Disc assay device with inoculation pad and methods of use

Details Disc assay device with inoculation pad and methods of use Disc assay device with inoculation pad and methods of use

1999-03-09

2001-01-16

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S283100, C422S050000

Reexamination Certificate

active

06174699

ABSTRACT:

FIELD
This invention relates to disc devices and methods of use for detecting and enumerating microorganisms present within a sample.
BACKGROUND
The detection and enumeration of microorganisms is practiced in numerous settings, including the food-processing industry (testing for the contamination of food by microorganisms such as
E. coli
and
S. aureus
), the health care industry (testing of patient samples and other clinical samples for infection or contamination), environmental testing industry, the pharmaceutical industry, and the cosmetic industry.
Growth-based detection and enumeration of microorganisms is commonly practiced using either liquid nutrient media (for example, most probable number analysis (MPN)) or semi-solid nutrient media (agar petri dishes). Enumeration using the liquid MPN method is typically achieved by placing serial 10-fold dilutions of a sample of interest in replicate sets of tubes containing selective media and chemical indicators. The tubes are incubated for 24-48 hours at elevated temperatures (30-37° C.) followed by examination for growth of organisms. A statistical formula, based on the volume of sample tested and the number of positive and negative tubes for each set, is used to estimate the number of organisms present in the initial sample.
This method of performing MPN analysis has several disadvantages. It is labor intensive because of the multiple diluting and pipetting steps necessary to perform the analysis. Typically, it is only practical to use replicate sets of about three to five tubes for each dilution. As a result, the 95% confidence limits for an MPN estimate for microbial concentration are extremely wide. For example, a three tube MPN estimate of 20 has 95% confidence limits ranging from 7 to 89. Furthermore, results typically are not obtainable in less than twenty-four hours.
Multi-well devices have been introduced for use in connection with MPN analysis. A user inoculates the device by introducing a sample of the item to be tested, such as food, onto the substrate containing the wells. Typically, the sample includes growth media and indicators. Once inoculated, the user incubates the device and then calculates the MPN based on the number of “positive” wells.
Multi-well devices present many potential problems. Inoculation may be hampered by air bubbles that form in the wells during introduction of the sample. Each well may not receive the same volume of sample. Furthermore, the method of inoculation or the device may promote bridging and cross-contamination among the wells, thereby potentially adversely affecting the MPN calculation.
Another potential problem with multi-well devices is that they may be inconvenient to use. For example, most multi-well devices are inoculated by either pipetting sample directly into the wells or pouring sample onto the multi-well substrate. Pippetting is labor and time intensive. Pouring requires that the wells be evenly filled and that excess sample be poured off. In either event, these devices may lend themselves to contamination by outside sources during the inoculation procedure.
SUMMARY
The present invention addresses many of the disadvantages of the prior art. The invention provides assay devices and methods for the rapid detection and enumeration of microorganisms. The device has a substrate including absorbent discs attached thereto and an inoculation vehicle attached to the device, positioned to inoculate the absorbent discs. Optionally, the device may include a coversheet.
In one aspect of the present invention, the substrate includes two different sizes of discs. Preferably, the substrate includes about fifty discs of one size and fifty discs of a different size. In a particularly preferred embodiment, the substrate includes discs capable of holding about two microliters of sample and discs of a different size capable of holding about sixteen microliters of sample and the sixteen microliter discs at least partially surround the two microliter discs. The substrate may further be hydrophobic to assist in preventing cross-contamination between discs.
The device may optionally include an outer edge or margin defining a space between the discs and the end of the substrate. The outer edge or margin may optionally be raised. Preferably, the outer edge or margin is sized to permit the optional coversheet to seal to the substrate when the device is in use.
In another aspect of the present invention, the inoculation vehicle is an absorbent inoculation pad with a backing attached thereto. Preferably, the backing is detachably attached to the device along a perforation.
In another aspect of the present invention, the coversheet is attached to the substrate to form a hinge. The inoculation vehicle may be adhesively attached to the hinge in such a way that the inoculation vehicle is capable of inoculating the absorbent discs.
In a method of the present invention, an assay device as described above is provided. The user places the sample to be tested on the inoculation vehicle and places the inoculation vehicle against the absorbent discs, thereby resulting in the discs being inoculated with sample. The user removes and discards the inoculation vehicle and incubates the device. The user then conducts detection of any target microorganisms that grew during incubation and may subsequently enumerate the microorganisms. In a preferred embodiment, the method may be conducted in twenty-four hours or less.
The present invention overcomes many of the deficiencies of the devices and methods of the prior art. The device may be inoculated quickly and evenly and virtually simultaneously, without concern about air bubbles forming. The detachable inoculation vehicle is easily detached and disposed of. Once detached, the inoculation vehicle does not remain with the device and therefore does not present a possible source of contamination. The coversheet provides additional protection against contamination and helps prevent drying out of the discs.
In the case of MPN analysis for the detection and enumeration of microorganisms, the approaches described herein allow for the use of water-soluble indicator species, and reduce or eliminate the need for the several dilutions typically required in current MPN analysis.
Other advantages of the invention will be apparent from the following detailed description and the figures.


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