Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-01-05
2001-09-04
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287700, C435S288300, C435S288400, C435S032000, C435S033000
Reexamination Certificate
active
06284526
ABSTRACT:
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.
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Beisner William H.
McCormick Paulding & Huber LLP
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