Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2002-03-19
2004-06-15
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S283100, C435S286500, C422S068100
Reexamination Certificate
active
06750039
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a filtration apparatus and method for the separation of microscopic entities from a fluid (liquid or gas) and subsequent visual or imaging microscopic analysis of the entities separated thereon either directly or after treatment of the entities whilst on the apparatus in order to enhance their subsequent visualization and/or imaging. Such treatment can comprise reaction with reagents contained in other solutions that can be made to imbibe and/or pass through the filtration apparatus and which can be washed with solutions that can be made to imbibe and/or pass through the filtration apparatus. An example of a specific area of application is in the microbiological testing of fluids in order to detect, identify and/or enumerate microorganisms contained in a fluid test sample.
This invention also relates to an apparatus upon which organic and/or biological molecules (or organisms) can be immobilized and reacted with reagents contained in other solutions that can be made to imbibe and/or pass through the filtration apparatus and which can be washed with solutions that can be made to imbibe and/or pass through the filtration apparatus. An example of a specific area of application is in the immobilization of a probe or probes onto the apparatus for the purpose of capturing, from a test solution, nucleic acids containing a specific nucleotide sequence represented by the complement to the nucleic acid or non-nucleic acid (e.g. Peptide Nucleic Acid) probe or probes immobilized onto the apparatus.
BACKGROUND OF THE INVENTION
One area of application pertains to the microbiological testing of fluids with the goal of detecting, identifying, and/or enumerating microorganisms contained in a test fluid, or defined volume of test fluid.
Traditionally, a sample suspected of containing contaminating bacteria, yeast, or other microorganism of interest, is filtered through a sterile, microporous membrane (typically 0.22 &mgr; or 0.45 &mgr;porosity). The membrane is then exposed to a selective growth medium (e.g. agar or broth-based) and incubated, usually at 37° C., until visible colonies appear on the membrane; usually 24-72 hours, depending on the species of microorganism. Incubation for a shorter time results in microscopic colonies that can very tediously be counted under a high power microscope objective. This process however, requires substantial technician time and is subject to significant counting error. When subjected to longer incubation, the visible colonies are manually counted without the use of a microscope and the original concentration of microorganisms can be calculated. Many apparati have been developed for this purpose (e.g. U.S. Pat. No. 4,317,726, U.S. Pat. No. 4,614,585, U.S. Pat. No. 4,777,137, U.S. Pat. No. 4,912,037, U.S. Pat. No. 5,202,262, U.S. Pat. No. 5,288,638, U.S. Pat. No. 4,036,698, U.S. Pat. No. 4,829,005, U.S. Pat. No. 5,409,832, U.S. Pat. No. 5,112,488, all of which are herein incorporated by reference). All of these are intended for the manual counting of colonies as described above.
A considerable amount of time can be saved if the incubation time is reduced to 4-6 hours and counting of the microscopic colonies is automated. Such a method for identification and enumeration of microorganisms has been developed and is termed Fluorescence In-Situ Hybridization (FISH) followed by automated image capture and analysis using an automated commercially available laser or charge coupled device (CCD)-based imaging system (Stender, et. al., J. Micro. Meth. 1449, 2001). This FISH method typically involves the following steps:
1. Filtration through a microporous membrane, trapping microorganisms on the membrane filter. The filter is part of a filtration apparatus with a fluid reservoir on one side and a waste receptacle on the other. Pressure can be applied to the reservoir or vacuum can be applied to the waste side in order to force the fluid through the membrane to the waste side, trapping any microorganisms on or within the filter.
2. Growth of the microorganisms on the filter by transferring the filter from the filtration apparatus to a cellulose pad or agar plug imbibed with a selective growth medium.
3. Fixation of the microorganisms on the filter with a fixative solution by transferring the filter from the growth medium to a cellulose pad imbibed with a fixative solution.
4. Hybridization on the filter with a Peptide Nucleic Acid (PNA) probe specific to the microorganism in question. The probe is labeled with a fluorescent molecule. The filter is transferred from the fixation medium to a glass slide, microorganism side up. A small amount of hybridization solution is pipetted onto the top of the membrane filter and a cover slip placed thereon in order to spread the hybridization solution over the membrane and minimize evaporation. The filter on the slide is incubated at an elevated temperature (45-65° C.) in a humidified chamber.
5. Washing excess and unbound reagent from the filter. The slide with the filter is placed in a standard slide tray and incubated in heated wash solution. The membrane is gently teased from the slide in order to wash both sides of the membrane and remove as much hybridization solution as possible.
6. Transferring the membrane to an imager for analysis. The membrane is removed from the wash solution and placed on a microscope slide for subsequent imaging and analysis.
This process involves manually handling the delicate microporous filter directly multiple times prior to the analysis step, increasing the chance of contamination and damage.
In addition to the analysis of microorganisms or particles, this invention also pertains to microarrays. Microarrays is a fast growing field that allows for deposition of ~100-200 micron spots of target molecules (in most cases, nucleic acid probes, antigens, or antibodies) that are immobilized onto a support substrate, which in most cases is a pretreated glass microscope slide. The spots are arrayed in a rectangular matrix on the surface of the slide allowing for thousands of experiments per slide. Reagents are flowed over the surface or the slides are immersed in reagent and rinse reservoirs utilizing standard equipment used for processing glass microscope slides in fields such as histochemistry, cytochemistry, immunohistochemistry, and cytogenetics. Recently, (solid) glass slides coated on one surface with a microporous matrix have become commercially available. The microporous matrix (for example, nitrocellulose, nylon) purportedly provides more surface area for immobilization and reaction with target molecules resulting in localized fluorescent, chemiluminescent, or radioactive product as an indicator that a target molecule has bound to the probe immobilized within a given “spot”.
Discussion of Certain Documents
U.S. Pat. No. 5,891,394 discusses an apparatus for detection and enumeration, but not identification, of microorganisms on a filter by fluorescence. Their fluorescence method is not capable of identifying a specific microorganism in the presence of other microorganisms. Furthermore, filtration and processing are done on standard 25 mm membranes. After processing, the filter is manually transferred to a well in the apparatus for analysis. There is no slide involved and the membrane is directly handled multiple times prior to analysis.
U.S. Pat. No. 4,124,449 describes a filter holder for mounting on the stage of a microscope. This apparatus allows for filtration of fluids through an integral microporous membrane, incubation of microorganisms retained on the filter with a dye to stain them, and visualization under a light or dark field microscope for the visual enumeration of microorganisms. It is not only restricted to visual observation with a microscope, but its overall dimensions far exceed those of a standard microscope slide, rendering it incompatible with standard apparatus for processing specimens on a microscope slide.
U.S. Pat. No. 5,252,293 describes a plastic disposable analytical slide with an integral microporous filter membrane. The
Bargoot Frederick G.
Hyldig-Nielsen Jens J.
Rising Donald B.
Stender Henrik
Birch Richard J.
Boston Probes, Inc.
Leary Louise N.
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