Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-04-02
2002-05-21
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S031000, C435S034000, C435S288300, C435S288400
Reexamination Certificate
active
06391578
ABSTRACT:
FIELD
This invention relates to methods and devices for partitioning biological samples into microvolume aliquots, based on the tendency for aqueous liquids to be retained within hydrophilic zones of the devices while being substantially excluded from hydrophobic areas of the devices, and detecting and enumerating microorganisms present within the samples.
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 (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 at elevated temperature (24-48 hours) 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. In addition, in practice 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.
In contrast to the method described above, a direct count of viable microorganisms in a sample can be achieved by spreading the sample over a defined area using nutrient media containing a gelling agent. The gelling agent (agar) prevents diffusion of the organisms during incubation (24-48 hours), producing a colony in the area where the original organism was deposited. There is, however, a limit to the number of colonies that can fit on a given area of nutrient media before fusion with neighboring colonies makes counting difficult. This usually necessitates performing several dilutions for each sample. In addition, the classes of chemical indicator molecules that can be used for identifying individual types of microorganisms present within a mixed population are limited to those that produce a product that is insoluble in the gelled media. Furthermore, rapid detection, i.e., in less than twenty-four hours, and enumeration is not feasible using this method.
In addition to these disadvantages, both the currently used MPN analysis and gel-based systems require a relatively long incubation time before a positive result can be detected.
SUMMARY
The invention is based on the discovery that biological liquid samples can be partitioned into discrete microvolumes with only minimal manipulation on the part of an operator. The method of partitioning employs devices that have hydrophilic liquid-retaining zones surrounded by hydrophobic “land” areas. The methods and devices provide a system for the detection and enumeration of microorganisms and other biological materials that solves the problems associated with currently used systems. The system is a liquid-based system, allowing efficient and effective partitioning of the sample into discrete microvolumes for testing, and allows for rapid detection and enumeration.
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.
In general, the invention features a method for partitioning an aqueous liquid sample, into discrete microvolumes, comprising
a) providing a device for culturing a microorganism, said device having an assay surface, the assay surface comprising hydrophilic liquid-retaining zones and a hydrophobic land area between the zones, each zone having a microvolume capacity of liquid retention; and
b) contacting the liquid sample with the assay surface such that the liquid sample is partitioned into the hydrophilic liquid-retaining zones.
The zones may comprise a coating or deposition of assay reagent, such as a nutrient medium or indicator substance. Appropriate indicator substances include without limitation chromogenic indicators, fluorescent indicators, luminescent indicators and electrochemical indicators.
The zones may be of uniform size, with each zone having a liquid retention capacity of about 0.01 to about 25 microliters, more preferably about 1 to about 2 microliters.
The culture device can have, for example, about 10 to about 10,000 hydrophilic liquid-retaining zones, more preferably about 400 to about 600 hydrophilic liquid-retaining zones.
The hydrophilic liquid-retaining zones may comprise microvolume wells surrounded by a hydrophobic land area. Alternatively, the culture device may have a land area comprising a treated nanostructured film. In further alternative embodiments, the hydrophilic liquid-retaining zones may comprise hydrophilic fiber material projecting from the assay surface. The fiber material can be constructed of hydrophilic absorbent discs or of hydrophilic nonwoven fiber loop material. Hydrophilic absorbent discs may have media provided thereon to facilitate growth of microorganisms. The media may be selective for one or more types of microorganisms. The discs are biocompatible with the microorganisms such that the materials do not substantially interfere with the growth or detection of the microorganisms.
In an alternative embodiment, the culture device may comprise a plurality of sets of hydrophilic liquid-retaining zones, each of the sets having zones of uniform size, the sets varying in liquid retention capacity, and the device having at least two sets of zones.
In another aspect, the invention features a culture device for detection or enumeration of microorganisms, the device comprising an assay surface, the assay surface comprising hydrophilic liquid-retaining zones and a hydrophobic land area between the zones, each zone having a microvolume capacity of liquid retention, and at least some of the zones comprising an assay reagent.
As used herein, the term “microorganism” includes all microscopic living organisms and cells, including without limitation bacteria, mycoplasmas, rickettsias, spirochetes, yeasts, molds, protozoans, as well as microscopic forms of eukaryotic cells, for example single cells (cultured or derived directly from a tissue or organ) or small clumps of cells. Microorganisms are detected and/or enumerated not only when whole cells are detected directly, but also when such cells are detected indirectly, such as through detection or quantitation of cell fragments, cell-derived biological molecules, or cell by-products.
As used herein, “microvolume” refers to a volume of less than about 25 microliters, and includes volumes in the sub-microliter range.
The terms “hydrophobic” and “hydrophilic” are herein given the meanings commonly understood in the art. Thus, a “hydrophobic” material has relatively little or no affinity for water or aqueous media, while a “hydrophilic” material has relatively strong affinity for water or aqueous media. The relative hydrophobicities and hydrophilicities of the devices described herein are such as to ensure partitioning of liquid samples substantially into the described hydrophilic liquid-retaining zones upon application of the sample. The required levels of hydrophobicity and hydrophilicity may vary dependi
Berg James G.
Calhoun Clyde D.
Debe Mark K.
Halverson Kurt J.
Krejcarek Gary E.
Gram Christopher D.
Lankford , Jr. Leon B.
Rogers James A.
Sprague Robert W.
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