Microbiological testing apparatus and method

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism

Reexamination Certificate

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C435S029000, C435S004000, C435S283100, C435S287100, C435S288300, C422S050000

Reexamination Certificate

active

06238879

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a microbiological testing apparatus and an associated method. More specifically, the present invention relates to an apparatus for use in the automated antibiotic susceptibility testing of samples, such as those from patients possibly infected by a microorganism.
Agar disk diffusion is a widely recognized microbiological assay for measuring susceptibility—a parameter effectively defined by the assay itself. The susceptibility of a microorganism to a given antibiotic is essentially a description of the size of the inhibitory zone resulting from placement of a permeable disk impregnated with the given antibiotic onto an agar surface inoculated with a sample culture of the microorganism. This parameter provides a measure of the ability of the antibiotic compound to stem growth of the target culture, but it is also a complex function of diffusion constants and other kinetic factors.
Early laboratory standards for the agar diffusion assay involved qualitative evaluation by a laboratory technician, characterizing the tested bacterium's interaction with the antimicrobial agent as “susceptible”, “moderately susceptible”, “intermediate” or “resistant”, depending on the size of the inhibition zone surrounding the antibiotic impregnated disk.
Of additional use to the clinician is a related quantitative measure of susceptibility, known as “minimum inhibitory concentration” (MIC). Although still requiring additional information to translate the parameter into a prescription for clinical practice, this quantitative measure eliminates some sources of complexity and uncertainty relative to qualitative susceptibility. A additional useful clinical parameter is the “inhibitory quotient”, which expresses the ratio of the drug concentration in a particular body tissue at a lowest clinical dose to the minimum inhibitory concentration.
The MIC is ideally determined by an assay appropriately called the dilution method, which straightforwardly involves inoculating a series of test tubes with the target culture, the test tubes containing a series of dilutions of the target antibiotic. One series of test tubes therefore tests only one culture and one antibiotic, in contradistinction to an agar diffusion assay petri dish, which may test a plurality of antibiotics simultaneously with less material and expense. The advantage of the dilution method is that it provides less ambiguously interpretable quantitative results relative to the agar diffusion method, while its disadvantage is primarily its expense, both in materials and labor.
It is therefore desirable to have a device which automatically translates a dimension of an inhibition zone on an agar diffusion assay plate into a more clinically useful quantitative measure of drug-bacterium interaction, such as the MIC. Such a device is disclosed by U.S. Pat. No. 4,701,850. It is further desirable to have a device which automates the process of reading the apposite linear dimension of the inhibition zone, such devices being revealed in subsequent United States patents. The relation of the diameter of the inhibition zone to the MIC for an unknown biological agent is approximated by a linear relation, the parameters for which assumed relation for a particular antibiotic being determined by statistical estimation based on the scatter of data points whose coordinates are inhibition zone diameters and actual minimum inhibitory concentrations determined by dilution assay for a particular microorganism, the relation being assumed linear and being assumed to persist for untested organisms.
Since a number of different antibiotics are simultaneously tested against an unknown culture on a single agar plate and since these antibiotics are characterized by different values of the linear parameters relating inhibition zone dimensions to estimated MIC, and by differing values of the measured dimension of the inhibition zone in a given test, it would be advantageous to have a method of associating the zone surrounding a given antibiotic disk with the subject antibiotic compound without further operator intervention or opportunity for human error.
OBJECTS OF THE INVENTION
An object of the present invention is to provide an improved apparatus and/or associated method for microbiological testing.
A further object of the present invention is to provide a microbiological testing apparatus and/or method which enables automatic determination of a susceptibility of a microorganism to an antibiotic agent.
An additional object of the present invention is to provide such a microbiological testing apparatus and/or method which is easy to use.
It is a supplemental object of the present invention to provide such a microbiological testing apparatus and/or method which is inexpensive to use.
A particular object of the present invention is to provide an essentially automated microbiological testing apparatus and/or method wherein a dimension of an inhibition zone associated with an antibiotic impregnated disk on an agar plate is measured automatically.
A more particular object of the present invention is to provide such a microbiological testing apparatus and/or method which facilitates association of a microorganism susceptibility measurement with a particular antibiotic agent.
These and other objects of the present invention will be apparent from the descriptions and illustrations provided herein.
SUMMARY
An apparatus for carrying out a microbiological assay in accordance with the present invention comprises a holder for holding an agar diffusion plate in operative proximity to a sensing system having one or more sensing devices, which in concert are able to read in a digital or digitizable form both pattern information created by the growth of microbial cultures on an agar gel, and in particular the patterns created by the diffusion of compounds inimical to the growth of said microorganism from the loci of circular disks impregnated with the compounds, and also identification codes imprinted on the disks prior to use of the disks in a multi-disk agar diffusion assay. In a preferred embodiment, this sensing system takes the form of a single digital camera, the pattern information being stored in an intermediate form as a single digital image. Other embodiments are possible without departing from the spirit of the invention, for example, a laser scanner able to simultaneously locate and measure regions of distinct reflectance on the agar plate corresponding reduced microbial growth zones, and able to read codes on the diffusion disks, such as a bar code.
The pattern information is transmitted from the sensing system to a processing system which uses pattern recognition and processing software in order to identify and measure the diameter of a growth inhibition zone, and decode the antibiotic identification code present on the impregnated disk which lies in and gives rise to the growth inhibition zone. The processing system then provides, in a machine readable form suitable for further processing, a list whose entries include pairs of a) identifiers of the compounds present on the disks and b) the numerical diameters of the associated inhibition zones. In a preferred embodiment, the processing system is further used to estimate quantitative susceptibility parameters using as inputs the paired inhibition zone diameters and antibiotic identifiers and also a database relating inhibition zone diameters to known values of quantitative susceptibility parameters. Such a database may most simply take the form of linear regression coefficients for each compound for which there is sufficient prior data.
It is to be noted that the antibiotic identification codes provided on the diffusion disks may take any of a number of different forms including, for instance, bar codes, color codes, alphanumeric characters, or other symbols. As noted above, the sensing system of the microbiological testing apparatus or method in accordance with the present invention may include devices which are equivalent to a digital camera, for example, a video ca

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