Method and apparatus for determining the sensitivity of a microorganism to a growth altering agent

A method and an apparatus for determining the concentration at which a growth-altering agent has an appreciable effect on the growth of a target microorganism are provided. The method comprises the steps of (a) providing a microorganism growth medium; (b) providing a sensible reagent, which includes a growth-altering agent mixed with a marker that has a signal with a magnitude proportional to the concentration of the marker; (c) incorporating the sensible reagent into the growth medium, in a manner that creates a gradient of growth-altering agent and marker concentrations within the growth medium; (d) inoculating the growth medium with the target microorganism; (e) incubating the inoculated growth medium for a period of time sufficient for the target microorganism to grow a detectable amount; (f) evaluating growth characteristics of the microorganism in a region containing the growth-altering agent, (g) measuring the magnitude of the marker signal in that region; and (h) determining the concentration of the growth-altering agent using the measured magnitude of the marker signal.

BACKGROUND OF THE INVENTION
 1. Technical Field
 The present invention relates to methods and apparatus for determining a
 microorganism's sensitivity to a growth altering substances in general,
 and to methods and apparatus for determining the minimum concentration at
 which a growth altering agent has an appreciable effect on a microorganism
 in particular.
 2. Background Information
 Growth-altering agents like antibiotics, antiseptics, drugs, hormones,
 mutagens, and nutrients can be used to alter, inhibit, or enhance
 microbial growth. Because the effect of a growth-altering agent is
 typically a function of its concentration, there is considerable utility
 in knowing the concentration at which a growth-altering agent will have an
 appreciable effect on a microorganism. Some existing methods and apparatus
 for evaluating the effect a growth-altering agent has on a microorganism
 expose the microorganism to a plurality of discrete concentrations of a
 growth-altering agent. The Kirby-Bauer test, for example, utilizes a
 number of disks placed on a layer of growth medium, each of which contains
 antibiotic in a specific concentration. Bacteria grown on the growth
 medium form a visible coating, except in the area around those disks
 having sufficient antibiotic concentration to inhibit bacterial growth. A
 disadvantage of the Kirby-Bauer test is that there are a number of
 variables which affect the antibiotic concentration at any given point in
 the growth medium, and thus do not readily allow for an accurate
 determination of the minimum inhibitory concentration of the antibiotic.
 Another method and apparatus for evaluating the effect a growth-altering
 agent has on a microorganism is the tube dilution method wherein an equal
 amount of target microorganism is placed in a plurality of wells (referred
 to as "tubes") disposed in a platter, and different concentrations of a
 growth-altering agent are added to each tube. At some concentration of
 growth-altering agent, there will be an appreciable change in the target
 microorganism; e.g., its growth will be altered, enhanced, or inhibited. A
 disadvantage of the tube dilution method is that its accuracy depends on
 the step size in concentration change between tubes. A small step size
 yields greater accuracy, but may require an impractical number of tubes
 and effort. In addition, preparing accurate dilutions is an expensive
 process that increases in cost with the number of tubes. Hence, increasing
 the accuracy of this method can also increase the cost and time required.
 What are needed are a method and an apparatus for determining the
 concentration at which a growth-altering agent has an appreciable effect
 on a target microorganism, and one that can do so accurately in a time
 effective manner.
 DISCLOSURE OF THE INVENTION
 It is, therefore, an object of the present invention to provide a method
 and an apparatus for determining the concentration at which a
 growth-altering agent has an appreciable effect on a target microorganism.
 According to the present invention, a method and an apparatus for
 determining the concentration at which a growth-altering agent has an
 appreciable effect on the growth of a target microorganism is provided.
 The method comprises the steps of
 (a) providing a microorganism growth medium;
 (b) providing a sensible reagent, which includes a growth-altering agent
 mixed with a marker that has a signal with a magnitude proportional to the
 concentration of the marker;
 (c) incorporating the sensible reagent into the growth medium, in a manner
 that creates a gradient of growth-altering agent and marker concentrations
 within the growth medium;
 (d) inoculating the growth medium with the target microorganism;
 (e) incubating the inoculated growth medium for a period of time sufficient
 for the target microorganism to grow a detectable amount;
 (f) evaluating one or more growth characteristics of the microorganism in a
 region containing the growth-altering agent;
 (g) measuring the magnitude of the marker signal in that region; and
 (h) determining the concentration of the growth-altering agent using the
 measured magnitude of the marker signal.
 As used herein, the term "growth-altering agent" includes agents that will
 alter, inhibit, or enhance microbial growth. Examples of growth-altering
 agents include, but are not limited to, antibiotics, antiseptics, drugs,
 chemical agents, hormones, mutagens, nutrients, or growth promoting
 agents. The phrase "growth of a [or the] target microorganism" is defined
 to include positive or negative growth, mutated growth, and atypical
 shaped growth.
 An advantage of the present invention is that a method for determining the
 concentration at which a growth-altering agent has an appreciable effect
 on the growth of a target microorganism is provided that gives accurate
 results in a minimum amount is of time. The present invention uses a
 sensible reagent that includes a marker having a signal with a magnitude
 that is proportional to the concentration of the marker. The concentration
 of the marker within the reagent is proportional to the concentration of
 the growth-altering agent. The concentration of the growth-altering agent
 in a region within the growth medium can therefore be determined by
 sensing the marker signal in that region. Accordingly, the exact
 concentration of the growth-altering agent can be determined rather than
 an approximation, and can be determined without a multitude of time
 consuming dilution steps.
 Another advantage of the present invention is that a cost-effective method
 for determining the concentration at which a growth-altering agent has an
 appreciable effect on the growth of a target microorganism is provided.
 The ability of the present invention method to provide accurate
 growth-altering agent information obviates the need for expensive multiple
 dilutions as are required in the tube dilution method. A person of skill
 in the art will recognize that minimizing expensive medical laboratory
 time and laboratory assets make the present method likely to be
 considerably less expensive than presently available methods.
 Another advantage of the present invention is that a "user-friendly"
 apparatus for determining the concentration at which a growth-altering
 agent has an appreciable effect on the growth of a target microorganism is
 provided. The present apparatus embodiments facilitate the testing
 process, minimize the opportunity for sample spillage, and can be readily
 disposed of after the test. These qualities and others make the present
 apparatus attractive as a disposable.
 These and other objects, features and advantages of the present invention
 will become apparent in light of the detailed description of the best mode
 embodiment thereof as illustrated in the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION
 The present method for determining the concentration at which a
 growth-altering agent has an appreciable effect on the growth of a target
 microorganism includes providing a microorganism growth medium capable of
 supporting the microorganism, an effective amount of target microorganism,
 and a sensible reagent. Growth mediums in gel, semi-solid, or
 permeable-solid form can be used. Dehydrated growth mediums that may be
 rehydrated during use are particularly favorable because they can be
 readily stored for extended periods of time. The target microorganism may
 consist of either first generation microbes taken from a sample of urine,
 cerebrospinal fluid, body cavity fluid, or a suspension of microbes taken,
 for example, from a colony grown on another growth medium. The target
 microorganism may also be acquired from environmental sources such as
 water, food, or food preparation surfaces.
 The sensible reagent includes the growth-altering agent and a marker. In a
 first embodiment, the sensible reagent contains an accurate quantity of
 the growth-altering agent mixed with a useful, but imprecisely measured,
 quantity of marker. In a second embodiment, the growth-altering agent and
 the marker of the reagent are mixed in known accurate proportion, and the
 overall quantity of the reagent may vary to suit the application. These
 reagent embodiments require only one parameter (growth-altering agent
 quantity or growth-altering agent to marker proportion) to be known
 accurately, thus minimizing the cost of manufacturing the sensible reagent
 and consequently the overall method.
 The marker may be any material that: 1) has an identifiable signal with a
 magnitude proportional to the concentration of the marker; 2) has a signal
 that is distinguishable from other elements within the test sample; 3) has
 a signal and signal magnitude that are not adversely affected by growth of
 the target microorganism; 4) does not substantially adversely effect
 growth of the target microorganism; 5) does not unpredictably or adversely
 affect the action of the growth-altering agent being evaluated; and 6) one
 which, if necessary, will co-diffuse with the growth-altering agent in the
 growth medium during the incubation period in a predictable manner so that
 the local marker concentration is proportional to the local
 growth-altering agent concentration. For example, a fluorescent marker
 having excitation or emission wavelengths outside the range of the
 excitation or emission wavelengths of the growth medium, and one that does
 not bind to the growth medium or the target microorganism may be used. The
 marker and the growth-altering agent preferably diffuse within the growth
 medium at the same rate, although a similar diffusion rate is not
 required. A marker and a growth-altering agent having different, but
 known, diffusion rates may be used alternatively. In another example, an
 identifiable dye that is absorbed by the growth-altering agent may be
 used. The magnitude of the marker signal emitted from the dye is
 proportional to the concentration of growth-altering agent since it is the
 growth-altering agent that is "carrying" the dye. The terms "proportion"
 and "proportional" as used within the present specification comprise any
 relationship that can be mathematically described; e.g., x:y, x:y.sup.2,
 x:.sup.1 /.sub.y, etc.
 The present method includes the further steps of a) incorporating the
 sensible reagent into the growth medium; b) inoculating the growth medium
 with the target microorganism; c) incubating the inoculated growth medium;
 d) evaluating one or more growth characteristics of the microorganism in a
 region of the growth medium; e) measuring the magnitude of the marker
 signal in that region; and f) determining the concentration of the
 growth-altering agent in the region using the magnitude of the marker
 signal measured in the region. In some applications it may be useful to
 evaluate the one or more growth characteristics of the microorganism in
 more than one region of the growth medium. The concentration of the
 growth-altering agent would be determined in those regions using the same
 method.
 In some applications the present method may include the further step of
 relating changes in the growth characteristics of the microorganism to the
 concentration of the growth-altering agent. The manner in which the
 microorganism growth changes relative to the concentration will depend on
 the application at hand; e.g., the relationship could be linear,
 exponential, a step function, etc. The ability of the present method to
 accurately determine the concentration along a gradient enables the
 relationship to be determined.
 The effects on the organism can be determined in several ways. For example,
 the growth medium can be examined for areas of contiguous growth where
 changes in the coverage or density of the growth will be indicative of the
 effects of the growth-altering agent. A more sensitive method is the
 examination of individual colonies for changes in characteristics such as
 growth rate, colony area, colony morphology, etc. Individual organisms or
 small clusters or microorganisms may also be examined for such changes.
 The sensible reagent is incorporated into the growth medium in a manner
 such that at least one gradient of reagent concentration forms within the
 growth medium The concentrations of the growth-altering agent and marker
 at the ends of the gradient are preferably such that the point at which
 the growth-altering agent has an appreciable effect on the growth of the
 target microorganism will always fall in between the two ends of the
 gradient. Incorporation can be accomplished directly, for example by
 inserting the reagent into the growth medium or by applying the reagent
 onto a surface of the growth medium. Incorporation can also be
 accomplished indirectly by applying the reagent onto a substrate and
 placing the substrate in contact with, or in close proximity to, the
 growth medium.
 The growth medium can be inoculated by any known method acceptable for use
 with the growth medium and the target microorganism. The number of target
 microorganism microbes inoculated into the growth medium should be
 sufficient to provide adequate coverage over the entire area of the growth
 medium incorporating the reagent. The sufficiency of inoculum
 concentration will depend on the parameters of the test at hand, including
 the type growth medium, target microorganism, growth-altering agent etc.
 The growth medium, incorporated with the sensible reagent and inoculated
 with the target microorganism, can be incubated under any conditions that
 are acceptable to the growth medium and the target microorganism.
 Because the point(s) at which the growth-altering agent has an appreciable
 effect on the growth of the target microorganism falls in between the two
 ends of the gradient, the gradient enables the determination of the
 effects of the growth-altering agent on the microorganism as a function of
 the concentration of the growth-altering agent. In most instances, there
 will be a first section of the growth medium having no appreciable effects
 from the growth-altering agent, a second section of the growth medium
 having appreciable effects from the growth-altering agent on one or more
 growth characteristics, and a boundary region located between the first
 and second sections. The extent of the boundary region will depend on
 circumstances at hand including the type growth-altering agent, the
 microorganism, the slope of the concentration gradient, etc. Depending
 upon the growth-altering agent and the microorganism, the changes across
 the boundary region may be mathematically related to the concentration of
 the growth-altering agent. In the case of a growth-altering agent that
 promotes growth, for example, an exact growth concentration curve
 producible from such a mathematical relationship could help choose the
 optimal cost/benefit ratio for the addition of such agents into a
 commercial process.
 If the growth-altering agent is an inhibiting agent, for example, the
 boundary region will define the border between a first section having
 substantially uninhibited development (where the concentration of the
 inhibiting agent was too low and therefore did not have an appreciable
 effect on the microorganism) and a second section having substantially
 inhibited development (where the concentration of the inhibiting agent was
 great enough to have an appreciable effect on the microorganism). The
 point on the gradient at which the boundary region falls is the minimum
 concentration at which the growth-altering agent will have an appreciable
 effect on the growth of the microorganism. If on the other hand the
 growth-altering agent is a nutrient, a first section (having an effective
 concentration of nutrient) might have appreciably advanced rate of
 development relative to a second section (having an ineffective
 concentration of nutrient).
 Another example of characteristic growth differences is an appreciable
 difference in the atypical forms of the microorganism. If the
 growth-altering agent can cause the microorganism to produce atypical
 forms of the microorganism, there will be a first section having
 substantially normal forms of the microorganism (where the concentration
 of the growth-altering agent was too low and therefore did not have an
 appreciable effect on the microorganism) and a second section having a
 statistically substantial population of atypical forms of the
 microorganism (where the concentration of the growth-altering agent was
 great enough to have an appreciable effect on the microorganism).
 The effects of the growth-altering agent on the microorganism and the
 position of the boundary region are usually determined by optical means,
 for example by changes in the optical density of the coat of
 microorganisms on the medium. In some instances, a second method for
 determining the effects of the growth-altering agent and the position of
 the boundary region may be used that involves a marker (which may be the
 same as, or independent of, the marker contained within the reagent) that
 interacts with, including but not limited to being metabolized by, the
 growing microorganism to produce a sensible product. The sensible product,
 which is present with the target microorganism growth, is sensed to
 establish the boundary. A third method for determining the effects of the
 growth-altering agent and the position of the boundary region includes
 evaluating the light scattering characteristics within the section(s)
 bearing target microorganism growth versus the light scattering
 characteristics in the section(s) bearing substantially no target
 microorganism growth. In all three methods, once the boundary region is
 determined, the signal from the marker within the reagent can be sensed
 and its magnitude measured. If individual colonies or organisms in those
 sections of the growth medium are to be evaluated, they may readily be
 viewed or photographed using a magnifier or a microscope.
 The marker mixed with the growth-altering agent in the sensible reagent
 provides the quantitative information in all sections of the growth
 medium, including the boundary region that enables the concentration of
 the growth-altering agent to be calculated. Specifically, the magnitude of
 the marker signal in the boundary region is proportional to the marker
 concentration in the boundary region, and the concentration of the
 growth-altering agent can be determined using the known proportional
 relationship between the concentrations of marker and growth-altering
 agent. The exact method for determining the growth-altering agent
 concentration will depend on the physical embodiments of the growth
 medium, how the sensible reagent is distributed, the proportional
 relationship between the marker and the growth-altering agent within the
 reagent, etc. The following examples illustrate how the growth-altering
 agent concentration may be calculated using the present invention method.
 EXAMPLE I
 Referring to FIGS. 1 and 2, in a first example the present invention method
 uses a Kirby-Bauer type apparatus 10 (shown in diagrammatic cross-section)
 which includes a plate 12, a layer of microorganism growth medium 14 of
 uniform thickness "T" inoculated with a target microorganism, and a disk
 16. The sensible reagent (with an accurately known quantity of
 growth-altering agent mixed with an imprecisely measured quantity of
 fluorescent marker) is applied to the disk 16 and the disk is placed in
 contact with the growth medium 14. The sensible reagent diffuses into the
 growth medium 14, creating a concentration gradient 18 as it travels
 radially (shown diagrammatically in FIG. 1). The inoculated growth medium
 14 is incubated and a section 20 having detectable microorganism growth
 develops contiguous with a section 22 having no detectable microorganism
 growth.
 Referring to FIGS. 1 and 2, the plate is placed in a commercially available
 scanning fluorometer (not shown) adjusted to sense the fluorescent signal
 characteristic of the marker. The fluorometer generates a curve 25 (FIG.2)
 representing signal magnitude as a function of radial distance across the
 plate. Marker signal magnitude is a function of the marker concentration
 in a given volume (V), and is determined by sensing an area (A) of the
 growth medium which has a uniform thickness (T; V=A*T). The marker signal
 magnitude in the boundary region 24 between the sections 20, 22, for
 example, is given as the signal magnitude measured within a volume (V) of
 growth medium located in the boundary region 24. As stated earlier, the
 position of the boundary region 24 between the sections 20, 22 may be
 determined optically, or by other means. In the growth medium 14 below the
 disk 16 and the section 20 of growth medium having detectable target
 microorganism growth, the signal from the marker may be obscured by
 interference. The total signal magnitude from the marker can be determined
 by estimating the marker signal magnitude in the obscured regions with a
 curve fitting mathematical analysis. For illustrative purposes, FIG. 2
 shows an example of a mathematically fit curve 27 in the obscured regions.
 The total signal magnitude from the marker is subsequently determined by
 integrating the area under the curve. As described above, the marker
 signal is scanned in a linear manner across the center of the test area.
 Since the sensible reagent actually diffuses radially in the round growth
 medium, it may be necessary to adjust the signal integration to reflect
 the radial diffusion of the reagent. The signal integration adjustment may
 be avoided, however, by scanning the entire area containing the radial
 diffusion of reagent.
 The concentration of marker in the given volume (V) in the boundary region
 24 can be expressed as the ratio of the magnitude of the marker signal in
 the boundary region 24 (sensed from volume V) over the total magnitude of
 the marker signal sensed within the total volume of the growth medium 14.
 The concentration of marker in the boundary region 24, in turn, is related
 to the concentration of the growth-altering agent in the boundary region
 24 (in the same volume V), by the ratio of diffusion rates of the marker
 and growth-altering agent. If the diffusion rates are equal, the
 growth-altering agent concentration in the boundary region can be
 determined as follows:
 ##EQU1##
 which can be rearranged to solve for the unknown growth-altering agent
 concentration in the boundary region 24:
 ##EQU2##
 If the diffusion rates of the growth-altering agent and marker differ, a
 correction factor representing the mathematical relationship between the
 two diffusion rates is used to correct for the difference. In addition,
 the above expressions require that the quantity of growth-altering agent
 in the total volume of growth medium 14 be accurately known. If all the
 sensible reagent (containing an accurately known quantity of
 growth-altering agent mixed with an imprecisely measured quantity of
 fluorescent marker) is incorporated into the growth medium, then the
 quantity of growth-altering agent is ascertainable from the reagent. Other
 methods of accurately determining the quantity of growth-altering agent
 within the total volume of growth medium 14 may be used alternatively.
 EXAMPLE II
 Referring to FIGS. 3-5, a trough 26 contains a layer of growth medium 28 of
 known uniform thickness "T" inoculated with a target microorganism. FIG. 3
 illustrates an embodiment where a quantity of sensible reagent containing
 a known accurate concentration ratio of growth-altering agent and marker
 is applied to a surface 30 of the growth medium 28 located at one end of
 the trough 26. FIG. 5 illustrates an alternative embodiment where a
 quantity of sensible reagent containing a known accurate concentration
 ratio of growth-altering agent and marker is applied to a substrate 32
 placed in contact with a surface 34 of the growth medium 28. In both
 embodiments, the ratio of growth-altering agent to marker initial
 concentrations is chosen to ensure that the growth-altering agent and the
 marker will diffuse into the growth medium 28 sufficiently enough so as to
 provide a readily detectable quantity of marker at the probable boundary
 region. The ratio of initial concentrations is expressed as the constant
 "k .sub.1 ":
EQU k.sub.1 =(growth-altering agent concentration).sub.initial /(marker
 concentration).sub.initial
 An accurate value representing the ratio of growth-altering agent to marker
 initial concentrations is determined at the time the sensible reagent is
 manufactured. A reference pad 36, independent of the layer of growth
 medium 28, is provided containing a known amount of marker that emits a
 known magnitude of fluorescent signal. The marker contained within the
 reference pad 36 can be different from that used within the reagent. If
 the markers are different, however, or if the response of the marker
 within the reference pad 36 differs from the response of the marker in the
 growth medium 28, the concentration to signal magnitude ratio of each
 marker must be known.
 The sensible reagent diffuses into the growth medium 28, creating a
 gradient 38 of decreasing concentration as it travels laterally (shown
 diagrammatically in FIGS. 3 and 5). The inoculated growth medium 28 is
 incubated and a section 40 having detectable microorganism growth develops
 contiguous with a section 42 having no detectable microorganism growth. A
 commercially available scanning fluorometer (not shown) adjusted to sense
 the fluorescent signal characteristic of the marker is used to measure the
 magnitude of the marker signal emitted from a given volume (V) located in
 the boundary region 44 between the sections 40, 42, where the volume (V)
 is defined as an area (A) of inoculated growth medium scanned, having a
 uniform thickness (T; V =A*T). As stated earlier, the position of the
 boundary region 44 between the sections 40, 42 may be determined
 optically, or by other means. The fluorometer generates a curve 43 (FIG.
 4) representing signal magnitude as a function of lateral distance across
 the trough 26.
 The concentration of the growth-altering agent in the boundary region 44
 can be calculated by first determining the marker concentration in a given
 volume (V) in the boundary region 44 using the following relationship:
 ##EQU3##
 which can be rearranged to solve for the unknown marker concentration since
 the marker signal in the boundary region 44 is known:
 ##EQU4##
 Once the marker concentration in the boundary region 44 is determined, the
 growth-altering agent concentration in the boundary region 44 can be
 determined by multiplying the ratio (k.sub.1) of growth-altering agent and
 marker initial concentrations times the marker concentration in the
 boundary region 44, provided the growth-altering agent and the marker
 within the sensible reagent have equal diffusion rates:
 k.sub.1 *[(am't of marker)/(V)].sub.b =Growth-Altering Agent Concentration
 in the Boundary Region
 If the diffusion rates of the growth-altering agent and marker differ, a
 correction factor representing the mathematical relationship between the
 two diffusion rates is used to correct for the difference.
 Referring to FIGS. 6 and 7, a preferred apparatus 46 for use in determining
 the concentration at which a growth-altering agent has an appreciable
 effect on the growth of a target microorganism includes a body 48 having a
 well 50, sensible reagent incorporated into a sheet of the growth medium
 52, a pair of absorbent strips 54, and a transparent well cap 56. The well
 50 includes a pair of channels 58 extending between walls of the well 50.
 The growth medium 52 is disposed in the well 50 and an absorbent strip 54
 is placed in each channel 58. The growth medium 52 shown in FIG. 7
 contains a number of distinct sections 60, each incorporating a gradient
 strip 62 of reagent. In instances where there is utility in evaluating a
 plurality of different growth-altering agents, each gradient strip 62 of
 reagent may contain a different growth-altering agent. The transparent
 well cap 56 attaches to the body 48 above the well 50 to protect and
 maintain the growth medium 52 and absorbent strips 54 inside the well 50.
 The apparatus body 48 may alternatively include a plurality of wells 50,
 each similar to that described above. The apparatus further includes a
 port 64 through which a target microorganism solution can be distributed
 to the growth medium 52.
 The apparatus 46 further includes a machine-readable information label 66
 and a user readable information label 68. The machine-readable information
 label 66 includes a data block 70 containing pertinent information such as
 the test to be performed, calibration constants, patient identification,
 or the like, in a machine-readable format such as a bar code or magnetic
 strip. Depending upon the analysis application, the machine-readable label
 66 may directly contain all of the information necessary to enable an
 analytical device to perform the analysis at hand. In other instances, the
 machine-readable label 66 may instruct the analytical device to access
 data files contained within the analytical device or remotely accessed by
 the analytical device. Hence, it can be said that the label 66 directly or
 indirectly contains the information necessary to enable an analytical
 device to perform the analysis at hand. The machine-readable information
 label 66 may also include a reference pad 72 containing a known amount of
 a sensible marker for use in analyzing the reagent marker signal within
 the growth medium. The user-readable information label 68 includes
 information that enables the apparatus 46 to be identified by the user
 without machine assistance.
 Although this invention has been shown and described with respect to the
 detailed embodiments thereof, it will be understood by those skilled in
 the art that various changes in form and detail thereof may be made
 without departing from the spirit and the scope of the invention.