Note: Descriptions are shown in the official language in which they were submitted.
CA 02237639 1998-0~-12
W O 97/18455 PCTrUS96/18119
DESCRIPTION
r
Method For Ouanti~ication of
Bioloqical Material in a Sample
Related A~lications
This application is a continuation-in-part of Croteau
et al., U.S. Serial No. 08/606,229 filed Feb~uary 23,
1996, which is a continuation-in-part of Croteau et al.,
U.S. Serial No. 08/557,529, ~iled November 13, 1995, both
entitled "Method ~or Quanti~ication o~ Biological Material
in a Sample" hereby incorporated herein by re~erence,
including drawings.
Field o~ the Invention
This invention relates to a method ~or quantification
o~ biological material in a sample.
Backqround o~ the Invention
Many industries need to detect and quanti~y the con-
centration and level o~ biological material in a sample.
For example, the determination o~ bacterial concentration
in food and water is an essential part o~ ~ood and water
quality testing. EPA regulations require that no Coli-
form such as Escherichia coli can be present in potable
water. The "presence/absence" ~ormat o~ a testing medium,
such as Colilert~ chemical mixture (IDEXX Laboratories,
ME) which is used as a testing medium for Escherichia coli
and all coli~orm bacteria, is very useful in making this
determination. Colilert~ chemical mixture is based on the
De~ined Substrate Technology described in Edberg, "Method
and Medium ~or use in Detecting Target Microbes In Si tu in
A Specimen Sample o~ A Possibly Contaminated Material,"
U.S. Patent Nos. 4,925,789 and 5,492,933. See also,
Townsend et al., U.S. Serial No. 08/484,593 ~iled June 7,
1995 entitled, "Method and Composition ~or Detecting
Bacterial Cont~m;nation in Food Productsn, hereby
CA 02237639 1998-0~-12
W O 97/18455 PCTAUS96/18119
incorporated by reference herein, describes a medium ~or
the detection of bacteria in food and water samples.
However, there are areas where the ~uantification, not
just the detection, of bacterial concentration is impor-
tant. Examples of such areas include waste water, inco-
ming water in water purification systems, surface water,
and food testing. For example, numerous restaurant chains
will only accept raw ground beef or poultry that contains
less than a certain concentration of bacterial contamina-
tion. Therefore, food processing plants must carry outthe necessary microbiological tests to determine the
bacterial concentration of these food items before they
can be released to customers.
The classical methods of quantification of biological
material are the standard plate count method or the multi-
ple tube fermentation (MTF) method. A quantity of sample
being tested for microbial contamination is first dis-
pensed in a Petri-dish. Then 15 ml of the appropriate
media is poured over the sample. The Petri-dish is then
swirled to mix the sample in the medium and the Petri-dish
is left to solidify at room temperature for approximately
20 minutes. The medium is then incubated at a specific
temperature for a specific time, and any resulting
colonies are counted.
The multiple tube fermentation method is described in
Recles et al., "Most Probable Number Techniques" published
in "Compendium of Methods for the Microbiological ~m; ~ -
tion of Foods", 3rd ed. 1992, at pages 105-199, and in
Greenberg et al., "Standard Methods For the Ex~m;n~tion of
Water and Wastewater" 8th ed. 1992). In this method, a
volume of sample is dispensed into several tubes repre-
senting this dilution range. The tubes are then incubated
at the appropriate temperature so that the bacteria in
each tube are allowed to grow. After incubation at a
specific temperature for a specific time, the number of
positive tubes is counted. The most probable number can
CA 02237639 1998-0~-12
W O 97/18455 PCT~US96/18119
be determined from the formula described in Recles et al.,
- supra.
Water testing is mostly done by membrane filtration,
where a certain volume of water is passed through the
membrane and the membrane is incubated in a medium for a
certain period of time. After appropriate incubation, the
colonies are counted.
Summarv of the Invention
The present invention provides a simple method for
more accurate quantification of the number of micro-
organism in a sample, or for quantification of any other
type of discrete particulate biological material within a
sample. Such biological materials include fungi or other
living organisms, as well as aggregates of proteins, such
as enzymes, or even co-factors, using reaction mixtures
well known to those in the art. The invention generally
makes use of a novel article which is designed to hold a
liquid sample in which chemical and/or microbiological
reactants are provided. For example, such chemical
reactants may be a specific growth medium for bacteria.
The device used is generally in the form of an incubation
plate having a multitude of wells able to hold separate
aliquots of liquid. Generally, the device i8 designed to
hold between 5 and 100 ml of liquid in total, and the
wells are designed to form separate incubation chambers
for each aliquot of sample. The wells can ~e of same size
or of different size and shape to increase counting range
and/or simulate dilution effects. See, Naqui et al., U.S.
Serial No. 08/201,110, filed February 23, 1994, entitled
"Apparatus and Method for Quantification of Biological
Material in a Liquid Sample", incorporated by reference
herein.
Thus, in a first aspect the invention features a
method for detection of a biological material in a sample.
The method includes the steps of liquefying the sample (if
necessary) and pouring the liquified sample and reagent
CA 02237639 1998-0~-12
W O 97/18455 PCTrJS96/18119
into the incubation plate. The incubation plate has a
generally flat horizontal sur~ace and the surface is
divided into a plurality of at least 20 recessed wells.
Each well is adapted to hold an aliquot of liquid and is
sized and shaped, and formed of a suitable material, to
hold the aliquot within the well by surface tension. Any
excess liquid from the liqui~ied sample is poured ~rom the
surface of the plate due to the hydrophobicity of the
material used to form the plate. The method then involves
incubating that incubation plate until the presence or
absence of the biological material is determined. In a
preferred embodiments the wells are cham~ered to allow
liquid, that is above the horizontal plane, to be poured
off easily (see Fig. 3).
As noted above, the biological material that can be
detected is any material that forms a discrete particle,
such as a microorganism, which may be quantified by
determining the presence or absence o~ such a biolo~ical
material within each well of the incubation plate. The
sample may be any biological sample or en~ironmental
sample such as waste water, food, a sur~ace swab, or swabs
from other surfaces, such as a throat, or other samples
well known to those in the art. This sample may be a
liquid sample, or may be dissolved in a liquid to form the
liquified sample. Thus, the term "liquefying" in the
above paragraph re~ers to providing the sample in a liquid
that once combined with a microbiological reagent can be
rapidly aliquoted within the incubation plate. The
liquidi~ied sample may remain as a liquid or may be
solidified in the wells.
The incubation plate may be ~ormed of any desired
material, but in particular it is desirable that a plastic
be used which allows separate aliquots o~ the liqui~ied
sample to be held by surface tension within each well
without cross contamination of the wells. Preferably, the
material is hydrophobic. The sur~ace can be untreated or
CA 02237639 1998-0~-12
W O 97/1845~ PCT~US96/18119
treated chemically or physically to enhance retention of
liquid within the wells.
The shape of the incubation plate is not relevant, and
in preferred embodiments is a generally circular shape
(such as that of a Petri-dish). Indeed, the incubation
plate can be used to take the place of a Petri-dish.
Specifically, the method of this invention can be used to
replace those existing tests that are generally run on
Petri dishes to score the number of bacterial colonies.
lo Since discrete aliquots of the sample are provided in the
plate, one of ordinary skill in the art need only score
the number o~ positive wells in the plate to define the
~uantity of biological material within the original
sample, as with the MPN test discussed above.
The generally flat horizontal surface is designed to
allow the li~uid to be aliquoted readily between the wells
and then excess liquid to be poured from the plate. In a
preferred embodiment, a lip or pouring spout is provided
for the plate. Those in the art will recognize that the
depth and shape of the wells, as well as the material used
to make the wells and the plate, are chosen such that
surface tension can be used to hold the aliquots within
each well dependent on the type of the liquid used in the
liquified sample.
In other preferred embodiments, the surface defines at
least 40, 60, 80 or even 200 or more recessed wells; the
plate is formed of any formable plastic; a lid is also
provided to prevent contamination of liquid within the
wells; and the plate is provided in a sterile form so that
no positive aliquots are noted unless at least one
biological material particle is present in the sample.
In yet other preferred embodiments, the incubation
plate is clear or colored, for example, white or yellow
(to enhance the appearance of color (e.g., blue)) within
the incubation plate) and the well has a diameter of about
0.15 inches, and the plate a diameter of about 3 or 5
inches.
CA 02237639 1998-0~-12
W O 97/18455 PCTnUS96/18119
In still other preferred embodiments, the incubation
plate has a "pour-of~ pocket" adjacent to the sur~ace of
the plate. The pocket has sufficient capacity to contain
the excess liquid to be removed from the plate surface.
As an aid in preventing the excess fluid from spilling
back onto the plate surface, it is preferable that the
pocket contain an absorbent material, e.g., a gauze-like
material. In a particular embodiment, the plate has both
a pour-of~ pocket and a "landing pad". Th~e "landing pad"
i9 described below.
In a related aspect, the invention features a sterile
incubation plate having a generally flat horizontal sur-
face. The surface de~ines a plurality of at least 20
recessed wells (in preferred embodiments, at least 40, 60,
90 or even 200 recessed wells are provided) and each well
is adapted as described above to hold aliquots of liquid
by surface tension.
In preferred embodiments, the invention features the
sterile incubation plate much as described above but
having incorporated therein a "landing pad", which is a
generally central area of the plate lacking wells, which
can receive the sample prior to that sample being diluted
in, for example, an incubation medium. Thus, a volume of
0.01 to 5 ml of sample liquid may be applied in the
"landing pad" area (depending on its size and shape) and
then that liquid dispensed into each well by applying the
diluent and growth supporting medium (e.g., the ColilertTM
chemical mixtures noted above) and that liquid will simul-
taneously dilute the sample and allow dispersion of the
sample throughout the wells.
In addition, a pour spout can be provided within the
incubation plate to allow pouring of~ excess liquid within
the plate. Such a pour spout can be matched with a suit-
able lid having a slit which allows liquid in the incuba-
tion plate to be poured from the incubation plate onlywhen the slit is lined up with the pour spout, as des-
cribed below.
CA 02237639 1998-0~-12
W O 97/18455 PCTAJS96/18119
As indicated for the method above, the incubation
- plate may also have a "pour-off pocket" adjacent to the
surface of the plate. The pocket has sufficient capacity
to contain the excess liquid to be removed from the plate
surface, and preferably the pocket contains an absorbent
material, e.g., a gauze-like material. In a particular
embodiment, the plate has both a "pour-off pocket" and a
"landing pad".
Applicant provides an extremely useful method which
allows unskilled personnel to rapidly determine the
quantity of biological material within a sample. Since
the sample is readily liquified by people without siyni-
ficant training in microbiology, and the materials for any
specific tests can be provided by the manufacturer, such
people can readily per~orm the tests with accuracy. The
incubation plate is generally provided in the sterile form
so that no }nappropriate detection of biological material
can occur.
While it is known to provide plastic containers which
can hold liquid within a plurality of recesses, applicant
believes that this device provides a new automatic aliquo-
ting method. This is an improvement over the existing
products used to detect and quantify microorganisms
because the liquid migrates to the individual wells
without individual dispensing.
The present device can replace the use of a Petri-dish
and can be used particularly in ~ood analysis and in
testing of clinical samples. The separation of the wells
of the present device prevents crosstalk or contamination
between each aliquot. Because of this, many of the tests
can be performed by observing fluorescence (which is not
readily performed in an agar-containing Petri-dish). The
device is particularly useful when there is a large quan-
tity of microorganisms present in a sample, such as more
than one organism per one ml or per ten ml.
CA 02237639 1998-0~-12
W O 97/18~5 PCTAUS96/18119
Other ~eatures and advantages o~ the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
Descri~tion of the Prefer~ed Embodiment
The drawing will first briefly be described.
Drawinqs
Figs. 1 and 2 are diagrammatic representations of a
formed plastic incubation plate.
Fig. 3 shows a cross section of a well, with or
without a chamfer.
Fig. 4 is a diagrammatic representation of a formed
plastic incubation plate having a pour spout and
corresponding slit as well as a "landing padn.
Fig. 5 is a diagrammatic representation of a formed
plastic incubation plate having a "landing pad" and a
"pour-off pocket".
Structure
Referring to Figs. 1 and 2 there is shown an incuba-
tion plate 10 having a plurality of wells 12 each having
a diameter of about 0.16 inches. The incubation plate 10
has a diameter of about 5 lnches. The incubation plate is
made of formed plastic. Wells 12 are spaced apart suffi-
ciently to prevent crosstalk between the wells. These
wells may have a chamfer (Fig. 3) if desired to prevent
liquid remaining at the upper edge of the well. Those in
the art will recognize that incubation plate 10 can be
readily formed by standard procedure and manufactured in
the general shape of a Petri-dish, with or without a lip
or pouring spout, and with or without a lid 14. This lid
is provided with a dimple 16 to prevent contact of the lid
with plate 10.
Referring to Fig. 4, there is shown in incubation
plate 10 having a plurality of wells much as described
above. The incubation plate also includes a "landing pad~
CA 02237639 1998-0~-12
W O 97/18455 PCTrUS96/18119
22 of size about one and one-half inch diameter which is
- simply an area abLe to hold a defined volume of liquid.
Within the incubation plate is also provided a pour spout
24 which allows excess liquid to be removed from the
incubation plate. Also provided is a corresponding lid 14
having a slit which can be matched with the pour spout to
allow liquid to be removed from the incubation plate.
When the slit is not aligned with the pour spout, evapora-
tion of liquid within the plate is decreased by reducing
lo air flow over the liquid in the wells. A dimple 16 may
also be provided in the lid to prevent the lid surface
contacting the wells and thus preventing cross contamina-
tion between the wells.
Referring to Fig. 5, there is shown an incubation
plate lO having a plurality of wells much as described
above, which also includes a "landing pad" 22 o~ size
about one inch diameter. Within the incubation plate is
also provided a "pour-off pocket" 26 adjacent to the sur-
face of the plate which allows excess liquid to be removed
from the incubation plate. As shown in the cross-
sectional view, the "pour-off pocket" is formed by an
elevated barrier 28 between the pocket and the plate
surface. The barrier has a lower barrier section 30,
which serves as a channel through which the excess fluid
from the plate surface may be poured into the pocket.
Typically, the pocket will contain an absorbent material
which will retain the fluid within the pocket, preventing
back spill onto the plate surface.
Use
In use, a reagent powder suitable for detection of a
biological material can be rehydrated with an appropriate
amount of sterile liquid and then inoculated with a known
volume of a test sample. For example, 20 ml of sterile
water can be inoculated with between lo and 1,000 micro-
liter of sample. The inoculated reagent can then be added
to incubation plate 10 and that liquid swirled within
CA 02237639 1998-0~-12
W O 97/18455 PCTrUS96/18119
incubation plate 10 to distribute the inoculated liquid
reagent to each of wells 12. Incubation plate 10 is then
held at an angle of approximately 90 degrees to allow
excess inoculated liquid reagent to be removed from the
plate. A lid may then be placed on the incubation plate
and that plate held in an incubator for the appropriate
length of time, for example 18-48 hours. After that
length of time, the presence or absence of a positive
result can be scored in each well 12 of the plate. In
addition, for plates having a "pour-off pocket", due to
the larger volume of fluid contained in~ the pocket, a
positive result in the pocket can serve as an early
indication o~ high bacterial counts.
~xample 1: ~se of Incubation Plate For Bulk Testina
For total plate count a plate as described above is
used for the detection and quantification of the total
bacterial concentration o~ food. It is based on a Multi-
ple Enzyme Technology (Townsend and Chen, Method and
Composition for Detecting Bacterial Contamination in Food
Products, U.S. Serial No. 08/484,593 hereby incorporated
by reference herein) which correlates enzyme activity to
the presence of viable bacteria in ~ood. It utilizes
multiple enzyme substrates that produce a blue ~luorescent
color when metabolized by bacteria. When the liquid
reagent is inoculated with a prepared food sample and
dispensed into a plate as described herein the total
viable bacterial concentration of that food product can be
determined after 24 hours of incubation. The actual
medium used herein is not critical to the invention, but
is provided only for illustrative purposes.
Storaqe and Dis~osal
Store bulk powder and unused Simplates at room temper-
ature (4 to 25~C) away from the light. After use, the
Simplate device will contain viable bacteria which must be
handled and discarded appropriately. Once the powder is
CA 02237639 1998-0~-12
W O 97/18455 PCT~US96/18119
rehydrated it is stable for up to 24 hours when stored at
- 4 to 25~C.
Test Procedure
1. Pour an appropriate amount of bulk powder to a
container of sterile deionized water. One vial contains
enough powder for 10 tests. Each test has a ~inal volume
o~ 10ml. For example: add 1 vial of powder to 100ml of
sterile water to make enough media for 10 tests.
2. Place test sample on the center "landing pad" 22
of the plate 10 shown in Fig. 4. At the completion of
this procedure half of the test sample will be poured off
and discarded, there~ore, the size o~ the inoculum must
take this into account. For example, if you wish to
measure the bacterial concentration o~ 0.lml of test
sample then you must place 0.2ml of test sample on the
"landing pad". Place no more than 2ml on the center
"landing pad".
3. Remove the lid from the plate and dispense 10ml
of TPC media in the plate making sure to direct the liquid
over the test sample on the center "landing pad". If the
test sample is greater than 0.lml add enough TPC to
achieve a final volume of 10ml in the plate. Note, if the
liquid is not dispensed on the "landing pad" it may
splatter.
4. Place the lid back on the plate. Note, to ensure
that the liquid remains in the Simplate make sure that the
slit on the lid is not lined up with the pour spout.
5. Distribute the liquid into the wells by swirling
the plate as you would a standard pour plate.
6. Line up the slit on the lid with the pour spout
and carefully pour off the excess liquid that did not end
up in the wells. Holding the plate at an angle of approx-
imately 90- from the work bench ensures proper pour off of
excess liquid. Make sure that all liquid "cross bridges"
between wells are removed by gently tapping the plate.
Dispose of excess liquid appropriately.
CA 02237639 1998-0~-12
W O 97/18455 PCT~US96/18119
7. Slide the lid away from the pour spout to avoid
drying the liquid in the wells during incubation and to
avoid contamination from outside through the opening.
8. Place the plate in an incubator for 24 hours.
Plates can be inverted if desired. Incubation
temperatures greater than 37 C are not recommended.
9. Count the number of fluorescent wells after 24
hours by placing a 6 watt 36nm W light within five inches
of the plate. Do not read plate before 24 hours..
Results are stable to 48 hours.
10. Compare the number of fluorescent wells to an MPN
chart to determine the most probable number of bacterial
present in the plate.
Test Procedure Usina Plate Havinq "Pour-Off Pocket"
1. Pour an appropriate amount of bulk powder to a
container of sterile deionized water. One vial contains
enough powder for 10 tests. Each test has a final volume
of 10ml. For example: add 1 vial of powder to 100ml of
sterile water to make enough media for 10 tests.
2. Place test sample on the center "landing pad" 22
of the plate 10 shown in Fig. 5. At the completion of
this procedure half of the test sample will be poured off
into the pocket, therefore, the size of the inoculum must
take this into account. For example, if you wish to
measure the bacterial concentration of 0.lml of test
sample then you must place 0.2ml of test sample on the
"landiny pad". Place no more than 2ml on the center
"landing pad".
3. Remove the lid from the plate and dispense 10ml
of media in the plate making sure to direct the liquid
over the test sample on the center "landing pad". If the
test sample is greater than 0.lml add enough media to
achieve a final volume of 10ml in the plate. Note, if the
li~uid is not dispensed on the "landing pad" it may
splatter.
CA 02237639 1998-0~-12
W O 97/18455 PCTrUS96/18119
4. Distribute the liquid into the wells by swirling
- the plate as you would a 5tandard pour plate, taking care
that the fluid does not enter the "pour-off pocket".
5. Care~ully pour off the excess liquid that did not
end up in the wells through the "pour-off pocket" barrier
channel. Holding the plate at an angle of approximately
go ~rom the work bench ensures proper pour off o~ excess
liquid. Make sure that all liquid "cross bridges" between
wells are removed by gently tapping the plate.
106. Place the plate in an incubator ~or 24 hours.
Plates can be inverted if desired if the "pour-off" pocket
contains an absorbent material.
7. Count the number of ~luorescent wells after 24
hours by placing a 6 watt 36nm W light within five inches
o~ the plate. Do not read plate before 24 hours. Results
are stable to 48 hours.
8. Compare the number of fluorescent wells to an MPN
chart to determine the most probable number of bacterial
present in the plate.
ExamPle 2: Use of Incubation Plate for Unit Dose Testinq
The plate and media described in Example 1 are used
for this test.
Test Procedure
1. Add 10ml of sterile water to the tube of predis-
pensed powder. If greater than 0~lml of ~ood sample is tobe inoculated into the test, reduce the volume of sterile
water appropriately to achieve a final volume of 10ml in
the tube.
2. Inoculate the liquid reagent with the food sample
being tested.
3. Shake tube several times to completely mix powder
and inoculated food sample. Avoid excessive mixing which
tends to foam up liquid reagent. Too much foam can
complicate the distribution of the liquid into the plate.
The rest o~ the procedure is as in Example 1.
Other embodiments are within the following claims.
