Note: Descriptions are shown in the official language in which they were submitted.
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Title: System for Rapid Analysis of
Microbiological Materials in Liquid Samples
Field of the invention
[0001] This invention relates to methods and apparatus for detecting
the presence and enumeration of microbiological materials in liquid samples,
and in particular, to methods and apparatus for the quantitative analysis of
pathogenic microbes in water samples.
Background of the invention
[0002] Drinking water and recreational water (water at be aches and
other swimming facilities) should be tested on a regular basis, and the test
results should be made available within a short period of time, in order to
protect the public from harmful and contagious deceases.
[0003]
Currently, most water sample tests are carried out in a
laboratory environment away from water facilities or locations. Many methods
and procedures currently used in routine microbiological anal lysis were
developed over 100 years ago. They are labor intensive and time¨consuming
procedures both in operation and data collection. The results of such tests
are typically not made available to the operators of these facilities for
about 36
to 72 hours. Consequently, it is often not possible for operato rs of water
facilities to take action to correct tainted water until long after the
tainted water
has been consumed or used.
[0004] In
addition, the transmission of water-borne diseases remains a
major concern despite worldwide attempt to curb the problem. This problem
is not confined to developing and under developed countries but is global in
nature. Some key reasons for this are:
(1) The
current testing frequencies are not sufficient to provide early
warning so that corrective action can be taken to prevent outbreak of
diseases; and
(2) The current testing methods are laborious and time consuming and
hence discourage frequent testing.
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[0005] In 1988, Edberg S.C., et al. developed a new technology based
on a chemically defined substrate MTF method known as Autoanalysis
Colilert (AC): "National filed evaluation of a defined substrate method for
the
simultaneous evaluation of total coliforms and Escherichia coli from drinking
water: comparison with standard multiple tube technique', Appl. Environ.
Microbiol., 54 1595 (1988)." This allowed the simultaneous detection and
identification of both total coliforms and E.coli in water at 1CFU per 100mIs
in
less than 24 hours. The Colilert , a chromogenic-fluorogenic reagent
medium, provided the specific nutrients, and enzyme substrates with
chromophores and fluorophores for the simultaneous detection of total
coliform and E.coli. In 1989, the US EPA approved this method as a means
of qualitative testing of total coliform in drinking water.
[0006] The development of chromogenic/fluorogenic reagents to
conduct microbial testing has opened the door to better and faster testing
protocols. In addition, these products provide the additional opportunity to
use technology such as optical spectroscopy to conduct biological,
microbiological and chemical analysis. Spectrophotometric analyses are very
sensitive and hence can detect the presence of a very low concentration of
color producing components of interest in liquid samples (in parts per
million).
Visually, the human eye can only detect the color when these components are
present in very high concentration, thus the need for incubation periods
ranging from 18 to 72 hours. The time required to identify or estimate the
presence of microbiological indicators in water, food and environmental
samples can be drastically reduced when combining incubation with
photometric analysis.
[0007] The use and advantage of spectrophotometric application to
microbial analysis in liquid samples have been cited in the literature.
However,
the tests have been done outside the incubation chamber by drawing an
aliquot of a sample from the incubation vessel to photometric tubes at various
intervals and measuring using standard spectrometers. This is not only time
consuming but requires separate incubators, spectrometers and technical
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personnel to conduct the test and in some cases robotic sampling systems.
There is also a potential risk of cross contamination and human error, if
proper care is not applied in conducting the analysis.
[0008] Accordingly, there is an urgent need for improved methods and
apparatus for testing microbiological materials in drinking water,
recreational
water and wastewater, to provide better management of water facilities and to
protect public health and the environment.
Summary of the Invention
[0009] The present invention relates to a system for the rapid
quantitative analysis of bacteria in fluid samples such as water. One aspect
of the present invention is a system comprising a specimen container for
containing a test sample and an apparatus having a spectrophotometer
system comprising an appropriate light emitting source and a detector
proximate to the specimen container within the housing of the apparatus. A
reagent that provides a detectable parameter (e.g., color, fluorescence etc.)
is
added to the test sample in the specimen container. While the sample
undergoes incubation, the detector monitors the light from the source passing
through the sample and the specimen container. The detector is connected to
a spectrophotometer processor that measures, processes, records and stores
the information. The processor can also be connected to an appropriate
measuring and recording device such as a computer, multimeter or any other
device, which can measure, and record the output signal from the detector.
This provides a non-intrusive continuous incubation and signal growth
measurement of the parameter under investigation.
[0010] Another aspect of the present invention is a system for the rapid
analysis of microbiological parameters in liquid samples. The system
comprises a specimen container for containing the liquid sample, the
specimen container being made from a material that allows for the
propagation of light, a housing defining an enclosable chamber for holding the
specimen container, an incubating system mounted within the housing for
incubating microbiological materials within the liquid sample, and a
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spectrophotometer system mounted within the housing for propagating light
within the specimen container and measuring light absorbed, emitted or
scattered by the liquid sample as the microbiological materials are incubated
by the incubating system over time.
[0011] A further
aspect of the present invention is an apparatus for
detection of microbiological materials in a liquid sample. The apparatus
comprises a housing having an enclosable chamber shaped for holding a
clear plastic container for containing a liquid sample, incubating apparatus
mounted within the housing for incubating any microbiological materials within
the liquid sample, and spectrophotometer apparatus mounted within the
housing for measuring light absorbed, emitted or scattered by the liquid
sample as the microbiological materials are incubated by the incubation
apparatus over time.
[0012] The
present invention is also directed to a method for the rapid
analysis of microbiological materials in a liquid sample, comprising the steps
of:
(a) mixing a liquid sample having an unknown initial population
of a microbiological material with a reagent in a specimen
container, the reagent providing a detectable parameter
indicative of the microbiological material, thereby creating a
sample/reagent mixture;
(b) placing the specimen container in an enclosable housing
and enclosing the housing;
(c) incubating the sample/reagent mixture in the enclosed
housing at a temperature within a pre-selected temperature
range over a period of time; and
(d) measuring changes in the detectable parameter as the
sample/reagent mixture is being incubated during the period
of time.
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[0013] The present invention is further directed to a method for the
rapid quantitative analysis of microbiological materials in a liquid sample.
The
method comprises the steps of:
(a) placing a liquid sample having an unknown initial population
of a microbiological material in a specimen container made
from a material that allows for the propagation of light;
(b) creating a sample/reagent mixture by mixing the liquid
sample with a reagent that provides a detectable parameter
indicative of the microbiological material upon exposure to
light;
(c) incubating the sample/reagent mixture in an enclosed
housing at a temperature within a pre-selected temperature
range over a period of time;
(d) measuring changes in the detectable parameters as the
sample/reagent mixture is being incubated by propagating
light of a known intensity within the sample/reagent mixture
and measuring changes in the intensity of the light over
time;
(e) recording the changes in the intensity of the light as a
function of time;
(f) recording a time of significant deviation at which there
occurs an exponential change in the detectable parameter;
and
(g) determining the initial population by correlating the time of
significant deviation with known times of exponential growth
for the detectable parameter for known initial populations of
the microbiological material.
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Brief description of the drawings
[0014] The invention will now be described, by way of example only,
with reference to the following drawings, in which:
[0015] Figure 1 is a schematic diagram of the systern of the present
invention;
[0016] Figure 2 is a perspective view of apparatus madle in
accordance
with a preferred embodiment of the subject invention;
[0017] Figure 3 is an exploded perspective view of the subject
apparatus, showing the cap off and the specimen container removed from the
base unit;
[0018] Figure 4 is a sectional view of the subject apparatus taken
along
line 4-4 of Figure 2;
[0019] Figure 5 is a sectional view of the subject apparatus taken
along
line 5-5 of Figure 4;
[0020] Figure 6 is a sectional top view of the subject apparatus taken
along line 6-6 of Figure 4;
[0021] Figure 7 is a sectional front view of an apparatus made in
accordance with an alternative embodiment of the present invention;
[0022] Figure 8 is a flow chart illustrating the method of the
subject
invention;
[0023] Figure 9 is a graph illustrating a typical time of growth
curve;
[0024] Figure 10 is a data graph displaying exempla ry growth curves
for two different microbiological parameters;
[0025] Figure 11 is a data table showing the results generated by
the
method of the present invention;
[0026] Figure 12 is an exemplary linear correlation curve used in
the
method of the subject apparatus;
[0027] Figure 13 is a test report generated by the subject method;
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[0028] Figure 14a is a flow chart illustrating the heating control
algorithm of the present invention; and
[0029] Figure 14b is a flow chart illustrating the ternperature
control and
data collection algorithm of the present invention.
Detailed description of the invention
[0030] Referring to Figure 1, illustrated therein is a system for
the rapid
analysis of microbial parameters using non-intrusive in-vessel incubation and
detection, made in accordance with the subject invention. The system 10
comprises an incubator-detector apparatus 12, a specimen container 14 for
containing a liquid sample 11 mixed with reagent 20, and external data
recorder 80. Incubator-detector apparatus 12 comprises a housing 15 having
a detection chamber 65 shaped for receiving specimen container 14, an
incubation system 60 mounted in housing 15 for incubating microbiological
materials within liquid sample 11, and a spectrophotometer system 62
mounted in housing 15. Spectrophotometer system 62 measures the amount
of light absorbed, emitted or scattered by the liquid sample 11 in specimen
container 14 as microbiological materials are incubateci by incubation system
60.
[0031] Incubation system 60 includes heating controller 92, and
spectrophotometer system 62 includes spectrophotometer controller 94.
Power source 90 provides power to incubation system 60 and
spectrophotometer system 62. External data recorder 80 preferably
comprises a computer 85 having a microprocessor 86 and memory device 88,
and an output device such as printer 82 that is connected to computer 85.
[0032] Referring now to Figures 2-6, illustrated therein is a preferred
embodiment of incubator-detector apparatus 12. Housing 15 of apparatus 12
is a generally cylindrical enclosure comprising a base 16, container holder 18
shaped to hold sample container 14, and a removable cap 50. Base 16
includes an upwardly extending cylindrical lip 55 shaped to receive cap 50.
Base 16 houses power source 90, heating controller 92 and
spectrophotometer controller 94. Power source 90 can be any suitable power
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source known in the art, such as a rechargeable battery located %within base
16 having power outlet 99 for connection to an external 120 or a20 volt AC
power source or a DC power source. Mounted on the exterior of base 16 are
power switch 13, status LEDs 97, and data port 98.
[0033] Container holder 18 comprises a base 21 and an co pen-ended
cylindrical wall 19 extending upwardly from base 21. Wall 19 is shaped to
surround the lower portion of specimen container 14 when specimen
container 14 is placed inside housing 15. Wall 19 includes a pair of inwardly
extending, diametrically opposing, generally rectangular indents 23.
[0034] Removable cap 50 is shaped to fit snugly around wall 19 of
container holder 18. Cap 50 preferably comprises a thermally efficient,
double wall cylindrical shell having an outer wall 51, inner wall 59,. closed
top
52 and open-ended bottom flange 53. The outside surface of bcyttom flange
53 is provided with a bead 66 shaped to thread into groove 67 on the inside
surface of lip 55 of base 16. The inner surface of inner wall 59 includes a
protrusion 68 shaped to sealingly engage ring 49 extending arou nd base 21
of container holder 18. Cap 50 may optionally be provided with vacuum or
inert gas between the walls 51 and 59.
[0035] When cap 50 is placed over container holder 18, ic ap 50 and
container holder 18 define a very efficient thermally insulated incubation-
detection chamber 65. The inside surface of wall 19 of specimen holder 18 is
preferably blackened to make chamber 65 an efficient black box (dark room)
for optical detection and measurement.
[0036] The incubation system 60 of apparatus 12 comprises heating
element 24, temperature sensor 25, and incubation controller 92. Heating
element 24 is mounted within heating finger 57 extending upwardly through
an aperture in base 21 of container holder 18. Temperature sensor 25 is
mounted inside a temperature finger 56 extending upwardly Iran base 21 of
container holder 18. The temperature sensor 25 may comprise a thermistor
26 placed near the top of finger 56.
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[0037] Heating controller 92 controls the heat to the heating
element 24
and monitors the temperature of liquid sample 11 through temperature sensor
25. Once the temperature reaches the optimum, heati ng controller 92
maintains the temperature of liquid sample 11 within. Heating controller 92
preferably comprises a timer (not shown) for measuring the incubation time
from the start and to deactivate the heating at the end of a preset time.
[0038] Specimen container 14 comprises a specimen cap 42 and a
specimen bottle 44. Specimen bottle 44 is generally cylindrical with bottom
cavity 45 to accommodate heating finger 57 and bottom cavity 46 to
accommodate temperature sensor finger 56. Specimen bottle 44 also has a
pair of diametrically opposed, generally rectangular side recesses 48 shaped
to register with indents 23 of wall 19 when specimen container 14 is
positioned within container holder 18. Specimen bottle 44 is made from a
material that allows for the propagation of light signals, and is preferably
made
of a clear plastic or other material that is optically transparent.
[0039] Spectrophotometer system 62 is a system for measuring light
absorbed, emitted or scattered by the liquid sample as the microbiological
materials are incubated over time. As best shovvn in Figure 4,
spectrophotometer system 62 preferably co mprises three
spectrophotometers, a first spectrophotometer comprising light emitting
source 30a and detector 35a, a second spectrophotometer comprising light
emitting source 30b and detector 35b, and a third spectrophotometer
comprising light emitting source 30c and detector 35c. Light emitting sources
30a,b,c propagate beams of light of a given intensity along selected optical
paths within specimen container 14. Light detectors 35a, b, c are positioned
to
detect changes in the intensity of the beams of light within a selected field
of
view related to the optical paths, resulting from light that is absorbed,
emitted
or scattered by the liquid sample 11 as the microbiological materials are
incubated by the incubator system over time.
[0040] Each of light sources 30a,b,c preferably comprises a light
emitting diode (LED) of a specific wavelength maxim um, and each of
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detectors 35a,b,c preferably comprises a phototransistor detector. Light
sources 30a,b,c, and detectors 35a,b,c are mounted on printed circuit boards
33a,b,c that are electrically connected to spectrophotometer controller 94.
[0041] Light emitting sources 30a, 30b extend through apertures 70a
and 70b in base of container holder 18. Light emitting sources 30a, 30b
placed in such a way that the light emitting from the sources travels through
apertures 70a and 70b, respectively, and upwardly along a selected optical
path within specimen container 14. In the case of light emitting source 30a,
30b, the optical paths are shown by arrow 1 and arrow 4, respectively.
Container holder 18 also has apertures 75a, 75b for signal detectors 35a,
35b. Detectors 35a, 35b are placed at a 90 degree angle with respect to light
emitting sources 30a, 30b, in such a way that the detector window face
towards specimen bottle 44 to receive any signal propagating towards
detectors 35a, 35b , having fields of view shown by arrow 2 and arrow 5,
respectively.
[0042] Container holder 18 also includes suitable apertures 70c and
75c for signal-emitting source 30c and signal detector 35c, respectively.
Light
emitting from light source 30c passes through aperture 70c along a horizontal
optical path, with the direction of the propagation shown by arrow 3 through
the specimen container 14 and aperture 75c to detector 35c. Detector 35c is
positioned so that its field of view is at a 1800 angle to the optical path of
light
source 30c.
[0043] Spectrophotometer controller 94 controls the operation of the
spectrophotometer system. Spectrophotometer, control ler 94 activates and
deactivates and may pulse signal emitting' sources 30a,b,c.
Spectrophotometer controller 94 also measures and p rocesses the output
signals generated by detectors 35a, 35b and 35c. Spectrophotometer
controller 94 includes a microprocessor 95 having a built in time clock that
functions as a data logger and stores the measured detector signal values
along with the corresponding temperatures of the liquid sample 11 and time of
the measurements in a specific memory location within microprocessor 95.
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Spectrophotometer controller 94 may indicate the end of the test by activating
one or more of status LEDs 97 or an audio signaling device (not shown).
Spectrophotometer controller 94 also communicates through data port 98 with
external data recording device 80, such as computer 85, a rriultimeter or
other
external signal manipulator.
[0044]
Microprocessor 95 of spectrophotometer controller 94 includes a
memory for storing software that implements the test method of the subject
invention. The test method provides the specifications and conditions required
to conduct the testing process. Through custom software the method can be
programmed and downloaded into the memory of microprocessor 95 through
data port 98. The software is also capable of erasing all rnemory locations
within the controller 94.
[0045] To
utilize apparatus 12 of the present invention to test liquid
samples, liquid sample 11 is placed in sample container 14. Liquid sample 11
is not limited to water, and may comprise other liquids or other liquid medium
containing suspensions such as food particles, filter papers and other solids.
An appropriate reagent 20 is then added to liquid sample 11 inside specimen
container 14. Reagent 20 may be chemical or biological in nature and provide
a detectable parameter such as color, fluorescence, turbidity etc. that
indicates the presence or absence of the microbiological material under
investigation.
[0046] The
detection of color, fluorescent or turbidity signal is time
dependent and the time of detection is related to the quantity of the bacteria
present at the start of the test. Thus quantification of the d etected
microbial
parameters such as total coliform and e.coli in water sample can be achieved
by measuring the signal due to color change or fluorescen ce signal and the
time at which they were detected in appreciable amount.
[0047] The
detection of the color or the detection of =the fluorescence
signal is measured using spectrophotometer system 62 of apparatus 12. In
the preferred embodiment, spectrophotometer system 62 comprises three
spectrophotometers that provide colorimetric detection for total coliform,
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fluorescence detection for e.coli and microbial growth turbidity by
nephelometry, respectively.
[0048] The built-in time clock of microprocessor 95 of
spectrophotometer controller 94 provides the time of growth while the
constant temperature of incubation system 60 provides both microbial growth
and optical reproducibility.
[0049] In a preferred embodiment of system 10 of the present
invention, spectrophotometer system 62 comprises three w'time-of-growth-
spectrophotometers" within a single constant temperature incubator, i.e.
spectrophotometers that record the growth of specified microbiological
parameters as a function of time. The type of spectrophotometric analysis
done by each spectrophotometer depends upon the configuration and
specification of the source and detector of the spectrophotometer. The 180-
degree configuration of source detector pair 30c, 35c provides a colorimetric
or turbidimetric analysis, while the 90-degree configuration of source-
detector
pairs 30a, 35a and 30b, 35b provides for either fluorometric or nephelometric
analysis.
[0050] Source 30c of colorimeter spectrophotometer preferably
comprises an LED with wavelength maximum at 620 nm, and detector 35c is
preferably a phototransistor detector having a signal response ranging across
the visible region including the 620 nm. Source 30a of fluorometer
spectrophotometer is preferably an UVLED with maximum wavelength at 380
nm, and detector 35a, placed strategically at 90 degrees to source 30b, is
preferably a phototransistor detector having a signal response in the visible
region including the 400-500 nm range. The nephelometer configuration is
similar to that of the fluorometer except that source 30b is p referably an
LED
with a maximum wavelength at 400 nm.
[0051] After reagent 20 is aseptically added to the sample, specimen
cap 42 is fastened to specimen bottle 44, and specimen container 14 is gently
shaken to dissolve the reagent, and form a sample/reagent mixture. For
simultaneous testing of total coliform and e.coli in water samples, typical
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reagents provides not only an optimum growth nutrient, but also a color
change for total coliform and fluorescence signal for e.coli, if they are
present
in any quantity in the sample. Examples of typical chromogenic/fluorogenic
reagents are:
Merck KGaA - Readycult
IDEXX- Colilert
[0052] The
specimen container 14 is then placed inside the container
holder 18 as best shown in Figure 2. Once the removable cap 50 is placed on
base 16, incubation-detection chamber 65 provides a black box (dark room)
condition ideal for microbial growth and spectrophotometric detection.
[0053]
Pressing the start button 13 on base 16 of apparatus 12
activates the incubation cycle and the detection process. Optionally, a
separated activation button can be used to activate the detection process at a
pre-determined time after the start of the incubation.
[0054] Activation of the detection process may include turning the
power to the signal emitting sources 30a,b,c and detectors 35a,b,c, pulsing
the signal emission and monitoring the signal output of detectors 35a, b, c.
[0055]
Heating controller 92 brings and maintains the temperature of
liquid sample 11 at a constant temperature within a preset temperature range.
For coliform and E.coli testing in water a temperature of 36 1 C is
preferred.
However, the temperature depends upon the reagent used and may vary from
one reagent to another. The temperature also depends upon the test method
specification. For example, E.coli can be tested at either 36 C or 41 C using
the same reagent.
[0056] Spectrophotometer controller 94 continuously monitors, records
and stores the output signals from the detectors 35a,b,c. In the method of the
preferred embodiment, spectrophotometer controller 94 also records and
stores the time and temperature of each output signal. Controller 94 may be
connected to external data recorder 80 that is programmed to record the
signal either continuously or at a pre determined intervals. External data
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recorder 80 may also record the time of each signal measured and the
corresponding temperature of the sample, and generate a "time dependent
growth signal pattern" (TDGSP) of the microbial parameters under
investigation.
[0057] The colorimetric TDGSP of total coliform and fluoronnetric
TDGSP of e.coli are preferably recorded simultaneously along with a
nephelometric TDGSP of increasing turbidity due to bacterial growth in water.
[0058] A significant deviation of the output signal from the initial
base
line is an indication of the presence of the parameter under investigation
while
the time needed to reach the significant deviation from the start provides an
indication of the original amount of the test parameter.
[0059] The time clock of microprocessor 95 provides the date and
time
test started, the time of each measured signal and the corresponding
temperature, the time at which the test completed or terminated. The end of
the test can be indicated through status LEDs 97 and/or audio signals or can
be controlled through a software program.
[0060] Referring now to Figure 7, illustrated therein is a schematic
view
of apparatus 112 made in accordance with an alternative embodiment of the
present invention. Apparatus 112 is generally similar to apparatus 12 of the
preferred embodiment as shown in Figures 2-6 except for a few modifications.
[0061] Apparatus 112 comprises a specimen container 114, and a
housing 115 comprising a base 116, a cylindrical container holder 118, and a
removable cap 150. Container holder 118 has a cylindrical base 155 and an
open-ended cylindrical wall 160. Container holder base 155 has a
temperature controller finger 156 extending upwards to accommodate
temperature controller 180. Temperature controller 180 may be a bimetal
switch or any other suitable device, which can activate and deactivate the
heating element.
[0062] A heating element 124 is mounted within the open ended
cylindrical wall 160 of the sample holder 150. As shown, heating element 124
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comprises a resistor wire 125. Alternatively, heating element 124 may be a
resistor coil, resistor foil etc. The length of the resistor wire is dictated
by the
resistor temperature and the ohm per foot rating of wire 125.
[0063] Specimen container 114 comprises a specimen cap 142 and a
specimen bottle 144. The specimen bottle is generally cylindrical with a
bottom cavity 145 shaped to accommodate temperature controller finger 156.
[0064] Heating controller 192 maintains a constant preset temperature
range within the sample. Optionally it may comprise a timer (not shown) for
measuring the incubation time from the start and to deactivate the heating at
the end of a preset time.
[0065] The spectrophotometer system of apparatus 112 is similar to
that of apparatus 12 of the preferred embodiment. Light emitting source 130a
emits light in the direction of arrow 1 and detector 135a detects emitted or
scattered light traveling in the direction of arrow 2. Light emitting source
130b
emits light in the direction of arrow 4 and detector 135b detects emitted or
scattered light traveling in the direction of arrow 5. Light emitting source
130c
emits light in the direction of arrow 3 and detector 135 detects light
traveling in
the direction of arrow 3. The spectrophotometer system also includes
spectrophotometer controller 194 for controlling the operation of light
sources
130a,b,c and detectors 35a,b,c, and power source 190.
[0066] Referring now to Figure 8-13, illustrated therein is a
preferred
embodiment of the quantitative analysis method of the present invention.
[0067] The quantitative analysis method of the present invention is
based on the recognition that there is a relationship between initial
population
and growth population with time. The time interval between the start of the
test (starting population) and a fixed growth population is a function of the
initial population, the incubation temperature and the growth media. Thus
keeping the incubation temperature and growth media as constants, the time
required to reach the fixed growth population is a direct function of the
initial
population.
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[0068] A chromogenic/fluorogenic reagent such as Readycult (Merck
KgaA) or CoInert (IDEXX) provides the mechanism by which the microbial
growth population (total coliform and e.coli) can be monitored and measured
in this invention through photometric detection process. The specific enzymes
produced by these organisms (for example _-galactosidase (total conform)
and _-glucuronidase (specific to e.coli)) will metabolize the nutrient-
indicator
and releases the chromophor or fluorophor into the liquid medium. The
concentration of the chromophors or flurophors, at a given time, in the
detection medium is proportional to the growth population at that time and
hence the change in the signal intensity due to the increase in concentration
of the colour components is a measure of the time based growth population.
[0069] The population detection time (tp0p), which is defined as the
time
taken to reach a detectable population size, has been used to estimate
bacterial growth parameters. The detection time has been shown to be
inversely proportional to the logarithm of the inoculum (initial population of
the
microbe) level.
tpop oc 1/log X0 {1}
where X0 = initial bacterial population.
[0070] The time of significant deviation (TSD) is the time at which
the
measured photometric signal quantity above the baseline signal is
statistically
significant. TSD also depends on the initial concentration of the bacteria in
the sample and higher the initial bacterial count shorter the TSD. Since the
increase in population size can be measured using increase in signal output,
the time required to obtain a significant deviation (TSD) of the signal output
from the baseline should corresponds to tpop if measured in the growth phase.
The method is
tpop = TSD {2}
And therefore,
TSD oc 1/log X0 {3}
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[0071] This linear correlation curve equation- LCCE between TSD and
initial population of the microbe under investigation (X0) provides, in
addition
to detecting the presence, quantitative information of the bacterial
population.
[0072] Figure 8 is a flow chart describing the steps of the subject
method. At block 200, reagent 20 is mixed with liquid sample 11 having an
unknown initial population of a microbiological material in specimen container
14, thereby creating a sample/reagent mixture. Reagent 20 provides a
detectable parameter, such as colour or fluorescence, indicative of the
microbiological material. At block 202, specimen container 14 is placed inside
housing 15 and cap 50 is placed on housing 15. At block 204, the incubation
process is initiated, and the sample/reagent mixture is incubated in the
enclosed housing at a constant temperature over a period of time.
[0073] At block 206, data collection is initiated, and the changes in
the
detectable parameter are measured as the sample/reagent mixture is
incubated over time. These changes are measured by propagating light of a
known intensity within the sample/reagent mixture in specimen container 14,
and detecting changes in the intensity of the light over the period of
incubation.
[0074] During the incubation period, data relating to the time,
temperature and photometric signal indicative of the changes in light
intensity
are collected by spectrophotometer controller 94. An increase in microbial
population with time is accompanied by the increase in photometric signal.
This generates a real-time microbial growth curve, such as that shown in
Figure 9. At block 208, the testing is terminated, and the collected data is
stored in the memory of microprocessor 95 of spectrophotometer controller
94.
[0075] At block 210, the data is downloaded to external data recorder
80, preferably computer 85 installed with custom software. At block 212,
computer 80 processes the data and displays the result both in graphical
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format and tabular format. Figure 10 illustrates the growth curves of all
selected parameters of a typical sample, as recorded on a real time basis or
downloaded from apparatus 12, as well as the incubation temperature profile.
The left hand side vertical values indicate signal intensity (arbitrary
numbers).
The right hand side is the temperatures in C (Celsius). The bottom
horizontal scale represents the time in hours. Figure 11 illustrates a data
table that contains the values of the parameter signals along with the time
and
temperature for a typical sample.
[0076] At
block 214, the software of computer 85 automatically
calculates the TSD based upon a pre-set value of the photometric signal
above the baseline signal value defined by the analyst. At block 216, a built
in
pre-defined linear correlation curve equation is used to calculate the initial
population (expressed in Colony Forming Unit (CFU) in a given sample
volume) of the microbial parameter under investigation. To obtain this linear
correlation curve equation, a series of split samples of varying initial
microbial
population (for example E.coli) are run both using the method of the present
invention and a standard method (Membrane Filtration). The pre-defined
linear correlation curve equation (LCCE) is generated by plotting TSD values
obtained from the present invention and the corresponding initial population
values (Xo) from the membrane filtration method. A sample linear correlation
curve is shown in Figure 12.
[0077] At
block 218, the initial population values are displayed on a
computer screen such as that shown in Figure 13.
[0078] The
method of the present invention accordingly provides a
continuous, non-intrusive monitoring and recording of one or more detectable
parameters as the incubation process progresses. A significant deviation of
the output signal is an indication of the presence of the detectable
parameter,
while the time taken to reach the significant deviation provides a
quantitative
analysis of the parameter.
[0079] Referring now to Figures 14a and 14b, illustrated therein are the
heating and temperature control and data collection algorithms of controller
92
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and 94. Pressing the power switch 13 at the command block 300 initiates the
controller 92 program.
[0080] At command block 305 the controllers 92 and 94 perform a
quality check to verify the incubation and detection systems and components
are functioning properly.
[0081] At command block 310 if the logic is "Yes" the controllers 92
and
94 will proceed to block 320. If the logic is "No" the controllers will
initiate the
appropriate LEDs to signal "Failed Unit".
[0082] At command block 320 the controller 92, through heating
element 24, heats the sample 11 in container 14 and at a pre-determined
interval monitors the temperature of the sample 11 through temperature
sensor 25.
[0083] If the logic at command block 330 is "No" then the controller
92
continues heating and monitoring the temperature of sample 11 in container
14.
[0084] If the logic at command block 330 is "Yes" then the
temperature
of the sample 11 has reached a pre-determined temperature value as
measured by the temperature sensor 25. The controller 92 will then set the
test time zero and continue monitoring the temperature of the sample 11 in
container 14. The controller 92 also starts monitoring the time.
[0085] If the logic in command block 350 is "No", the controller 95
will
continue heating and monitoring the temperature of sample 11.
[0086] If the logic in command block 350 is "Yes", the controller 92
moves to block 355 and stops heating sample 11 in container 14 and moves
to command block 365.
[0087] At command block 365 controller 92 starts collecting
temperature and time data while controller 94 starts collecting the signal
data.
Controller 92 also initiates the temperature control Loop 1 to maintain the
incubation temperature at the pre-set range. If the logic at block 400 within
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Loop 1 is "Yes" then the controller falls back to block 355 and continues the
loop.
[0088] If the logic at block 400 is "No" then the controller will go
to
command block 410 and initiate heating of sample 11 in container 14. Loop 2
will continue until the logic at block 400 becomes "Yes" and falls back to
Loop
1.
[0089] Irrespective of the logic loops 1 and 2, both controllers 92
and
94 will collect their respective data at a pre-set time interval and store the
data
in the processor memory.
[0090] At block 370 the status LEDs 97 are updated to indicate the test
in progress.
[0091] If the logic at command block 375 is "No" the controllers 92
and
94 will go back to command block 365 and continue collecting data, thus
initiating a data collection loop ¨ Loop 3. If the logic at block 375 is "Yes"
then
the test is deemed completed and the controllers 92 and 94 stop monitoring
and collecting data and shut down both incubation and detection systems and
go into idle (stand by) mode at command block 385 and await further
instruction from the user or analyst.
[0092] The methods and apparatus of the subject invention provide a
number of advantages over standard membrane filtration methods. The
subject methods and apparatus provide for a rapid but simple, reliable and
accurate onsite testing of microbiological material in various types of liquid
samples, including drinking water and recreational water. Other advantages
include less interference from turbidity, no need for dilution because of
large
dynamic analytical range, simplified operation through total automation, and
build in quality control (QC) providing auto QC for every test.
[0093] It should be appreciated that various modifications can be
made
to the embodiments of the methods and apparatus described herein. While
the spectrophotometer system of the preferred embodiment comprises.three
spectrophotometers, it should be understood that the apparatus could
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comprise a different number of spectrophotometers. As well, the spatial
configuration of the source-detectors could be altered significantly without
departing from the present invention. Also, each spectrophotometer can be
configured for detecting different test parameters and can be operated
independently or simultaneously.
[0094] It should also be appreciated that the light emitting
sources are
not limited to LEDs (as they could be lasers, or laser diodes), and the
dtectors are not limited to phototransistors, (as they could be photodiodes,
photoresistors, CCDs, etc.)
[0095] Furthermore, the method of the present invention is not limited
to the detectable parameters of the preferred embodiment, as the present
method could be used to detect light emission resulting from bioluminescence
or chemiluminescence processes resulting from a biological or chemical
component in reagent 20 within the sample container 14. This would allow
the method and apparatus of the present invention to be used for toxicity
studies using bioluminescence bacteria.
[0096] Also, the light emitting source and the detector of any
or all of
the spectrophotometers could be placed outside of the incubation-detection
chamber 65 but within apparatus 10 and used to monitor the signal growth
through fiber optics placed strategically within the chamber 65.
[0097] The scope of the claims should not be limited by the
preferred embodiments set forth in the Examples, but should be given
the broadest interpretation consistent with the description as a whole.
