Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-11-03
2002-10-15
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S040500, C435S042000, C435S004000
Reexamination Certificate
active
06465207
ABSTRACT:
BACKGROUND OF THE INVENTION
The presence of microorganisms, such as spirochetes or bacteria, for instance Helicobacter pylori, in tissue samples can be detected by histological stains such as the Warthin-Starry silver stain. The stain generally involves combining a silver nitrate solution with gelatin and hydroquinone. Since gelatin is typically a solid at room temperature, the manual stain protocol generally includes liquifying the gelatin, typically by warming it, such as in a microwave oven, prior to its use in the staining process. Generally temperatures as high as 56° C. are used.
One existing automated histo-cytochemistry slide staining system dispenses a solid gelatin matrix. Upon heating, the gelatin matrix forms a solution. In a number of automated staining systems, however, it is preferred to dispense staining reagents that are liquid at room temperature. Such systems cannot currently employ the Warthin Starry staining approach because of the high temperature required to liquify gelatin. As a result, alternative protocols which do not require gelatin but which often are complex and time consuming are currently being used.
Therefore a need exists for simplified automated staining procedures based on the selective deposition of silver metal which can employ liquid dispensers to dispense room temperature staining reagents.
SUMMARY OF THE INVENTION
The methods of the present invention are directed to staining processes whose protocols include the use of gelatin. One specific example of such a staining process is the Warthin Starry method generally used to detect the presence of microorganisms, such as spirochetes, or other bacteria in a biological specimen. The methods described herein are particularly suited for performing automated staining processes using delivery of liquid reagents at room temperature and preferably from liquid dispensers. The methods described herein also provide advantages for staining procedures that are performed manually.
The methods of the invention include replacing gelatin, which generally requires heating to liquify or dissolve in water with non-gelling gelatin, a partially hydrolyzed gelatin. Non-gelling gelatin is soluble in water at room temperature, i.e., about 19° Celsius (C) to about 25° C. Furthermore, aqueous solutions of non-gelling gelatin in water are not viscous at concentrations such as those employed in the staining procedures described herein.
In a preferred embodiment of the invention, a biological specimen is stained by the selective deposition of silver metal. The staining method includes treating the biological specimen with an aqueous solution of non-gelling gelatin, a solution including a silver salt, e.g., silver nitrate, and a solution including a reducing agent, e.g., hydroquinone. The reaction of the reducing agent with the silver salt results in the formation of silver metal which is selectively deposited in some spirochetes, microorganisms or tissue abnormalities and visualizes their presence in the biological specimen.
The invention has many advantages. For example, it can be used to simplify existing manual staining protocols. It also allows the use of existing automated slide staining systems which employ liquid dispensers and room temperature dispensing to carry out staining procedures which could not be previously automated for such systems.
DETAILED DESCRIPTION OF THE INVENTION
The invention is related to staining procedures of biological specimens.
Examples of biological specimens include, but are not limited to, tissue sections, cell cultures, nasal, vaginal, urethral smears, control samples and cytospins. In one embodiment of the invention, the biological specimen is a tissue sample, for instance a tissue sample suitable for histological staining. The biological specimen is prepared as known in the art. In one embodiment of the invention, the biological specimen is a paraffin embedded tissue section after fixation with 10% neutral buffered formalin or other fixative.
Numerous staining procedures, also referred to herein as staining protocols, staining processes, staining methods, stains or staining, have been developed to visualize cell or tissue abnormalities and to detect, identify or characterize microorganisms present in a biological specimen. During histological, cytological or histopathological staining protocols a biological specimen is contacted with staining reagents, also referred to herein as staining solutions or solutions. The sequence and amounts in which the staining reagents are added to the biological specimen depend on the particular staining procedure, as known in the art. Special stains generally include numerous steps and often are some of the most complex tests performed in the laboratory. A number of special stains have been developed for the histological analysis of tissue samples. For example, special stains exists for determining the presence of microorganisms, such as would occur in the context of pathogen invasion, colonization or contamination of a biological specimen, for instance a tissue sample. Such stains are referred to herein as histologic stains.
The invention is generally related to staining procedures which employ gelatin. In one embodiment of the invention, the staining procedure includes the selective deposition, also referred to herein as selective impregnation, with silver. In another embodiment of the invention, the staining procedure is related to detecting a microorganism, a cell or tissue abnormality or a cell or tissue component which is argyrophilic. By argyrophilic it is meant herein that the microorganism, cell or tissue abnormality or component has the property of selectively absorbing silver from a silver salt solution. Generally, staining techniques which rely on the selective impregnation with silver show, against a lighter background not impregnated with silver, a metallic silver image, typically black, which indicates and helps visualize the presence of a pathogen, microorganism, cell or tissue component or abnormality.
Stain protocols which rely on visualizing silver metal that has been selectively deposited often require combining a silver salt solution with a gelatin solution. Aqueous solutions are preferred. One suitable silver salt is silver nitrate. Other silver salts that can be employed in such procedures include but are not limited to silver acetate, silver chlorate and silver fluoride.
Without wishing to be bound by any particular mechanism, it is believed that the role gelatin plays in these stains is somewhat similar to its role in black and white photography. For example, it is believed, that gelatin serves as a protective colloid and has good coating and adhesion properties when used in photographic processes. It is also believed that some of the chemical reactions taking place during stain processes which rely on the formation of metallic silver also parallel chemical reactions which occur in developing black and white photographs. For example, such stain protocols include the addition of a reducing agent, often hydroquinone, a phenolic compound widely employed in photographic processes. As silver is reduced to its metallic state, it is believed that hydroquinone is oxidized to quinone. Examples of other suitable reducing agents include, but are not limited to catechol and other o- and p-dihydroxy or hydroxyamine aromatic compounds.
In a preferred embodiment, the staining procedure is a procedure for detecting spirochetes or bacteria. One particular organism that can be detected using the methods of the invention is
Helicobacter pylori
, (
H. pylori
), a bacterium associated with active chronic gastritis and peptic ulcers.
Helicobacter pylori
is discussed, for example, by Rhatigan-Drexler, K., “A Comparison of Staining Methods for Helicobacter pylori,” Histo-Logic, 30: 3-8 (1999), published by Sakura Finetek Inc., Torrance, Calif.
Examples of other specific microorganisms that can be detected by the methods of the invention include, but are not limited to,
Staphylococcus aureus, Neisseria meningitidis
, Neisseria Gonorrhoeae, Lact
CytoLogix Corporation
Hamilton, Brook, Smith and Reynolds, P.C.
Leary Louise N.
LandOfFree
Silver-based staining processes employing non-gelling gelatin does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Silver-based staining processes employing non-gelling gelatin, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Silver-based staining processes employing non-gelling gelatin will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2985567