for use in carrying out the concentration process of the invention,
the kit comprising (a) at least one said concentration device of
the invention; and (b) at least one testing container or testing
reagent for use in carrying out the above-described concentration
process.
[0016] In yet another aspect, the invention provides a process for
preparing a concentration device comprising (a) providing a mixture
comprising (1) at least one particulate, sinterable polymer
(preferably, in the form of a powder) and (2) at least one
particulate concentration agent that comprises diatomaceous earth
bearing, on at least a portion of its surface, a surface treatment
comprising a surface modifier comprising ferric oxide, titanium
dioxide, fine-nanoscale gold or platinum, or a combination thereof;
and (b) heating the mixture to a temperature sufficient to sinter
the polymer, so as to form a sintered porous polymer matrix
comprising the particulate concentration agent.
BRIEF DESCRIPTION OF DRAWING
[0017] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying drawing,
wherein:
[0018] FIG. 1 shows, in side sectional view, an apparatus that was
used in preparing concentration agents for use in carrying out the
embodiments of the process of the invention described in the
examples section below.
[0019] FIG. 2 shows, in perspective view, the apparatus of FIG.
1.
[0020] These figures, which are idealized, are not drawn to scale
and are intended to be merely illustrative and nonlimiting.
DETAILED DESCRIPTION
[0021] In the following detailed description, various sets of
numerical ranges (for example, of the number of carbon atoms in a
particular moiety, of the amount of a particular component, or the
like) are described, and, within each set, any lower limit of a
range can be paired with any upper limit of a range.
Definitions
[0022] As used in this patent application:
"concentration agent" means a composition for concentrating
microorganisms; "detection" means the identification of at least a
component of a microorganism, which thereby determines that the
microorganism is present; "genetic detection" means the
identification of a component of genetic material such as DNA or
RNA that is derived from a target microorganism; "immunologic
detection" means the identification of an antigenic material such
as a protein or a proteoglycan that is derived from a target
microorganism; "microorganism" means any cell or particle having
genetic material suitable for analysis or detection (including, for
example, bacteria, yeasts, viruses, and bacterial endospores);
"microorganism strain" means a particular type of microorganism
that is distinguishable through a detection method (for example,
microorganisms of different genera, of different species within a
genera, or of different isolates within a species); "sample" means
a substance or material that is collected (for example, to be
analyzed); "sample matrix" means the components of a sample other
than microorganisms; "sinter" (in reference to a mass of polymer
particles) means to cause inter-particle binding or adhesion of at
least some of the polymer particles through application of heat,
without causing complete particle melting (for example, by heating
the mass of polymer particles to a temperature between the glass
transition temperature and the melting point of the polymer to
effect particle softening); "sinterable" (in reference to a
polymer) means a polymer that can be sintered; "sintered" (in
reference to a matrix) means formed by sintering; "target
microorganism" means any microorganism that is desired to be
detected; "through pore" (in reference to a porous matrix) means a
pore that comprises a passageway or channel (with separate inlet
and outlet) through the matrix; and "tortuous path matrix" means a
porous matrix having at least one tortuous through pore.
Concentration Agent
[0023] Concentration agents suitable for use in carrying out the
process of the invention include those that comprise diatomaceous
earth bearing, on at least a portion of its surface, a surface
treatment comprising a surface modifier comprising ferric oxide,
titanium dioxide, fine-nanoscale gold or platinum, or a combination
thereof (preferably, titanium dioxide, fine-nanoscale gold or
platinum, or a combination thereof). Concentration or capture using
such concentration agents is generally not specific to any
particular strain, species, or type of microorganism and therefore
provides for the concentration of a general population of
microorganisms in a sample. Specific strains of microorganisms can
then be detected from among the captured microorganism population
using any known detection method with strain-specific probes. Thus,
the concentration agents can be used for the detection of microbial
contaminants or pathogens (particularly food-borne pathogens such
as bacteria) in clinical, food, environmental, or other
samples.
[0024] In carrying out the process of the invention, the
concentration agents can be used in essentially any particulate
form (preferably, a relatively dry or volatiles-free form) that is
amenable to blending with particulate polymer to form the
concentration device used in the process. For example, the
concentration agents can be used in powder form or can be applied
to a particulate support such as beads or the like. Preferably, the
concentration agents are used in the form of a powder.
[0025] When dispersed or suspended in water systems, inorganic
materials exhibit surface charges that are characteristic of the
material and the pH of the water system. The potential across the
material-water interface is called the "zeta potential," which can
be calculated from electrophoretic mobilities (that is, from the
rates at which the particles of material travel between charged
electrodes placed in the water system). At least some of the
concentration agents used in carrying out the process of the
invention have zeta potentials that are at least somewhat more
positive than that of untreated diatomaceous earth, and the
concentration agents can be surprisingly significantly more
effective than untreated diatomaceous earth in concentrating
microorganisms such as bacteria, the surfaces of which generally
tend to be negatively charged. Preferably, the concentration agents
have a negative zeta potential at a pH of about 7 (more preferably,
a zeta potential in the range of about -5 millivolts to about -20
millivolts at a pH of about 7; even more preferably, a zeta
potential in the range of about -8 millivolts to about -19
millivolts at a pH of about 7; most preferably, a zeta potential in
the range of about -10 millivolts to about -18 millivolts at a pH
of about 7).
[0026] Thus, it has been discovered that concentration agents
comprising certain types of surface-treated or surface-modified
diatomaceous earth (namely, bearing a surface treatment comprising
a surface modifier comprising ferric oxide, titanium dioxide,
fine-nanoscale gold or platinum, or a combination thereof) can be
surprisingly more effective than untreated diatomaceous earth in
concentrating microorganisms. The surface treatment preferably
further comprises a metal oxide selected from ferric oxide, zinc
oxide, aluminum oxide, and the like, and combinations thereof (more
preferably, ferric oxide). Although noble metals such as gold have
been known to exhibit antimicrobial characteristics, the
gold-containing concentration agents used in the process of the
invention surprisingly can be effective not only in concentrating
the microorganisms but also in leaving them viable for purposes of
detection or assay.
[0027] Useful surface modifiers include fine-nanoscale gold;
fine-nanoscale platinum; fine-nanoscale gold in combination with at
least one metal oxide (preferably, titanium dioxide, ferric oxide,
or a combination thereof); titanium dioxide; titanium dioxide in
combination with at least one other (that is, other than titanium
dioxide) metal oxide; ferric oxide; ferric oxide in combination
with at least one other (that is, other than ferric oxide) metal
oxide; and the like; and combinations thereof. Preferred surface
modifiers include fine-nanoscale gold; fine-nanoscale platinum;
fine-nanoscale gold in combination with at least ferric oxide or
titanium dioxide; titanium dioxide; titanium dioxide in combination
with at least ferric oxide; ferric oxide; and combinations
thereof.
[0028] More preferred surface modifiers include fine-nanoscale
gold; fine-nanoscale platinum; fine-nanoscale gold in combination
with ferric oxide or titanium dioxide; titanium dioxide; titanium
dioxide in combination with ferric oxide; and combinations thereof
(even more preferably, fine-nanoscale gold; fine-nanoscale gold in
combination with ferric oxide or titanium dioxide; titanium dioxide
in combination with ferric oxide; and combinations thereof).
Fine-nanoscale gold, fine-nanoscale gold in combination with ferric
oxide or titanium dioxide, and combinations thereof are most
preferred.
Gold and/or Platinum
[0029] The concentration agents comprising fine-nanoscale gold or
platinum can be prepared by depositing gold or platinum on
diatomaceous earth by physical vapor deposition (optionally, by
physical vapor deposition in an oxidizing atmosphere). As used
herein, the term "fine-nanoscale gold or platinum" refers to gold
or platinum bodies (for example, particles or atom clusters) having
all dimensions less than or equal to 5 nanometers (nm) in size.
Preferably, at least a portion of the deposited gold or platinum
has all dimensions (for example, particle diameter or atom cluster
diameter) in the range of up to (less than or equal to) about 10 nm
in average size (more preferably, up to about 5 nm; even more
preferably, up to about 3 nm).
[0030] In most preferred embodiments, at least a portion of the
gold is ultra-nanoscale (that is, having at least two dimensions
less than 0.5 nm in size and all dimensions less than 1.5 nm in
size). The size of individual gold or platinum nanoparticles can be
determined by transmission electron microscopy (TEM) analysis, as
is well known in the art.
[0031] The amount of gold or platinum provided on the diatomaceous
earth can vary over a wide range. Since gold and platinum are
expensive, it is desirable not to use more than is reasonably
needed to achieve a desired degree of concentration activity.
Additionally, because nanoscale gold or platinum can be highly
mobile when deposited using PVD, activity can be compromised if too
much gold or platinum is used, due to coalescence of at least some
of the gold or platinum into large bodies.
[0032] For these reasons, the weight loading of gold or platinum on
the diatomaceous earth preferably is in the range of about 0.005
(more preferably, 0.05) to about 10 weight percent, more preferably
about 0.005 (even more preferably, 0.05) to about 5 weight percent,
and even more preferably from about 0.005 (most preferably, 0.05)
to about 2.5 weight percent, based upon the total weight of the
diatomaceous earth and the gold or platinum.
[0033] Gold and platinum can be deposited by PVD techniques (for
example, by sputtering) to form concentration-active,
fine-nanoscale particles or atom clusters on a support surface. It
is believed that the metal is deposited mainly in elemental form,
although other oxidation states may be present.
[0034] In addition to gold and/or platinum, one or more other
metals can also be provided on the same diatomaceous earth supports
and/or on other supports intermixed with the gold- and/or
platinum-containing supports. Examples of such other metals include
silver, palladium, rhodium, ruthenium, osmium, copper, iridium, and
the like, and combinations thereof. If used, these other metals can
be co-deposited on a support from a target source that is the same
or different from the gold or platinum source target that is used.
Alternatively, such metals can be provided on a support either
before or after the gold and/or platinum is deposited. Metals
requiring a thermal treatment for activation advantageously can be
applied to a support and heat treated before the gold and/or
platinum is deposited.
[0035] Physical vapor deposition refers to the physical transfer of
metal from a metal-containing source or target to a support medium.
Physical vapor deposition can be viewed as involving atom-by-atom
deposition, although in actual practice the metal can be
transferred as extremely fine bodies constituting more than one
atom per body. The deposited metal can interact with the surface of
the support medium physically, chemically, ionically, and/or
otherwise.
[0036] Physical vapor deposition preferably occurs under
temperature and vacuum conditions in which the metal is quite
mobile and will tend to migrate on the surface of the support
medium until immobilized in some fashion (for example, by adhering
to a site on or very near the support surface). Sites of adhering
can include defects such as surface vacancies, structural
discontinuities such as steps and dislocations, and interfacial
boundaries between phases or crystals or other metal species such
as small metal clusters. Metal deposited by PVD apparently is
sufficiently immobilized that the metal can retain a high level of
activity. In contrast, conventional methodologies often allow the
metal to coalesce into such large bodies that activity can be
compromised or even lost.
[0037] Physical vapor deposition can be carried out in various
different ways. Representative approaches include sputter
deposition (preferred), evaporation, and cathodic arc deposition.
Any of these or other PVD approaches can be used in preparing the
concentration agents used in carrying out the process of the
invention, although the nature of the PVD technique can impact the
resulting activity.
[0038] For example, the energy of the physical vapor deposition
technique can impact the mobility of the deposited metal and hence
its tendency to coalesce. Higher energy tends to correspond to an
increased tendency of the metal to coalesce. Increased coalescence,
in turn, tends to reduce activity. Generally, the energy of the
depositing species is lowest for evaporation, higher for sputter
deposition (which can include some ion content in which a small
fraction of the impinging metal species are ionized), and highest
for cathodic arc deposition (which can include several tens of
percents of ion content). Accordingly, if a particular PVD
technique yields deposited metal that is more mobile than desired,
it can be useful to use a PVD technique of lesser energy
instead.
[0039] Physical vapor deposition preferably is performed while the
support medium to be treated is being well-mixed (for example,
tumbled, fluidized, milled, or the like) to ensure adequate
treatment of support surfaces. Methods of tumbling particles for
deposition by PVD are described in U.S. Pat. No. 4,618,525
(Chamberlain et al.), the descriptions of which are incorporated
herein by reference.
[0040] When carrying out PVD on fine particles or fine particle
agglomerates (for example, less than about 10 micrometers in
average diameter), the support medium is preferably both mixed and
comminuted (for example, ground or milled to some degree) during at
least a portion of the PVD process. This can assist in maintaining
the separation and free flow of the particles or agglomerates
during the deposition. In the case of fine particles or fine
particle agglomerates, it can be advantageous for the mixing of the
particles to be as vigorous and rapid as possible while still
retaining controlled deposition of the metal.
[0041] PVD can be carried out by using any of the types of
apparatus that are now used or hereafter developed for this
purpose. A preferred apparatus 10 is shown, however, in FIGS. 1 and
2. The apparatus 10 includes a housing 12 defining a vacuum chamber
14 containing a particle agitator 16. The housing 12, which can be
made from an aluminum alloy if desired, is a vertically oriented
hollow cylinder (for example, 45 cm high and 50 cm in diameter).
The base 18 contains a port 20 for a high vacuum gate valve 22
followed by a six-inch diffusion pump 24 as well as a support 26
for the particle agitator 16. The vacuum chamber 14 is capable of
being evacuated to background pressures in the range of 10.sup.-6
Torr.
[0042] The top of the housing 12 includes a demountable, rubber
L-gasket-sealed plate 28 that is fitted with an external mount,
three-inch diameter direct current (dc) magnetron sputter
deposition source 30 (a US Gun II, US, INC., San Jose, Calif.).
Into the sputter deposition source 30 is fastened a gold or
platinum sputter target 32 (for example, 7.6 cm (3.0 inch)
diameter.times.0.48 cm ( 3/16 inch) thick). The sputter deposition
source 30 is powered by an MDX-10 Magnetron Drive (Advanced Energy
Industries, Inc, Fort Collins, Colo.) fitted with a Sparc-1e 20 arc
suppression system (Advanced Energy Industries, Inc, Fort Collins,
Colo.).
[0043] The particle agitator 16 is a hollow cylinder (for example,
12 cm long.times.9.5 cm diameter horizontal) with a rectangular
opening 34 (for example, 6.5 cm.times.7.5 cm). The opening 34 is
positioned about 7 cm directly below the surface 36 of the sputter
target 32, so that sputtered metal atoms can enter the agitator
volume 38. The agitator 16 is fitted with a shaft 40 aligned with
its axis. The shaft 40 has a rectangular cross section (for
example, 1 cm.times.1 cm) to which are bolted four rectangular
blades 42 which form an agitation mechanism or paddle wheel for the
support particles being tumbled. The blades 42 each contain two
holes 44 (for example, 2 cm diameter) to promote communication
between the particle volumes contained in each of the four
quadrants formed by the blades 42 and particle agitator 16. The
dimensions of the blades 42 are selected to give side and end gap
distances of either 2.7 mm or 1.7 mm with the agitator walls
48.
[0044] Physical vapor deposition can be carried out at essentially
any desired temperature(s) over a very wide range. However, the
deposited metal can be more active (perhaps due to more defects
and/or lower mobility and coalescence) if the metal is deposited at
relatively low temperatures (for example, at a temperature below
about 150.degree. C., preferably below about 50.degree. C., more
preferably at ambient temperature (for example, about 20.degree. C.
to about 27.degree. C.) or less). Operating under ambient
conditions can be generally preferred as being effective and
economical, as no heating or chilling is required during the
deposition.
[0045] The physical vapor deposition can be carried out in an inert
sputtering gas atmosphere (for example, in argon, helium, xenon,
radon, or a mixture of two or more thereof (preferably, argon)),
and, optionally, the physical vapor deposition can be carried out
in an oxidizing atmosphere. The oxidizing atmosphere preferably
comprises at least one oxygen-containing gas (more preferably, an
oxygen-containing gas selected from oxygen, water, hydrogen
peroxide, ozone, and combinations thereof; even more preferably, an
oxygen-containing gas selected from oxygen, water, and combinations
thereof; most preferably, oxygen). The oxidizing atmosphere further
comprises an inert sputtering gas such as argon, helium, xenon,
radon, or a mixture of two or more thereof (preferably, argon). The
total gas pressure (all gases) in the vacuum chamber during the PVD
process can be from about 1 mTorr to about 25 mTorr (preferably,
from about 5 mTorr to about 15 mTorr). The oxidizing atmosphere can
comprise from about 0.05 percent to about 60 percent by weight
oxygen-containing gas (preferably, from about 0.1 percent to about
50 percent by weight; more preferably, from about 0.5 percent to
about 25 percent by weight), based upon the total weight of all
gases in the vacuum chamber.
[0046] The diatomaceous earth support medium can optionally be
calcined prior to metal deposition, although this can increase its
crystalline silica content. Since gold and platinum are active
right away when deposited via PVD, there is generally no need for
heat treatment after metal deposition, unlike deposition by some
other methodologies. Such heat treating or calcining can be carried
out if desired, however, to enhance activity.
[0047] In general, thermal treatment can involve heating the
support at a temperature in the range of about 125.degree. C. to
about 1000.degree. C. for a time period in the range of about 1
second to about 40 hours, preferably about 1 minute to about 6
hours, in any suitable atmosphere such as air, an inert atmosphere
such as nitrogen, carbon dioxide, argon, a reducing atmosphere such
as hydrogen, and the like. The particular thermal conditions to be
used can depend upon various factors including the nature of the
support.
[0048] Generally, thermal treatment can be carried out below a
temperature at which the constituents of the support would be
decomposed, degraded, or otherwise unduly thermally damaged.
Depending upon factors such as the nature of the support, the
amount of metal, and the like, activity can be compromised to some
degree if the system is thermally treated at too high a
temperature.
Titanium Dioxide, Ferric Oxide, and/or Other Metal Oxides
[0049] The concentration agents comprising metal oxide can be
prepared by depositing metal oxide on diatomaceous earth by
hydrolysis of a hydrolyzable metal oxide precursor compound.
Suitable metal oxide precursor compounds include metal complexes
and metal salts that can be hydrolyzed to form metal oxides. Useful
metal complexes include those comprising alkoxide ligands, hydrogen
peroxide as a ligand, carboxylate-functional ligands, and the like,
and combinations thereof. Useful metal salts include metal
sulfates, nitrates, halides, carbonates, oxalates, hydroxides, and
the like, and combinations thereof.
[0050] When using metal salts or metal complexes of hydrogen
peroxide or carboxylate-functional ligands, hydrolysis can be
induced by either chemical or thermal means. In chemically-induced
hydrolysis, the metal salt can be introduced in the form of a
solution into a dispersion of the diatomaceous earth, and the pH of
the resulting combination can be raised by the addition of a base
solution until the metal salt precipitates as a hydroxide complex
of the metal on the diatomaceous earth. Suitable bases include
alkali metal and alkaline earth metal hydroxides and carbonates,
ammonium and alkyl-ammonium hydroxides and carbonates, and the
like, and combinations thereof. The metal salt solution and the
base solution can generally be about 0.1 to about 2 M in
concentration.
[0051] Preferably, the addition of the metal salt to the
diatomaceous earth is carried out with stirring (preferably, rapid
stirring) of the diatomaceous earth dispersion. The metal salt
solution and the base solution can be introduced to the
diatomaceous earth dispersion separately (in either order) or
simultaneously, so as to effect a preferably substantially uniform
reaction of the resulting metal hydroxide complex with the surface
of the diatomaceous earth. The reaction mixture can optionally be
heated during the reaction to accelerate the speed of the reaction.
In general, the amount of base added can equal the number of moles
of the metal times the number of non-oxo and non-hydroxo
counterions on the metal salt or metal complex.
[0052] Alternatively, when using salts of titanium or iron, the
metal salt can be thermally induced to hydrolyze to form the
hydroxide complex of the metal and to interact with the surface of
the diatomaceous earth. In this case, the metal salt solution can
generally be added to a dispersion of the diatomaceous earth
(preferably, a stirred dispersion) that has been heated to a
sufficiently high temperature (for example, greater than about
50.degree. C.) to promote the hydrolysis of the metal salt.
Preferably, the temperature is between about 75.degree. C. and
100.degree. C., although higher temperatures can be used if the
reaction is carried out in an autoclave apparatus.
[0053] When using metal alkoxide complexes, the metal complex can
be induced to hydrolyze to form a hydroxide complex of the metal by
partial hydrolysis of the metal alkoxide in an alcohol solution.
Hydrolysis of the metal alkoxide solution in the presence of
diatomaceous earth can result in metal hydroxide species being
deposited on the surface of the diatomaceous earth.
[0054] Alternatively, the metal alkoxide can be hydrolyzed and
deposited onto the surface of the diatomaceous earth by reacting
the metal alkoxide in the gas phase with water, in the presence of
the diatomaceous earth. In this case, the diatomaceous earth can be
agitated during the deposition in either, for example, a fluidized
bed reactor or a rotating drum reactor.
[0055] After the above-described hydrolysis of the metal oxide
precursor compound in the presence of the diatomaceous earth, the
resulting surface-treated diatomaceous earth can be separated by
settling or by filtration or by other known techniques. The
separated product can be purified by washing with water and can
then be dried (for example, at 50.degree. C. to 150.degree.
C.).
[0056] Although the surface-treated diatomaceous earth generally
can be functional after drying, it can optionally be calcined to
remove volatile by-products by heating in air to about 250.degree.
C. to 650.degree. C. generally without loss of function. This
calcining step can be preferred when metal alkoxides are utilized
as the metal oxide precursor compounds.
[0057] In general, with metal oxide precursor compounds of iron,
the resulting surface treatments comprise nanoparticulate iron
oxide. When the weight ratio of iron oxide to diatomaceous earth is
about 0.08, X-ray diffraction (XRD) does not show the presence of a
well-defined iron oxide material. Rather, additional X-ray
reflections are observed at 3.80, 3.68, and 2.94 .ANG.. TEM
examination of this material shows the surface of the diatomaceous
earth to be relatively uniformly coated with globular
nanoparticulate iron oxide material. The crystallite size of the
iron oxide material is less than about 20 nm, with most of the
crystals being less than about 10 nm in diameter. The packing of
these globular crystals on the surface of the diatomaceous earth is
dense in appearance, and the surface of the diatomaceous earth
appears to be roughened by the presence of these crystals.
[0058] In general, with metal oxide precursor compounds of
titanium, the resulting surface treatments comprise nanoparticulate
titania. When depositing titanium dioxide onto diatomaceous earth,
XRD of the resulting product after calcination to about 350.degree.
C. can show the presence of small crystals of anatase titania. With
relatively lower titanium/diatomaceous earth ratios or in cases
where mixtures of titanium and iron oxide precursors are used, no
evidence of anatase is generally observed by X-ray analysis.
[0059] Since titania is well-known as a potent photo-oxidation
catalyst, the titania-modified diatomaceous earth concentration
agents can be used to concentrate microorganisms for analysis and
then optionally also be used as photoactivatable agents for killing
residual microorganisms and removing unwanted organic impurities
after use. Thus, the titania-modified diatomaceous earth can both
isolate biomaterials for analysis and then be photochemically
cleaned for re-use. These materials can also be used in filtration
applications where microorganism removal as well as antimicrobial
effects can be desired.
Diatomaceous Earth
[0060] Diatomaceous earth (or kieselguhr) is a natural siliceous
material produced from the remnants of diatoms, a class of
ocean-dwelling microorganisms. Thus, it can be obtained from
natural sources and is also commercially available (for example,
from Alfa Aesar, A Johnson Matthey Company, Ward Hill, Mass.).
Diatomaceous earth particles generally comprise small, open
networks of silica in the form of symmetrical cubes, cylinders,
spheres, plates, rectangular boxes, and the like. The pore
structures in these particles can generally be remarkably
uniform.
[0061] Diatomaceous earth can be used in carrying out the process
of the invention as the raw, mined material or as purified and
optionally milled particles. Preferably, the diatomaceous earth is
in the form of milled particles with sizes in the range of about 1
micrometer to about 50 micrometers in diameter (more preferably,
about 3 micrometers to about 10 micrometers).
[0062] The diatomaceous earth can optionally be heat treated prior
to use to remove any vestiges of organic residues. If a heat
treatment is used, it can be preferable that the heat treatment be
at 500.degree. C. or lower, as higher temperatures can produce
undesirably high levels of crystalline silica.
Concentration Device
[0063] Concentration devices suitable for use in carrying out the
process of the invention include those that comprise a sintered
porous polymer matrix comprising at least one of the
above-described concentration agents. Such concentration devices
can be prepared, for example, by mixing or blending at least one
particulate, sinterable polymer (preferably, in the form of a
powder) and at least one particulate concentration agent, and then
heating the resulting mixture to a temperature sufficient to sinter
the polymer. This process, as well as other known or
hereafter-developed sintering processes, can be used to provide,
upon cooling, a sintered porous polymer matrix comprising the
particulate concentration agent.
[0064] For example, sintering can cause the polymer particles to
soften at their points of contact, and subsequent cooling can then
cause fusion of the particles. A solidified or self-supporting,
porous polymer body comprising particulate concentration agent can
result (for example, with the concentration agent being embedded in
or on the surface of the polymer body). This can provide a
concentration device having a relatively complex pore structure
(preferably, a concentration device comprising a tortuous path
matrix) and relatively good mechanical strength.
[0065] Polymers suitable for use in preparing the concentration
device include sinterable polymers and combinations thereof.
Preferred sinterable polymers include thermoplastic polymers and
combinations thereof. More preferably, the thermoplastic polymers
can be selected so as to have relatively high viscosities and
relatively low melt flow rates. This can facilitate particle shape
retention during the sintering process.
[0066] Useful sinterable polymers include polyolefins (including
olefin homopolymers and copolymers, as well as copolymers of
olefins and other vinyl monomers), polysulfones, polyethersulfones,
polyphenylene sulfide, and the like, and combinations thereof.
Representative examples of useful polymers include ethylene vinyl
acetate (EVA) polymers, ethylene methyl acrylate (EMA) polymers,
polyethylenes (including, for example, low density polyethylene
(LDPE), linear low density polyethylene (LLDPE), high density
polyethylene (HDPE), and ultra-high molecular weight polyethylene
(UHMWPE)), polypropylenes, ethylene-propylene rubbers,
ethylene-propylene-diene rubbers, polystyrene, poly(1-butene),
poly(2-butene), poly(1-pentene), poly(2-pentene),
poly(3-methyl-1-pentene), poly(4-methyl-1-pentene),
1,2-poly-1,3-butadiene, 1,4-poly-1,3-butadiene, polyisoprene,
polychloroprene, poly(vinyl acetate), poly(vinylidene chloride),
poly(vinylidene fluoride), poly(tetrafluoroethylene), and the like,
and combinations thereof.
[0067] Preferred polymers include olefin homopolymers and
copolymers (especially, polyethylenes, polypropylenes, ethylene
vinyl acetate polymers, and combinations thereof). More preferred
polymers include olefin homopolymers and combinations thereof (even
more preferably, polyethylenes and combinations thereof; most
preferably, ultra-high molecular weight polyethylenes (UHMWPE) and
combinations thereof). Useful ultra-high molecular weight
polyethylenes include those having a molecular weight of at least
about 750,000 (preferably, at least about 1,000,000; more
preferably, at least about 2,000,000; most preferably, at least
about 3,000,000).
[0068] A wide range of polymer particle sizes can be utilized,
depending upon the pore (for example, hole, depression, or,
preferably, channel) sizes desired in the sintered porous polymer
matrix. Finer particles can provide finer pore sizes in the
sintered matrix. Generally, the polymer particles can be
microparticles (for example, ranging in size or diameter from about
1 micrometer to about 800 micrometers; preferably, from about 5
micrometers to about 300 micrometers; more preferably, from about 5
micrometers to about 200 micrometers; most preferably, from about
10 micrometers to about 100 or 200 micrometers), so as to provide
pore sizes on the order of micrometers or less. Varying average
(mean) and/or median particle sizes can be utilized (for example,
average particle sizes of about 30 micrometers to about 70
micrometers can be useful.) If desired, the porosity of the
sintered matrix can also be varied or controlled by using blends of
higher and lower melt flow rate polymers.
[0069] The polymer particles and the particulate concentration
agent (and any optional additives, such as wetting agents or
surfactants) can be combined and mechanically blended (for example,
using commercial mixing equipment) to form a mixture (preferably, a
homogeneous mixture). Generally, the particulate concentration
agent can be present in the mixture at a concentration of up to
about 90 weight percent (preferably, about 5 to about 85 weight
percent; more preferably, about 10 to about 80 weight percent; most
preferably, about 15 to about 75 weight percent), based upon the
total weight of all particles in the mixture. Conventional
additives (for example, wetting agents, surfactants, or the like)
can be included in the mixture in small amounts (for example, up to
about 5 weight percent), if desired.
[0070] The resulting mixture can be placed in a mold or other
suitable container or substrate. Useful molds can be made of carbon
steel, stainless steel, brass, aluminum, or the like, and can have
a single cavity or multiple cavities. The cavities can be of
essentially any desired shape, provided that their sintered
contents can be removed from the mold after processing is
completed. Preferably, mold filling can be assisted by using
commercial powder handling and/or vibratory equipment.
[0071] Thermal processing can be carried out by introducing heat to
the mold (for example, through electrical resistance heating,
electrical induction heating, or steam heating). The mold can be
heated to a temperature sufficient to sinter the polymer (for
example, by heating to a temperature slightly below the melting
point of the polymer). Sintering methods are known and can be
selected according to the nature and/or form of the polymer(s)
utilized. Optionally, pressure can be applied to the mixture during
the heating process. After thermal processing, the mold can be
allowed to cool to ambient temperature (for example, a temperature
of about 23.degree. C.) naturally or through use of essentially any
convenient cooling method or device.
[0072] A preferred concentration device can be prepared by using
the polymer particles and processing methods described in U.S. Pat.
Nos. 7,112,272, 7,112,280, and 7,169,304 (Hughes et al.), the
descriptions of which particles and methods are incorporated herein
by reference. Two different types of ultra-high molecular weight
polyethylene (UHMWPE) particles can be blended together, one being
"popcorn-shaped" (having surface convolutions) and the other being
substantially spherical. Preferred "popcorn-shaped" and spherical
UHMWPEs are available from Ticona (a division of Celanese,
headquartered in Frankfurt, Germany) as PMX CF-1 (having a bulk
density of 0.25-0.30 g/cubic centimeter and an average diameter of
about 30 to 40 micrometers, with a range from about 10 micrometers
to about 100 micrometers) and PMX CF-2 (having a bulk density of
0.40-0.48 g/cubic centimeter and an average diameter of about 55 to
65 micrometers, with a range from about 10 micrometers to about 180
micrometers), respectively. UHMWPEs from other manufacturers having
comparable morphologies, bulk densities, and particle sizes and
having molecular weights in the range of about 750,000 to about
3,000,000 can also be utilized. The two types of UHMWPE particles
can be selected to be of the same or different molecular weight(s)
(preferably, both have the same molecular weight within the stated
range; more preferably, both have molecular weights of about
3,000,000).
[0073] The two types of UHMWPE particles can be combined in varying
relative amounts (for example, equal amounts) and then further
combined with concentration agent in the ratios described above.
Either type of UHMWPE can be used in lesser amount than the other,
or can even be omitted from the mixture, depending upon the desired
characteristics of the concentration device.
[0074] The selected particles can be blended together to form a
mixture that is preferably homogeneous. For example, a ribbon
blender or the like can be used. The resulting mixture can then be
placed in a mold cavity while preferably being simultaneously
vibrated using essentially any standard mechanical vibrator. At the
end of the filling and vibration cycle, the mold can be heated to a
temperature that is sufficient to sinter the polymer(s) (generally,
a temperature in the range of about 225.degree. F. to about
375.degree. F. or higher, depending upon the molecular weight(s) of
the polymer(s)).
Upon cooling, a self-supporting, sintered porous polymer matrix can
be obtained. The matrix can exhibit a complex internal structure
comprising interconnected, multi-directional through pores of
varying diameters and can thus comprise a preferred tortuous path
matrix for use as a concentration device in the concentration
process of the invention. If desired, the concentration device can
further comprise one or more other components such as, for example,
one or more pre-filters (for example, to remove relatively large
food particles from a sample prior to passage of the sample through
the porous matrix), a manifold for applying a pressure differential
across the device (for example, to aid in passing a sample through
the porous matrix), and/or an external housing (for example, a
disposable cartridge to contain and/or protect the porous
matrix).
Sample
[0075] The process of the invention can be applied to a variety of
different types of samples, including, but not limited to, medical,
environmental, food, feed, clinical, and laboratory samples, and
combinations thereof. Medical or veterinary samples can include,
for example, cells, tissues, or fluids from a biological source
(for example, a human or an animal) that are to be assayed for
clinical diagnosis. Environmental samples can be, for example, from
a medical or veterinary facility, an industrial facility, soil, a
water source, a food preparation area (food contact and non-contact
areas), a laboratory, or an area that has been potentially
subjected to bioterrorism. Food processing, handling, and
preparation area samples are preferred, as these are often of
particular concern in regard to food supply contamination by
bacterial pathogens.
[0076] Samples obtained in the form of a liquid or in the form of a
dispersion or suspension of solid in liquid can be used directly,
or can be concentrated (for example, by centrifugation) or diluted
(for example, by the addition of a buffer (pH-controlled)
solution). Samples in the form of a solid or a semi-solid can be
used directly or can be extracted, if desired, by a method such as,
for example, washing or rinsing with, or suspending or dispersing
in, a fluid medium (for example, a buffer solution). Samples can be
taken from surfaces (for example, by swabbing or rinsing).
Preferably, the sample is a fluid (for example, a liquid, a gas, or
a dispersion or suspension of solid or liquid in liquid or
gas).
[0077] Examples of samples that can be used in carrying out the
process of the invention include foods (for example, fresh produce
or ready-to-eat lunch or "deli" meats), beverages (for example,
juices or carbonated beverages), potable water, and biological
fluids (for example, whole blood or a component thereof such as
plasma, a platelet-enriched blood fraction, a platelet concentrate,
or packed red blood cells; cell preparations (for example,
dispersed tissue, bone marrow aspirates, or vertebral body bone
marrow); cell suspensions; urine, saliva, and other body fluids;
bone marrow; lung fluid; cerebral fluid; wound exudate; wound
biopsy samples; ocular fluid; spinal fluid; and the like), as well
as lysed preparations, such as cell lysates, which can be formed
using known procedures such as the use of lysing buffers, and the
like. Preferred samples include foods, beverages, potable water,
biological fluids, and combinations thereof (with foods, beverages,
potable water, and combinations thereof being more preferred).
[0078] Sample volume can vary, depending upon the particular
application. For example, when the process of the invention is used
for a diagnostic or research application, the volume of the sample
can typically be in the microliter range (for example, 10
microliters or greater). When the process is used for a food
pathogen testing assay or for potable water safety testing, the
volume of the sample can typically be in the milliliter to liter
range (for example, 100 milliliters to 3 liters). In an industrial
application, such as bioprocessing or pharmaceutical formulation,
the volume can be tens of thousands of liters.
[0079] The process of the invention can isolate microorganisms from
a sample in a concentrated state and can also allow the isolation
of microorganisms from sample matrix components that can inhibit
detection procedures that are to be used. In all of these cases,
the process of the invention can be used in addition to, or in
replacement of, other methods of microorganism concentration. Thus,
optionally, cultures can be grown from samples either before or
after carrying out the process of the invention, if additional
concentration is desired. Such cultural enrichment can be general
or primary (so as to enrich the concentrations of most or
essentially all microorganisms) or can be specific or selective (so
as to enrich the concentration(s) of one or more selected
microorganisms only).
Contacting
[0080] The process of the invention can be carried out by any of
various known or hereafter-developed methods of providing contact
between two materials. For example, the concentration device can be
added to the sample, or the sample can be added to the
concentration device. The concentration device can be immersed in a
sample, a sample can be poured onto the concentration device, a
sample can be poured into a tube or well containing the
concentration device, or, preferably, a sample can be passed over
or through (preferably, through) the concentration device (or vice
versa). Preferably, the contacting is carried out in a manner such
that the sample passes through at least one pore of the sintered
porous polymer matrix (preferably, through at least one through
pore).
[0081] The concentration device and the sample can be combined
(using any order of addition) in any of a variety of containers or
holders (optionally, a capped, closed, or sealed container;
preferably, a column, a syringe barrel, or another holder designed
to contain the device with essentially no sample leakage). Suitable
containers for use in carrying out the process of the invention
will be determined by the particular sample and can vary widely in
size and nature. For example, the container can be small, such as a
10 microliter container (for example, a test tube or syringe) or
larger, such as a 100 milliliter to 3 liter container (for example,
an Erlenmeyer flask or an annular cylindrical container).
[0082] The container, the concentration device, and any other
apparatus or additives that contact the sample directly can be
sterilized (for example, by controlled heat, ethylene oxide gas, or
radiation) prior to use, in order to reduce or prevent any
contamination of the sample that might cause detection errors. The
amount of concentration agent in the concentration device that is
sufficient to capture or concentrate the microorganisms of a
particular sample for successful detection will vary (depending
upon, for example, the nature and form of the concentration agent
and device and the volume of the sample) and can be readily
determined by one skilled in the art.
[0083] Contacting can be carried out for a desired period (for
example, for sample volumes of about 100 milliliters or less, up to
about 60 minutes of contacting can be useful; preferably, about 15
seconds to about 10 minutes or longer; more preferably, about 15
seconds to about 5 minutes; most preferably, about 15 seconds to
about 2 minutes). Contact can be enhanced by mixing (for example,
by stirring, by shaking, or by application of a pressure
differential across the device to facilitate passage of a sample
through its porous matrix) and/or by incubation (for example, at
ambient temperature), which are optional but can be preferred, in
order to increase microorganism contact with the concentration
device.
[0084] Preferably, contacting can be effected by passing a sample
at least once (preferably, only once) through the concentration
device (for example, by pumping). Essentially any type of pump (for
example, a peristaltic pump) or other equipment for establishing a
pressure differential across the device (for example, a syringe or
plunger) can be utilized. Sample flow rates through the device of
up to about 100 milliliters per minute or more can be effective.
Preferably, flow rates of about 10-20 milliliters per minute can be
utilized.
[0085] A preferred contacting method includes such passing of a
sample through the concentration device (for example, by pumping)
and then incubating (for example, for about 3 hours to about 24
hours; preferably, about 4 hours to about 20 hours) a
microorganism-containing sample (preferably, a fluid) with the
concentration device (for example, in one of the above-described
containers). If desired, one or more additives (for example, lysis
reagents, bioluminescence assay reagents, nucleic acid capture
reagents (for example, magnetic beads), microbial growth media,
buffers (for example, to moisten a solid sample), microbial
staining reagents, washing buffers (for example, to wash away
unbound material), elution agents (for example, serum albumin),
surfactants (for example, Triton.TM. X-100 nonionic surfactant
available from Union Carbide Chemicals and Plastics, Houston,
Tex.), mechanical abrasion/elution agents (for example, glass
beads), and the like) can be included in the combination of
concentration device and sample during contacting.
[0086] The process of the invention can optionally further comprise
separating the resulting microorganism-bound concentration device
and the sample. Separation can be carried out by numerous methods
that are well-known in the art (for example, by pumping, decanting,
or siphoning a fluid sample, so as to leave the microorganism-bound
concentration device in the container or holder utilized in
carrying out the process). It can also be possible to isolate or
separate captured microorganisms (or one or more components
thereof) from the concentration device after sample contacting (for
example, by passing an elution agent or a lysis agent over or
through the concentration device).
[0087] The process of the invention can be carried out manually
(for example, in a batch-wise manner) or can be automated (for
example, to enable continuous or semi-continuous processing).
Detection
[0088] A variety of microorganisms can be concentrated and,
optionally but preferably, detected by using the process of the
invention, including, for example, bacteria, fungi, yeasts,
protozoans, viruses (including both non-enveloped and enveloped
viruses), bacterial endospores (for example, Bacillus (including
Bacillus anthracis, Bacillus cereus, and Bacillus subtilis) and
Clostridium (including Clostridium botulinum, Clostridium
difficile, and Clostridium perfringens)), and the like, and
combinations thereof (preferably, bacteria, yeasts, viruses,
bacterial endospores, fungi, and combinations thereof; more
preferably, bacteria, yeasts, viruses, bacterial endospores, and
combinations thereof; even more preferably, bacteria, viruses,
bacterial endospores, and combinations thereof; most preferably,
gram-negative bacteria, gram-positive bacteria, non-enveloped
viruses (for example, norovirus, poliovirus, hepatitis A virus,
rhinovirus, and combinations thereof), bacterial endospores, and
combinations thereof). The process has utility in the detection of
pathogens, which can be important for food safety or for medical,
environmental, or anti-terrorism reasons. The process can be
particularly useful in the detection of pathogenic bacteria (for
example, both gram negative and gram positive bacteria), as well as
various yeasts, molds, and mycoplasmas (and combinations of any of
these).
[0089] Genera of target microorganisms to be detected include, but
are not limited to, Listeria, Escherichia, Salmonella,
Campylobacter, Clostridium, Helicobacter, Mycobacterium,
Staphylococcus, Shigella, Enterococcus, Bacillus, Neisseria,
Shigella, Streptococcus, Vibrio, Yersinia, Bordetella, Borrelia,
Pseudomonas, Saccharomyces, Candida, and the like, and combinations
thereof. Samples can contain a plurality of microorganism strains,
and any one strain can be detected independently of any other
strain. Specific microorganism strains that can be targets for
detection include Escherichia coli, Yersinia enterocolitica,
Yersinia pseudotuberculosis, Vibrio cholerae, Vibrio
parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes,
Staphylococcus aureus, Salmonella enterica, Saccharomyces
cerevisiae, Candida albicans, Staphylococcal enterotoxin ssp,
Bacillus cereus, Bacillus anthracis, Bacillus atrophaeus, Bacillus
subtilis, Clostridium perfringens, Clostridium botulinum,
Clostridium difficile, Enterobacter sakazakii, Pseudomonas
aeruginosa, and the like, and combinations thereof (preferably,
Staphylococcus aureus, Salmonella enterica, Saccharomyces
cerevisiae, Bacillus atrophaeus, Bacillus subtilis, Escherichia
coli, human-infecting non-enveloped enteric viruses for which
Escherichia coli bacteriophage is a surrogate, and combinations
thereof.
[0090] Microorganisms that have been captured or bound (for
example, by adsorption or by sieving) by the concentration device
can be detected by essentially any desired method that is currently
known or hereafter developed. Such methods include, for example,
culture-based methods (which can be preferred when time permits),
microscopy (for example, using a transmitted light microscope or an
epifluorescence microscope, which can be used for visualizing
microorganisms tagged with fluorescent dyes) and other imaging
methods, immunological detection methods, and genetic detection
methods. The detection process following microorganism capture
optionally can include washing to remove sample matrix components,
slicing or otherwise breaking up the sintered porous polymer matrix
of the concentration device, staining, or the like.
[0091] Immunological detection is detection of an antigenic
material derived from a target organism, which is commonly a
biological molecule (for example, a protein or proteoglycan) acting
as a marker on the surface of bacteria or viral particles.
Detection of the antigenic material typically can be by an
antibody, a polypeptide selected from a process such as phage
display, or an aptamer from a screening process.
[0092] Immunological detection methods are well-known and include,
for example, immunoprecipitation and enzyme-linked immunosorbent
assay (ELISA). Antibody binding can be detected in a variety of
ways (for example, by labeling either a primary or a secondary
antibody with a fluorescent dye, with a quantum dot, or with an
enzyme that can produce chemiluminescence or a colored substrate,
and using either a plate reader or a lateral flow device).
[0093] Detection can also be carried out by genetic assay (for
example, by nucleic acid hybridization or primer directed
amplification), which is often a preferred method. The captured or
bound microorganisms can be lysed to render their genetic material
available for assay. Lysis methods are well-known and include, for
example, treatments such as sonication, osmotic shock, high
temperature treatment (for example, from about 50.degree. C. to
about 100.degree. C.), and incubation with an enzyme such as
lysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase
E, and viral enolysins.
[0094] Many commonly-used genetic detection assays detect the
nucleic acids of a specific microorganism, including the DNA and/or
RNA. The stringency of conditions used in a genetic detection
method correlates with the level of variation in nucleic acid
sequence that is detected. Highly stringent conditions of salt
concentration and temperature can limit the detection to the exact
nucleic acid sequence of the target. Thus microorganism strains
with small variations in a target nucleic acid sequence can be
distinguished using a highly stringent genetic assay. Genetic
detection can be based on nucleic acid hybridization where a
single-stranded nucleic acid probe is hybridized to the denatured
nucleic acids of the microorganism such that a double-stranded
nucleic acid is produced, including the probe strand. One skilled
in the art will be familiar with probe labels, such as radioactive,
fluorescent, and chemiluminescent labels, for detecting the hybrid
following gel electrophoresis, capillary electrophoresis, or other
separation method.
[0095] Particularly useful genetic detection methods are based on
primer directed nucleic acid amplification. Primer directed nucleic
acid amplification methods include, for example, thermal cycling
methods (for example, polymerase chain reaction (PCR), reverse
transcriptase polymerase chain reaction (RT-PCR), and ligase chain
reaction (LCR)), as well as isothermal methods and strand
displacement amplification (SDA) (and combinations thereof;
preferably, PCR or RT-PCR). Methods for detection of the amplified
product are not limited and include, for example, gel
electrophoresis separation and ethidium bromide staining, as well
as detection of an incorporated fluorescent label or radio label in
the product. Methods that do not require a separation step prior to
detection of the amplified product can also be used (for example,
real-time PCR or homogeneous detection).
[0096] Bioluminescence detection methods are well-known and
include, for example, adensosine triphosphate (ATP) detection
methods including those described in U.S. Pat. No. 7,422,868 (Fan
et al.), the descriptions of which are incorporated herein by
reference. Other luminescence-based detection methods can also be
utilized.
[0097] Since the process of the invention is non-strain specific,
it provides a general capture system that allows for multiple
microorganism strains to be targeted for assay in the same sample.
For example, in assaying for contamination of food samples, it can
be desired to test for Listeria monocytogenes, Escherichia coli,
and Salmonella all in the same sample. A single capture step can
then be followed by, for example, PCR or RT-PCR assays using
specific primers to amplify different nucleic acid sequences from
each of these microorganism strains. Thus, the need for separate
sample handling and preparation procedures for each strain can be
avoided.
Diagnostic Kit
[0098] A diagnostic kit for use in carrying out the concentration
process of the invention comprises (a) at least one above-described
concentration device; and (b) at least one testing container or
testing reagent (preferably, a sterile testing container or testing
reagent) for use in carrying out the concentration process of the
invention. Preferably, the diagnostic kit further comprises
instructions for carrying out the process.
[0099] Useful testing containers or holders include those described
above and can be used, for example, for contacting, for incubation,
for collection of eluate, or for other desired process steps.
Useful testing reagents include microorganism culture or growth
media, lysis agents, elution agents, buffers, luminescence
detection assay components (for example, luminometer, lysis
reagents, luciferase enzyme, enzyme substrate, reaction buffers,
and the like), genetic detection assay components, and the like,
and combinations thereof. A preferred lysis agent is a lytic enzyme
or chemical supplied in a buffer, and preferred genetic detection
assay components include one or more primers specific for a target
microorganism. The kit can optionally further comprise sterile
forceps or the like.
EXAMPLES
[0100] Objects and advantages of this invention are further
illustrated by the following examples, but the particular materials
and amounts thereof recited in these examples, as well as other
conditions and details, should not be construed to unduly limit
this invention. All microorganism cultures were purchased from The
American Type Culture Collection (ATCC; Manassas, Va.).
Preparation of Concentration Agents
[0101] Kieselguhr (diatomaceous earth) was purchased from Alfa
Aesar (A Johnson Matthey Company, Ward Hill, Mass.) as a white
powder (325 mesh; all particles less than 44 micrometers in size).
This material was shown by X-ray diffraction (XRD) to contain
amorphous silica along with crystalline .alpha.-cristobalite and
quartz. Calcined diatomaceous earth was purchased from Solvadis,
GmbH, Frankfurt, Germany (and observed to comprise small rods about
5 to about 80 micrometers in length and about 3 to about 8
micrometers in width, along with porous disks and disk fragments up
to about 60 micrometers in primary length and asymmetrical
fragments about 3 to about 60 micrometers in primary length). This
material was shown by XRD to comprise predominantly
.alpha.-cristobalite.
[0102] Concentration agents comprising various different surface
modifiers (namely, titanium dioxide; titanium dioxide in
combination with ferric oxide; ferric oxide; platinum; gold; and
gold in combination with ferric oxide) were prepared by surface
treating the diatomaceous earth in the manner described below:
Gold Deposition
[0103] About 57-60 g of dried diatomaceous earth or metal
oxide-modified diatomaceous earth support media (about 300 mL
volume of powder) was further dried in an oven at 150.degree. C.
for 24 hours to remove residual water. The resulting dried sample
was placed while hot into the PVD apparatus described above in the
detailed description with the PVD apparatus having a particle
agitator with a blade gap of 2.7 mm. The vacuum chamber of the
apparatus was then evacuated to a background pressure of about
5.times.10.sup.-5 Torr, and gas comprising argon sputtering gas was
admitted to the chamber at a pressure of about 10 mTorr.
[0104] The metal deposition process was then carried out by
applying power to the cathode of the apparatus for a pre-set period
of time, with its particle agitator shaft and holed blades being
rotated at 4 rpm during DC magnetron sputter coating of metal at a
controlled power of 0.02 kW. The duration of sputter coating was 5
hours. After the sputter coating was completed, the vacuum chamber
was vented with air to ambient conditions, and the resulting
metal-coated sample was removed from the PVD apparatus and sieved
through a 25 mesh (0.707 mm) screen to separate fine particulates
generated during the process. The amount of metal that had been
deposited on the sample was determined by weighing (both before and
after the deposition process) the metal sputtering target that was
utilized. In general, about 18 percent of the weight loss of the
target represented metal deposited on the sample (based on
inductively coupled plasma analysis). From this information, the
resulting amount of gold on the support medium was calculated to be
about 0.9 weight percent.
Platinum Deposition
[0105] The above-described gold deposition process was essentially
repeated, with the exception that a 7.62-cm (3-inch) platinum
target was substituted for the 7.62-cm (3-inch) gold target that
had been used, the power was set at 0.03 kW, and the time of
deposition was 1 hour. The resulting amount of platinum on the
support medium was calculated to be about 0.25 weight percent.
Deposition of Titanium Dioxide
[0106] A 20 weight percent titanium (IV) oxysulfate dehydrate
solution was prepared by dissolving 20.0 g of
TiO(SO.sub.4).2H.sub.2O (Noah Technologies Corporation, San
Antonio, Tex.) in 80.0 g of deionized water with stirring. 50.0 g
of this solution was mixed with 175 mL of deionized water to form a
titanium dioxide precursor compound solution. A dispersion of
diatomaceous earth was prepared by dispersing 50.0 g of
diatomaceous earth in 500 mL of deionized water in a large beaker
with rapid stirring. After heating the diatomaceous earth
dispersion to about 80.degree. C., the titanium dioxide precursor
compound solution was added dropwise while rapidly stirring over a
period of about 1 hour. After the addition, the beaker was covered
with a watch glass and its contents heated to boiling for 20
minutes. An ammonium hydroxide solution was added to the beaker
until the pH of the contents was about 9. The resulting product was
washed by settling/decantation until the pH of the wash water was
neutral. The product was separated by filtration and dried
overnight at 100.degree. C.
[0107] A portion of the dried product was placed into a porcelain
crucible and calcined by heating from room temperature to
350.degree. C. at a heating rate of about 3.degree. C. per minute
and then held at 350.degree. C. for 1 hour.
Deposition of Iron Oxide
[0108] Iron oxide was deposited onto diatomaceous earth using
essentially the above-described titanium dioxide deposition
process, with the exception that a solution of 20.0 g of
Fe(NO.sub.3).sub.3.9H.sub.2O (J. T. Baker, Inc., Phillipsburg,
N.J.) dissolved in 175 mL of deionized water was substituted for
the titanyl sulfate solution. A portion of the resulting iron
oxide-modified diatomaceous earth was similarly calcined to
350.degree. C. for further testing.
Deposition of Iron Oxide and Titanium Dioxide
[0109] A mixture of iron oxide and titanium dioxide was deposited
onto diatomaceous earth using essentially the above-described
titanium dioxide deposition process, with the exception that a
solution of 10.0 g of Fe(NO.sub.3).sub.3.9H.sub.2O (J. T. Baker,
Inc., Phillipsburg, N.J.) and 25.0 g of TiO(SO.sub.4).2H.sub.2O
(Noah Technologies Corporation, San Antonio, Tex.) dissolved in 175
mL of deionized water was substituted for the titanyl sulfate
solution. A portion of the resulting iron oxide- and titanium
dioxide-modified diatomaceous earth was similarly calcined to
350.degree. C. for further testing.
Concentration Agent Screening: Microorganism Concentration Test
Method
[0110] An isolated microorganism colony was inoculated into 5 mL
BBL.TM. Trypticase.TM. Soy Broth (Becton Dickinson, Sparks, Md.)
and incubated at 37.degree. C. for 18-20 hours. This overnight
culture at -10.sup.9 colony forming units per mL was diluted in
adsorption buffer (containing 5 mM KCl, 1 mM CaCl.sub.2, 0.1 mM
MgCl.sub.2, and 1 mM K.sub.2HPO.sub.4) at pH 7.2 to obtain 10.sup.3
microorganisms per mL dilution. A 1.1 mL volume of the
microorganism dilution was added to separate, labeled sterile 5 mL
polypropylene tubes (BD Falcon.TM. Becton Dickinson, Franklin
Lakes, N.J.) containing 10 mg of concentration agent, each of which
was capped and mixed on a Thermolyne Maximix Plus.TM. vortex mixer
(Barnstead International, Iowa). Each capped tube was incubated at
room temperature (25.degree. C.) for 15 minutes on a Thermolyne
Vari Mix.TM. shaker platform (Barnstead International, Iowa). After
the incubation, each tube was allowed to stand on the lab bench for
10 minutes to settle the concentration agent. Control sample tubes
containing 1.1 mL of the microorganism dilution without
concentration agent were treated in the same manner. The resulting
settled concentration agent and/or supernatant (and the control
samples) were then used for analysis.
[0111] The settled concentration agent was re-suspended in 1 mL
sterile Butterfield's Buffer solution (pH 7.2.+-.0.2; monobasic
potassium phosphate buffer solution; VWR Catalog Number 83008-093,
VWR, West Chester, Pa.) and plated on 3M.TM. Petrifilm.TM. Aerobic
Count Plates culture medium (dry, rehydratable; 3M Company, St.
Paul., Minn.) according to the manufacturer's instructions. Aerobic
count was quantified using a 3M.TM. Petrifilm.TM. Plate Reader (3M
Company, St. Paul., Minn.). Results were calculated using the
following formula:
Percent CFU/mL in Re-suspended Concentration Agent=(number of
colonies from plated re-suspended concentration agent)/(number of
colonies from plated untreated control sample).times.100
(where CFU=Colony Forming Unit, which is a unit of live or viable
microorganisms). Results were then reported in terms of percent
capture of microorganisms by the concentration agent using the
formula below:
Capture Efficiency or Percent Capture=Percent CFU/mL in
Re-suspended Concentration Agent
[0112] For comparison purposes, in at least some cases 1 mL of the
supernatant was removed and plated undiluted or diluted 1:10 in
Butterfield's Buffer solution and plated onto 3M.TM. Petrifilm.TM.
Aerobic Count Plates culture medium. Aerobic count was quantified
using a 3M.TM. Petrifilm.TM. Plate Reader (3M Company, St. Paul.,
Minn.). Results were calculated using the following formula:
Percent CFU/mL in Supernatant=(number of colonies from plated
supernatant)/(number of colonies from plated untreated control
sample).times.100
(where CFU=Colony Forming Unit, which is a unit of live or viable
microorganisms). When the microorganism colonies and the
concentration agent were similar in color (providing little
contrast for the plate reader), results were based upon the
supernatant and were then reported in terms of percent capture of
microorganisms by the concentration agent using the formula
below:
Capture Efficiency or Percent Capture=100-Percent CFU/mL in
Supernatant
Concentration Agent Screenings 1-12 and Comparative Screenings 1
and 2
[0113] Using the above-described microorganism concentration test
method, 10 mg of various different surface-treated diatomaceous
earth or surface-treated calcined diatomaceous earth concentration
agents (prepared as described above) and 10 mg of untreated
diatomaceous earth (hereinafter, DE) were tested separately for
bacterial concentration against target microorganisms, the
gram-negative bacterium Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 35987) and the gram-positive bacterium
Staphylococcus aureus (ATCC 6538). The results are shown in Table 1
below.
TABLE-US-00001 TABLE 1 Screening Concentration Percent Capture .+-.
No. Microorganism Agent Standard Deviation C-1 Staphylococcus DE 54
.+-. 13 1 Staphylococcus TiO.sub.2-DE 94 .+-. 4 2 Staphylococcus
Fe.sub.2O.sub.3--TiO.sub.2-DE 96 .+-. 1 3 Staphylococcus calcined
100 .+-. 0 Fe.sub.2O.sub.3--TiO.sub.2-DE 4 Staphylococcus
Pt-calcined DE 99 .+-. 0 5 Staphylococcus Au--Fe.sub.2O.sub.3-DE 99
.+-. 0 6 Staphylococcus Au-calcined DE 99 .+-. 0 C-2 Salmonella DE
45 .+-. 1 7 Salmonella TiO.sub.2-DE 86 .+-. 3 8 Salmonella
Fe.sub.2O.sub.3--TiO.sub.2-DE 88 .+-. 1 9 Salmonella calcined 89
.+-. 5 Fe.sub.2O.sub.3--TiO.sub.2-DE 10 Salmonella Pt-calcined DE
72 .+-. 1 11 Salmonella Au--Fe.sub.2O.sub.3-DE 91 .+-. 6 12
Salmonella Au-calcined DE 100 .+-. 0 13 Salmonella Au-DE 89 .+-.
2
Concentration Agent Screenings 14-19 and Comparative Screening
3
[0114] Using the above-described microorganism concentration test
method, 10 mg of various different surface-treated diatomaceous
earth or surface-treated calcined diatomaceous earth concentration
agents (prepared as described above) and 10 mg of untreated
diatomaceous earth (hereinafter, DE) were tested separately for
yeast concentration of the target microorganism, Saccharomyces
cerevisiae (10.sup.2 CFU/mL; ATCC 201390). The resulting materials
were plated on 3M Petrifilm.TM. Yeast and Mold Count Plate culture
medium (dry, rehydratable; 3M Company, St. Paul, Minn.) and
incubated for 5 days according to the manufacturer's instructions.
Isolated yeast colonies were counted manually, and percent capture
was calculated as described above. The results are shown in Table 2
below (standard deviation for all samples less than 10
percent).
TABLE-US-00002 TABLE 2 Screening Concentration Percent No.
Microorganism Agent Capture C-3 Saccharomyces DE 42 14
Saccharomyces TiO.sub.2-DE 99 15 Saccharomyces
Fe.sub.2O.sub.3--TiO.sub.2-DE 93 16 Saccharomyces calcined 100
Fe.sub.2O.sub.3--TiO.sub.2-DE 17 Saccharomyces Pt-calcined DE 100
18 Saccharomyces Au--Fe.sub.2O.sub.3-DE 100 19 Saccharomyces
Au-calcined DE 99
Concentration Agent Screenings 20-22 and Comparative Screenings
4-6
[0115] Food samples were purchased from a local grocery store (Cub
Foods, St. Paul). Ham slices, lettuce, and apple juice samples (11
g) were weighed in sterile glass dishes and added to sterile
Stomacher.TM. polyethylene filter bags (Seward Corp, Norfolk, UK).
The food samples were spiked with bacterial cultures at a 10.sup.2
CFU/mL concentration using an 18-20 hour overnight culture (stock)
of Salmonella enterica subsp. enterica serovar Typhimurium (ATCC
35987). This was followed by the addition of 99 mL of Butterfield's
Buffer solution to each spiked sample. The resulting samples were
blended for a 2-minute cycle in a Stomacher.TM. 400 Circulator
laboratory blender (Seward Corp. Norfolk, UK). The blended samples
were collected in sterile 50 mL conical polypropylene centrifuge
tubes (BD Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) and
centrifuged (Eppendorf.TM. centrifuge 5804; Westbury, N.Y.) at 2000
revolutions per minute (rpm) for 5 minutes to remove large debris.
The resulting supernatants were used as samples for further
testing.
[0116] Using the above-described microorganism concentration test
method, each 1 mL test sample prepared as above was added
separately to a test tube containing 10 mg of surface-treated
diatomaceous earth and to a control test tube containing 10 mg of
untreated diatomaceous earth and tested for bacterial concentration
of the target microorganism, Salmonella enterica subsp. enterica
serovar Typhimurium (ATCC 35987). For testing in lettuce, samples
were placed in sterile 100.times.20 mm tissue culture petridishes
(Sarstedt, Newton, N.C.) and incubated under ultraviolet (UV)
lights in an Alphalmager.TM. MultiImage.TM. light cabinet (Alpha
Innotech Corporation, San Leandro, Calif.) for 1 hour to eliminate
background flora. Such UV-treated samples were confirmed for
absence of native flora (by plating and counting a 1 mL sample
essentially as described above) and then used for concentration
experiments. The results are shown in Table 3 below (standard
deviation for all samples less than 10 percent).
TABLE-US-00003 TABLE 3 Screening Concentration Percent No.
Microorganism Agent Sample Capture C-4 Salmonella DE Apple 43 Juice
20 Salmonella Au--Fe.sub.2O.sub.3-DE Apple 94 Juice C-5 Salmonella
DE Ham 75 21 Salmonella Au--Fe.sub.2O.sub.3-DE Ham 94 C-6
Salmonella DE Lettuce 55 22 Salmonella Au--Fe.sub.2O.sub.3-DE
Lettuce 80
Concentration Agent Screenings 23-24 and Comparative Screenings
7-8
[0117] Following the procedure of Examples 20-22 and Comparative
Examples 4-6 above with a turkey sample (using 25 g of sliced
turkey and 225 mL Butterfield's Buffer solution), surface treated
diatomaceous earth and untreated diatomaceous earth were separately
tested for concentration of the target microorganism Salmonella
enterica subsp. enterica serovar Typhimurium (ATCC 35987) from
large-volume samples (300 mg concentration agent per 30 mL sample
volume). Also tested was potable water (100 mL) from a drinking
fountain, which was collected in a sterile 250 mL glass bottle
(VWR, West Chester, Pa.) and inoculated with the target
microorganism Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 35987) at 10.sup.2 CFU/mL. The resulting
inoculated water was mixed manually end-to-end 5 times and
incubated at room temperature (25.degree. C.) for 15 minutes.
[0118] 30 mL samples prepared as described above were added to
sterile 50 mL conical polypropylene centrifuge tubes (BD
Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) containing 300
mg of concentration agent and were tested by using the
above-described microorganism concentration test method. The
resulting settled concentration agent was re-suspended in 30 mL
sterile Butterfield's Buffer solution and plated on 3M.TM.
Petrifilm.TM. Aerobic Count Plates culture medium. The results are
shown in Table 4 below (standard deviation for all samples less
than 10 percent).
TABLE-US-00004 TABLE 4 Screening Concentration Percent No.
Microorganism Agent Sample Capture C-7 Salmonella DE Potable 79
Water 23 Salmonella Au--Fe.sub.2O.sub.3-DE Potable 97 Water C-8
Salmonella DE Turkey 52 24 Salmonella Au--Fe.sub.2O.sub.3-DE Turkey
88
Concentration Agent Screenings 25-34
[0119] 10 mg samples of various different surface-treated
diatomaceous earth concentration agents (prepared as described
above) were tested separately for concentration of the target
bacterial endospores Bacillus atrophaeus (ATCC 9372) and Bacillus
subtilis (ATCC 19659). The above-described microorganism
concentration test method was utilized with the following
modifications: the overnight cultures had 1.4.times.10.sup.3 CFU/mL
Bacillus atrophaeus and 6.times.10.sup.2 CFU/mL Bacillus subtilis,
respectively; the resulting supernatants were plated undiluted; and
the settled concentration agent with bound microorganism was
resuspended in 5 mL sterile Butterfield's Buffer solution and
plated in duplicate (1 mL each). Capture efficiencies were
calculated based on counts from the plated supernatants, and the
results are shown in Table 5 below (standard deviation for all
samples less than 10 percent).
TABLE-US-00005 TABLE 5 Screening Concentration Percent No.
Microorganism Agent Capture 25 Bacillus atrophaeus TiO.sub.2-DE 79
26 Bacillus atrophaeus Fe.sub.2O.sub.3-DE 97 27 Bacillus atrophaeus
Pt-DE 100 28 Bacillus atrophaeus Au-DE 81 29 Bacillus atrophaeus
Au--Fe.sub.2O.sub.3-DE 100 30 Bacillus subtilis TiO.sub.2-DE 97 31
Bacillus subtilis Fe.sub.2O.sub.3-DE 97 32 Bacillus subtilis Pt-DE
100 33 Bacillus subtilis Au-DE 99 34 Bacillus subtilis
Au--Fe.sub.2O.sub.3-DE 99
Concentration Agent Screenings 35-38
[0120] 10 mg samples of two different surface-treated diatomaceous
earth concentration agents (namely, Pt-DE and Au--
Fe.sub.2O.sub.3-DE) were tested separately for concentration of the
target non-enveloped, bacteria-infecting virus, Escherichia coli
bacteriophage MS2 (ATCC 15597-B1; which is often used as a
surrogate for various human-infecting, non-enveloped enteric
viruses). A double layer agar method (described below) was used to
assay for capture of the Escherichia coli bacteriophage MS2 (ATCC
15597-B1) using Escherichia coli bacteria (ATCC 15597) as host.
[0121] Escherichia coli bacteriophage MS2 stock was diluted
ten-fold serially in sterile 1.times. adsorption buffer (containing
5 mM KCl, 1 mM CaCl.sub.2, 0.1 mM MgCl.sub.2, and 1 mM
K.sub.2HPO.sub.4) at pH 7.2 to obtain two dilutions with 10.sup.3
and 10.sup.2 plaque forming units per milliliter (PFUs/mL),
respectively. A 1.0 mL volume of resulting bacteriophage dilution
was added to a labeled sterile 5 mL polypropylene tube (BD
Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) containing 10
mg of concentration agent and mixed on a Thermolyne Maximix
Plus.TM. vortex mixer (Barnstead International, Iowa). The capped
tube was incubated at room temperature (25.degree. C.) for 15
minutes on a Thermolyne Vari Mix.TM. shaker platform (Barnstead
International, Iowa). After the incubation, the tube was allowed to
stand on the lab bench for 10 minutes to settle the concentration
agent. A control sample tube containing 1.0 mL of the bacteriophage
dilution without concentration agent was treated in the same
manner. The resulting settled concentration agent and supernatant
(and the control sample) were then used for analysis.
[0122] 100 microliters of the supernatant was removed and assayed
for bacteriophage using the double layer agar method described
below. An additional 800 microliters of supernatant was removed and
discarded. One hundred microliters of the settled concentration
agent was also assayed for bacteriophage.
Double Layer Agar Method:
[0123] A single colony of Escherichia coli bacteria (ATCC 15597)
was inoculated into 25 mL sterile 3 weight percent tryptic soy
broth (Bacto.TM. Tryptic Soy Broth, Becton Dickinson and Company,
Sparks, Md.; prepared according to manufacturer's instructions) and
incubated at 37.degree. C. in a shaker incubator (Innova.TM. 44,
New Brunswick Scientific Co., Inc., Edison, N.J.) set at 250
revolutions per minute (rpm) overnight. 750 microliters of this
overnight culture was used to inoculate 75 mL sterile 3 weight
percent tryptic soy broth. The resulting culture was incubated at
37.degree. C. in the shaker incubator set at 250 rpm to obtain
Escherichia coli cells in the exponential phase as measured by
absorbance at 550 nm (absorbance values 0.3-0.6) using a SpectraMax
M5 spectrophotometer (Molecular Devices, Sunnyvale, Calif.). The
cells were incubated on ice until used for assay.
[0124] One hundred microliters of the above-described bacteriophage
test samples were mixed with 75 microliters of the ice-incubated
Escherichia coli (host bacteria) cells and incubated at room
temperature (25.degree. C.) for 5 minutes. The resulting samples
were mixed with 5 mL sterile molten top agar (3 weight percent
tryptic soy broth, 1.5 weight percent NaCl, 0.6 weight percent
agar; prepared that day and maintained in a 48.degree. C.
waterbath). The mixture was then poured on top of bottom agar (3
weight percent tryptic soy broth, 1.5 weight percent NaCl, 1.2
weight percent agar) in petridishes. The molten agar component of
the mixture was allowed to solidify for 5 minutes, and the
petridishes or plates were inverted and incubated at 37.degree. C.
The plates were visually inspected after overnight incubation, and
those plates containing settled concentration agent (as well as the
control plate) showed the presence of bacteriophage plaques.
Capture efficiencies were calculated based on counts from the
plated supernatants and determined to be 96 percent and 97 percent
for Pt-DE (for the 10.sup.3 and 10.sup.2 PFU/mL dilutions,
respectively) and 94 percent and 95 percent for Au--
Fe.sub.2O.sub.3-DE (for the 10.sup.3 and 10.sup.2 PFU/mL dilutions,
respectively) (standard deviation less than 10 percent).
Concentration Agent Screenings 39-41
[0125] Apple juice was purchased from a local grocery store (Cub
Foods, St. Paul). Apple juice (11 g) was weighed in a sterile glass
dish and added to 99 mL sterile Butterfield's Buffer. The resulting
combination was mixed by swirling for 1 minute and was spiked with
two bacterial cultures, each at a 1 CFU/mL concentration, using
18-20 hour overnight cultures (bacterial stocks) of Salmonella
enterica subsp. enterica serovar Typhimurium (ATCC 35987) and
Escherichia coli (ATCC 51813). Serial dilutions of the bacterial
stocks had been made in 1.times. adsorption buffer as described
above.
[0126] Using the above-described microorganism concentration test
method, a 10 mL volume of the spiked apple juice sample was added
to a sterile 50 mL conical polypropylene centrifuge tube (BD
Falcon.TM., Becton Dickinson, Franklin Lakes, N.J.) containing 100
mg of a surface-treated diatomaceous earth concentration agent
(namely, Fe.sub.2O.sub.3-DE, TiO.sub.2-DE, or Au--
Fe.sub.2O.sub.3-DE) and incubated for 15 minutes for bacterial
capture/concentration of the target microorganism, Salmonella (in
the presence of the Escherichia coli, a competitor microorganism).
The resulting supernatant was removed, and the settled
concentration agent was transferred to another sterile 50 mL tube
containing 2 mL sterile 3 weight percent tryptic soy broth
(Bacto.TM. Tryptic Soy Broth, Becton Dickinson and Company, Sparks,
Md.; prepared according to manufacturer's instructions). The tube
was loosely capped, and its contents were mixed and incubated at
37.degree. C. After overnight incubation, the resulting broth
mixture was tested for the presence of Salmonella using a
RapidChek.TM. Salmonella lateral flow immunoassay test strip from
SDI (Strategic Diagnostics, Inc., Newark, Del.). Visual inspection
of the test strip showed it to be positive for Salmonella.
[0127] Nucleic acid detection by polymerase chain reaction (PCR)
was also carried out for the microorganism-containing broth
mixture. 1 mL of the above-described overnight-incubated,
concentration agent-containing broth was assayed as a test sample
for the presence of Salmonella by using a TaqMan.TM. ABI Salmonella
enterica Detection Kit from Applied Biosystems (Foster City,
Calif.). As a control sample, 1 mL of the 18-20 hour overnight
culture (stock) of Salmonella enterica subsp. enterica serovar
Typhimurium (ATCC 35987) was also assayed. PCR testing was
conducted in a Stratagene Mx3005P.TM. QPCR (quantitative PCR)
System (Stratagene Corporation, La Jolla, Calif.) by using the
following cycle conditions per cycle for 45 cycles: 25.degree. C.
for 30 seconds, 95.degree. C. for 10 minutes, 95.degree. C. for 15
seconds, and 60.degree. C. for 1 minute. An average (n=2) cycle
threshold value (CT value) of 17.71 was obtained for the control
sample. Average (n=2) CT values of 18.59, 20.44, and 16.53 were
obtained for the test samples containing Fe.sub.2O.sub.3-DE,
TiO.sub.2-DE, or Au-- Fe.sub.2O.sub.3-DE, respectively, indicating
a positive PCR reaction and confirming the presence of
Salmonella.
Preparation of Concentration Devices
[0128] Two different ultra high molecular weight polyethylene
(UHMWPE) powders were obtained from Ticona (a division of Celanese
headquartered in Frankfurt, Germany) as PMX1 (product number
GUR.TM. 2126, irregularly shaped, size range of 50-100 micrometers)
and PMX2 (product number GUR.TM. 4150-3, spherical, median particle
size of about 40 micrometers). The powders were combined in a 4:1
ratio of PMX1:PMX2. The resulting combination (hereinafter, UHMWPE
mixture) was used to prepare three types of concentration
devices.
[0129] For Concentration Device Type A, a mixture of 40 percent by
weight concentration agent comprising ferric oxide (prepared
essentially as described above; designated Fe.sub.2O.sub.3-DE) was
combined with 60 weight percent of the UHMWPE mixture. For
Concentration Device Type B, a mixture of 40 percent by weight
concentration agent comprising titanium dioxide (prepared
essentially as described above; designated TiO.sub.2-DE) was
combined with 60 weight percent of the UHMWPE mixture. For
Concentration Device Type C (Control), the UHMWPE mixture was used
without added concentration agent. For each concentration device,
the selected components were weighed out into a one-liter
cylindrical container or jar. The jar was then placed on a
rollermill spinning at a low speed (about 10-15 revolutions per
minute (rpm)) for at least two hours to produce a homogenous blend
or floc.
[0130] A portion (about 6 g) of the floc was then used to fill a 50
mm diameter cylindrical mold, which had a depth of 5 mm and also
had 0.05 mm (2 mil) thick disks of
polytetrafluoroethylene-impregnated fiberglass placed in its bottom
and in its lid to prevent sticking of the floc and to retard heat
transfer through the faces of the mold. The floc was compressed
into the mold, and the lid of the mold was then pressed into
position to close the mold.
[0131] The filled mold was placed on a vortex mixer (IKA.TM. MS3
Digital Vortexer, available from VWR Scientific, West Chester, Pa.)
for 10 seconds to eliminate voids and cracks in its contents. The
mold was then placed in a vented convection oven (Thelco Precision
Model 6555, available from Thermo Fisher Scientific, Inc., Waltham,
Mass.) set at 180-185.degree. C. for one hour to sinter the floc.
After cooling to room temperature (about 23.degree. C.), the
resulting sintered floc was removed from the mold and trimmed using
a punch die to a 47 mm diameter for use as a concentration
device.
Examples 1-2 and Comparative Examples C-1-C-4
[0132] Food samples (pasteurized orange juice (pulp-free), sliced
ham luncheon meat, and sliced chicken luncheon meat) were purchased
from a local grocery store (Cub Foods, St Paul, Minn.). 25 mL of
the orange juice and 25 grams of each sliced meat were weighed
separately in sterile Stomacher.TM. polyethylene filter bags (PE-LD
Model 400, Seward Corp, Norfolk, UK). The resulting samples were
inoculated with both Salmonella enterica subsp enterica serovar
Typhimurium bacteria (ATCC 35987) and Escherichia coli bacteria
(ATCC 51813) at about 1 CFU/mL concentrations of each from
overnight tryptic soy broth cultures (prepared essentially as
described above under the heading "Concentration Agent Screening:
Microorganism Concentration Test Method" except using Bacto.TM.
Tryptic Soy Broth (Becton Dickinson, Sparks, Md.)).
[0133] After incubating the spiked samples at room temperature
(about 23.degree. C.) for about 10 minutes, test samples were made
by adding 225 mL of sterile Butterfield's Buffer (pH 7.2, VWR, West
Chester, Pa.) to each spiked sample to provide the approximate
total sample microorganism concentrations shown in Table 6 below.
The resulting orange juice-based samples (each having a total
volume of about 250 mL) were mixed by swirling and then used for
testing. The resulting luncheon meat-based samples (each having a
total volume of about 250 mL) were blended for a 2-minute cycle in
a Stomacher.TM. 400 Circulator laboratory blender (Seward Corp.
Norfolk, UK) at 230 revolutions per minute (rpm) and were then
processed through a sterile 100 micrometer mesh filter (Cell
Strainer, BD, San Jose, Calif.) prior to testing.
[0134] The samples were pumped through selected concentration
devices at a flow rate of 10 mL/minute for 25 minutes using a
custom made sample holder for the concentration device (the holder
consisting of upper and lower flow distribution plates with a
plastic tube machined out to provide a friction fit for the 47 mm
diameter concentration device; O-rings were used to prevent leakage
on the upstream and downstream sides; throughbolts provided closure
pressure), a peristaltic pump (Heidolph.TM. Pump Drive 5201
peristaltic pump, available from VWR Scientific, West Chester,
Pa.), and 3.1 mm internal diameter tubing. A digital pressure
sensor (SSI Technologies Model MGI-30-A-9V, Cole-Parmer, Vernon
Hills, Ill.) was placed upstream of the sample holder to monitor
pressure drop.
[0135] The concentration devices included those of Types A
(Fe.sub.2O.sub.3-DE) and C (no concentration agent), both prepared
essentially as described above, as well as comparative commercial
absolute micron filters (0.8 micrometer polyether sulfone and 0.45
micrometer nylon filter membranes from Pall Corporation, East
Hills, N.Y.). Corresponding food samples that were never spiked
with microorganisms (and never run through the concentration
devices) were plated undiluted on 3M.TM. Petrifilm.TM. Aerobic
Count Plates (3M Company, St. Paul, Minn.). The plated samples were
incubated at 37.degree. C. for 18-20 hours and were quantified the
next day (per the manufacturer's instructions) as controls and were
found to be negative for the presence of microorganisms.
[0136] After pumping, the concentration device was removed from its
holder using sterile forceps and was incubated overnight in a
sterile Stomacher.TM. polyethylene filter bag (PE-LD Model 400,
Seward Corp, Norfolk, UK) containing 25 mL of sterile Bacto.TM.
Tryptic Soy Broth (Becton Dickinson, Sparks, Md.). The bag was
closed with rubber bands and incubated in a 37.degree. C. shaker
incubator (VWR Signature.TM. Benchtop Shaking Incubator Model
1575R, VWR Scientific, West Chester, Pa.) at 75 rpm for 18-20
hours. After the overnight incubation, the resulting cultures were
tested for the presence of Salmonella by using commercial
immunoassays (for example, 3M.TM. TECRA.TM. Unique Salmonella
immunoassay, available from 3M Company, St. Paul, Minn., or
RapidChek.TM. Salmonella lateral flow immunoassay test strip,
available from Strategic Diagnostics, Inc. (SDI), Newark, Del.).
Results are shown in Table 6 below. Concentration devices of the
invention were generally able to process somewhat larger volumes of
particulate-containing samples prior to clogging than were the
commercial absolute micron filters.
TABLE-US-00006 TABLE 6 Sample Volume Total Micro- Passed Salmonella
organism (mL)/ Immuno- Concentration Total assay in 250 mL Sample
Result Example Micro- Sample Concentration Volume (Post No. Sample
organisms (CFUs) Device (mL) Incubation) C-1 Orange E. coli and 650
Commercial 8/250 Positive Juice Salmonella Filter (0.45 micrometer)
C-2 Orange E. coli and 670 Type C 100/250 Positive Juice Salmonella
(Control) 1 Orange E. coli and 850 Type A 200/250 Positive Juice
Salmonella (Fe.sub.2O.sub.3-DE) C-3 Ham E. coli and 650 Commercial
2/250 Positive Salmonella Filter (0.8 micrometer) C-4 Ham E. coli
and 670 Type C 250/250 Positive Salmonella (Control) 2 Chicken E.
coli and 280 Type A 12/250 Positive Salmonella
(Fe.sub.2O.sub.3-DE)
Example 3
[0137] Pasteurized apple juice was purchased from a local grocery
store (Cub Foods, St. Paul, Minn.). 25 mL of the apple juice was
mixed with 225 mL sterile Butterfield's Buffer (pH 7.2, VWR, West
Chester, Pa.) and was inoculated with Salmonella enterica subsp.
enterica serovar Typhimurium (ATCC 35987) at a concentration of
about 100 CFU/mL. The resulting sample was incubated at room
temperature (about 23.degree. C.) for 10 minutes and then was
pumped (essentially as described above) through a Type B
(TiO.sub.2-DE) concentration device (prepared essentially as
described above) at a flow rate of 10 mL/minute for 25 minutes.
Flow through sample fractions (1 mL) were collected in labeled
sterile 5 mL polypropylene tubes (BD Falcon.TM., Becton Dickinson,
Franklin Lakes, N.J.) every five minutes for 25 minutes and were
plated onto 3M.TM. Petrifilm.TM. Aerobic Count Plates culture
medium (dry, rehydratable; 3M Company, St. Paul, Minn.) according
to the manufacturer's instructions.
[0138] After the sample was passed through the concentration
device, the device was placed (using sterile forceps essentially as
described above) in a sterile Stomacher.TM. polyethylene filter bag
(PE-LD Model 400, Seward Corp, Norfolk, UK) containing 100 mL
sterile 3 weight percent tryptic soy broth (Bacto.TM. Tryptic Soy
Broth, Becton Dickinson and Company, Sparks, Md., prepared
according to the manufacturer's instructions). The bag was loosely
tied and incubated at 37.degree. C. for 18-20 hours. After 6 hours
incubation, 100 microliters were removed from the bag and plated
undiluted on 3M.TM. Petrifilm.TM. Aerobic Count Plates (3M Company,
St. Paul., Minn.), which were also incubated at 37.degree. C. for
18-20 hours (along with the flow through sample plates) and
quantified the next day according to the manufacturer's
instructions.
[0139] Capture efficiency was calculated based on counts obtained
from the plated flow through sample fractions by using the formula
below (where CFU=Colony Forming Unit, which is a unit of live or
viable microorganisms):
Percent CFUs in Fraction=(number of colonies from plated
fraction)/(total number of colonies in sample).times.100
Capture Efficiency or Percent Capture=100-Percent CFUs in
Fraction
A capture efficiency of greater than 99 percent was obtained. The
plate containing the 6-hour time point sample (resulting from
concentration device incubation for 6 hours) was analyzed and found
to contain colonies too numerous to count, indicating that the
captured bacteria were viable.
[0140] The overnight cultured broth containing the concentration
device was diluted in Butterfield's Buffer and plated onto 3M.TM.
Petrifilm.TM. Aerobic Count Plates (3M Company, St. Paul., Minn.),
which were incubated at 37.degree. C. for 18-20 hours and
quantified the next day. Plate counts indicated that the captured
Salmonella in the concentration device had increased in number to a
concentration of about 2.7.times.10.sup.9 CFU/mL.
Example 4
[0141] An isolated bacterial colony of Salmonella enterica subsp.
enterica serovar Typhimurium (ATCC 35987) was inoculated into 5 mL
BBL.TM. Trypticase.TM. Soy Broth (Becton Dickinson, Sparks, Md.)
and incubated at 37.degree. C. for 18-20 hours. This overnight
culture at a concentration of about 1.times.10.sup.9 CFU/mL was
diluted in Butterfield's Buffer (pH 7.2.+-.0.2; monobasic potassium
phosphate buffer solution; VWR Catalog Number 83008-093, VWR, West
Chester, Pa.) to obtain an approximately 1.times.10.sup.3 CFU/mL
inoculum.
[0142] A volume of 250 mL potable water (from a drinking fountain)
was spiked with a 1:100 dilution of the approximately
1.times.10.sup.3 CFU/mL inoculum, resulting in a sample having a
concentration of about 16 CFU/mL (total of about 4000 CFUs in the
250 mL sample). The sample was pumped (essentially as described
above) through a Type A (Fe.sub.2O.sub.3-DE) concentration device
(prepared essentially as described above) at a flow rate of 10
mL/minute for 25 minutes. Flow through sample fractions (1 mL) were
collected and plated essentially as described above.
[0143] After the sample was passed through the concentration
device, the concentration device was `flushed` with
filter-sterilized 20 mL Butterfield's Buffer containing 500
micrograms/mL BSA (Bovine Serum Albumin, stock of 1 mg/mL in water,
powder purchased from Sigma Chemicals, St Louis, Mo.) for elution
of bacteria by reversing the flow (5 mL/min). The resulting eluate
was collected in a sterile 50 mL polypropylene tube and plated
essentially as described above.
[0144] The concentration device was removed, bagged, incubated
overnight (along with the plated flow through sample fractions and
plated eluate), and the incubated plates were quantified the next
day essentially as described above. After 4.5 hours of incubation,
a 100 microliter sample was removed from the bag containing the
concentration device, the sample was diluted in 900 microliters of
Butterfield's Buffer, and the resulting total 1 mL volume was
plated essentially as described above.
[0145] Capture efficiency was calculated based on counts obtained
from the plated flow through sample fractions (essentially as
described above), and a capture efficiency of greater than 99
percent was obtained. Elution (by reversing the flow) released
approximately 17 percent (680/4000 CFU) of the captured inoculum.
The plate containing the 4.5-hour time point sample (resulting from
concentration device incubation for 4.5 hours) was analyzed and
found to contain an average of about 1300 CFU/mL, indicating that
the captured bacteria were viable.
[0146] The overnight cultured broth containing the concentration
device was tested for the presence of Salmonella using a SDI
RapidChek.RTM. Salmonella lateral flow immunoassay strip from SDI
(Strategic Diagnostics Inc., Newark, Del.), and a positive result
was obtained. The overnight cultured broth containing the
concentration device was diluted, plated, and the plates incubated
overnight and quantified the next day essentially as described
above. The resulting plate counts indicated that the captured
Salmonella in the concentration device had increased in number to a
concentration of about 2.5.times.10.sup.9 CFU/mL.
Comparative Example C-5
[0147] The procedure of Example 4 was essentially repeated using a
Type C (control) concentration device (no concentration agent)
instead of the Type A (Fe.sub.2O.sub.3-DE) concentration device,
and using a spiked potable water sample having about 13 CFU/mL
(total of about 3300 CFUs in the approximately 250 mL sample). A
capture efficiency of greater than 99 percent was obtained, and
elution (by reversing the flow) released approximately 2.4 percent
(80/3300 CFUs) of the captured inoculum (almost an order of
magnitude less than the percent eluted in Example 4 above). This
elution result suggests that a concentration device of the
invention can provide advantages over the comparative device in the
isolation or separation of captured microorganisms to facilitate
further analysis.
Example 5
[0148] An isolated bacterial colony of Escherichia coli (ATCC
51813) was inoculated into 5 mL BBL.TM. Trypticase.TM. Soy Broth
(Becton Dickinson, Sparks, Md.) and incubated at 37.degree. C. for
18-20 hours. This overnight culture at a concentration of about
1.times.10.sup.9 CFU/mL was diluted in Butterfield's Buffer (pH
7.2.+-.0.2; monobasic potassium phosphate buffer solution; VWR
Catalog Number 83008-093, VWR, West Chester, Pa.) to obtain an
approximately 1.times.10.sup.4 CFU/mL inoculum.
[0149] A volume of 9.6 liters of deionized water (18 megaohm,
Milli-Q.TM. Biocel water purification system, Millipore, MA) was
spiked with a 1:100 dilution of the approximately 1.times.10.sup.4
CFU/mL inoculum, resulting in a sample having a concentration of
about 140 CFU/mL (total of about 1.3.times.10.sup.6 CFUs in the
sample). The sample was pumped (essentially as described above)
through a Type A (Fe.sub.2O.sub.3-DE) concentration device
(prepared essentially as described above) at a flow rate of 20
mL/minute for about 8 hours.
[0150] Flow through sample fractions (1 mL) were collected every 15
minutes for the first hour and then every hour for the next 7 hours
and were plated essentially as described above. Capture efficiency
was calculated based on counts obtained from the plated flow
through sample fractions (essentially as described above), and a
capture efficiency of greater than 99 percent was obtained.
Example 6
[0151] An isolated bacterial colony of Escherichia coli (ATCC
51813) was inoculated into 5 mL BBL.TM. Trypticase.TM. Soy Broth
(Becton Dickinson, Sparks, Md.) and incubated at 37.degree. C. for
18-20 hours. This overnight culture at a concentration of about
1.times.10.sup.9 CFU/mL was diluted in Butterfield's Buffer (pH
7.2.+-.0.2; monobasic potassium phosphate buffer solution; VWR
Catalog Number 83008-093, VWR, West Chester, Pa.) to obtain an
approximately 1.times.10.sup.4 CFU/mL inoculum.
[0152] A volume of 2.7 liters of deionized water (18 megaohm,
Milli-Q.TM. Biocel water purification system, Millipore, MA) was
spiked with a 1:100 dilution of the approximately 1.times.10.sup.4
CFU/mL inoculum, resulting in a sample having a concentration of
about 120 CFU/mL (total of about 3.1.times.10.sup.5 CFUs in the
sample). The sample was pumped (essentially as described above)
through a Type B (TiO.sub.2-DE) concentration device (prepared
essentially as described above) at a flow rate of 15 mL/minute for
about 3 hours.
[0153] Flow through sample fractions (1 mL) were collected every 15
minutes and were plated essentially as described above. Capture
efficiency was calculated based on counts obtained from the plated
flow through sample fractions (essentially as described above), and
a capture efficiency of 97 percent was obtained.
[0154] The referenced descriptions contained in the patents, patent
documents, and publications cited herein are incorporated by
reference in their entirety as if each were individually
incorporated. Various unforeseeable modifications and alterations
to this invention will become apparent to those skilled in the art
without departing from the scope and spirit of this invention. It
should be understood that this invention is not intended to be
unduly limited by the illustrative embodiments and examples set
forth herein and that such examples and embodiments are presented
by way of example only, with the scope of the invention intended to
be limited only by the claims set forth herein as follows:
* * * * *