Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1999-12-22
2002-10-22
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
Reexamination Certificate
active
06468764
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods and apparatus for the diagnostic staining of biological material. More particularly, the present invention relates to methods and apparatus for staining biological material on microscope slides in an automated fashion.
2. Background and Summary of Prior Art
Biological material, whether viruses, bacteria, or various smears or samples of blood, mucus, and the like, have been analyzed for centuries, since Van Leeuvenhoek invented the microscope. Typically the sample of material is applied to a microscope slide, stained or otherwise rendered into an analyzable state, and analyzed by a human technician or scientist.
As laboratory services for hospitals, physician's offices, veterinarians, and other life-science-based enterprises becomes increasingly “outsourced,” the laboratory's ability to analyze slides of biological material rapidly and accurately becomes increasingly important. Several steps have been made toward automating these processes, but it seems unlikely that the skilled human technician will be almost entirely removed from the process in the foreseeable future.
A fairly typical and important laboratory staining technique is known as Gram's Stain, which was devised by H. C. J. Gram. The Gram's Stain is a “gateway” test that indicates to the technician the presence (or absence) of certain bacteria in a sample of biological material and gives the technician or scientist information necessary or helpful to make further analysis. For instance, Gram's Stain can be used to determine which pathogens are suspected and lead to an antibiotic prescription until further identification can be conducted.
Like several staining or analysis methods, Gram's Stain involves treating the biological material, usually applied to a conventional microscope slide, with a number of reagents or stains. The reagents or stains emphasize or highlight the presence (or absence) of certain types or features of bacteria or other biological material that is helpful to the technician. In the Gram's Stain, the bacteria are treated first with gentian violet, and then with a formulation of iodine conventionally known as Gram's iodine. This stains almost all of the bacteria a deep blue or violet. “Gram positive” bacteria absorb the gentian violet and Gram's iodine into their cellular structure, while “Gram negative” bacteria are stained only superficially. The sample is then washed with acid alcohol, which “decolorizes” or washes the color from Gram negative bacteria. Thus, when adequate decolorization has occurred, the blue or violet Gram positive bacteria can be distinguished from the colorless (or less deeply blue or violet) Gram negative bacteria. A “counter-stain,” of fuchsine for example, may be applied to turn the blue or violet bacteria to a reddish shade to improve their visibility. Decolorization is critical to the Gram's Stain because too little decolorization can yield false Gram positives and too much decolorization can yield false Gram negatives.
Several past attempts at providing automating the slide preparation and staining process have met with limited success. For example, U.S. Pat. No. 4,029,470, Jun. 14, 1977 to Wilkins et al. provides an apparatus for automatically staining a single microscope slide without a lab technician touching the slide. This patent addresses the decolorization issue by timing the application of decolorizing agent in selected volume. The time/volume control of decolorization is insufficient to accurately decolorize a Gram Stain. The decolorization process is simply too dependent upon observation and manual work to be so easily controlled.
GG&B Technology, Inc., of Wichita Falls, Tex., sells a more sophisticated slide stainer under the registered trademark Quick Slide®. This device fully automates the preparation of slides for analysis and is a useful tool in the modern medical laboratory. Nevertheless, the Quick Slides® device is not capable of accurately decolorizing slides for a staining process such as the Gram's Stain.
U.S. Pat. Nos. 5,545,535; 4,665,024; and 4,639,421 all disclose flourescent gram stains and methods of analyzing bacteria stained with the flourescent dyes in which a spectral analysis of the fluorescence of the stained bacteria is used to analyze the Gram positive and negative bacteria in the sample. These inventions do not address the decolorization issue because it seems that decolorization is less important (or unimportant) where flourescent dyes or stains are used in lieu of the conventional Gram's Stain of gentian violet.
A need exists, therefore, for an automated method and apparatus for staining biological material and accurately decolorizing the stained sample prior to analysis.
SUMMARY OF THE INVENTION
It is a general object of the present invention to provide an improved method and apparatus for staining samples of biological material for accurate analysis of the sample. These and other objects of the invention are achieved by applying the biological material to a substrate, preferably a microscope slide. The biological material then is then stained with a selected staining composition, which may be gentian violet for a Gram's Stain analysis. The stained biological material is at least partially decolorized and the level of decolorization is analyzed electro-optically. If necessary, the decolorizing step and the optical analysis steps are repeated until a selected level of decolorization is obtained.
Other objects, features, and advantages of the present invention will become apparent with reference to the drawings and description, which follow.
REFERENCES:
patent: 4029470 (1977-06-01), Wilkins et al.
patent: 4639421 (1987-01-01), Sage
patent: 4665024 (1987-05-01), Mansour
patent: 5081017 (1992-01-01), Longoria
patent: 5137810 (1992-08-01), Sizemore et al.
patent: 5340719 (1994-08-01), Hajek et al.
patent: 5545535 (1996-08-01), Roth et al.
patent: 5554505 (1996-09-01), Hajek et al.
patent: 5593886 (1997-01-01), Gaddy
patent: 5633722 (1997-05-01), Washinger et al.
Joklik et al., Zinsser Microbiology, 18th edition (1984), pp 19-20. Prentice-Hall, Inc., USA.
Gibbs Jeffrey Dwight
Gibbs Walden Lewis
Flood Michele C.
Perdue Mark D.
Tate Christopher R.
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