Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2001-10-02
2004-03-23
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S040500
Reexamination Certificate
active
06709836
ABSTRACT:
The present invention relates to an improved mounting medium that can be used in the preparation of slides for investigation by microscopy techniques.
The mounting medium of the invention is particularly suited for use in histochemistry, immunochemistry and/or cytochemistry, i.e. to provide slides of samples of blood, cells tissue or other biological fluids or materials, including but not limited to samples/material which have been stained to facilitate microscopic examination, e.g. for scientific and/or diagnostic purposes.
Further aspects of the invention will become clear from the description given hereinbelow.
Generally, in microscopy, a slide of a material or sample to be investigated is prepared by putting said sample or material onto a (bottom) slide and then covering said sample with a (top) cover. Generally, for light microscopy or similar techniques, slides and covers of glass or another suitable transparent materials are used.
Also, in the preparation of such slides, the cover glass is usually adhered to/fixated onto the bottom slide. This “mounting” assures that the sample and the cover glass stay in place on the bottom slide, which eases handling, storage and/or transportation of the slides. Also, such mounting assures that the slides are preserved for prolonged periods of time—i.e. for up to five years or more—without substantial deterioration of the sample or material.
Generally, in histochemistry-, cytochemistry-, immunochemistry- or similar medical or biological microscopy techniques, the mounting of the slides is the last step before examination of the specimen by microscope. Thus, in order to provide a better understanding of the present invention, the conventional techniques for the preparation of such slides will now be discussed in more detail,
First, one or more samples of the material to be investigated, which may for instance be samples of blood, cells, tissue, or other biological fluids or materials of interest, are put onto the bottom slide. The sample(s) is/are then stained, i.e. to (better) make visible one or more of the components of the sample or material, such as cell nuclei, cell cytoplasm, tissue and cell structures. Such staining techniques and compositions/agents for use therein will be clear to the skilled person, and may for instance include:
stains such as Hematoxylin and eosin (histochemistry),
immunoperoxidase (immunochemistry),
Papanicolaou (cytochemistry) and other histo-, immuno- and cytochemistry stains.
Thereafter, excess staining agent is washed off, e.g. using one or more washing steps with tap and/or distilled/deionized water, alcohol (ethanol, iso-propyl alcohol, benzyl alcohol etc, . . . ) or another suitable medium, Thereafter, the slides are generally washed with an organic solvent or solvent mixture, before the cover glass is fixated onto the bottom slide using the mounting medium. These last washing steps are also intended to remove/replace any water and/or alcohol (ethanol, iso-propyl alcohol, benzyl alcohol etc. . . . ) remaining from the first washing steps with the organic medium, i.e. in order to prepare the sample for the application of the mounting medium. In doing so, the organic solvent acts as an “intermediate” between the water/alcohol and the apolair organic mounting medium.
For instance, the regimen for preparing/mounting a slide may be essentially as described in Table 1
TABLE 1
scheme of mounting a slide (non-limiting):
Sequence
Time in minutes
Staining the specimen on the slide
1
Aqua dest.
1
Ethanol 100%
1
Ethanol 100%
1
Ethanol 100%
1
UltraClear/Xylene/Toluene
1
UltraClear/Xylene/Toluene
1
UltraClear/Xylene/Toluene
1
Embedding with mounting medium
Conventionally, as indicated in Table 1, xylene or aromatic organic solvents have been used for the last washing steps. However, because the use of such solvents is no longer desired—e.g. for reasons of health and safety—nowadays often (mixtures of) saturated hydrocarbons are used, a commercially available example of which is UltraClear® sold by Mallinokrodt Baker B.V., Deventer, Netherlands, which is a mixture of nonanes, decanes, undecanes and dodecanes.
After washing with the organic solvent (mixture), a few drops of mounting medium is applied to the sample or material, as shown in
FIG. 1
(three drops in FIG.
1
). Then, the cover glass is applied, which is fixed/adhered (on) to the bottom slide by the mounting medium. Mounting of a coverglass is applied either manually or by a coverslipping machine.
Conventional mounting media usually comprise a resin, such as an acrylate resin, in an aromatic hydrocarbon such as xylene or toluene as organic solvent. However, in practice, it is often found that the conventional mounting media—and in particular the acrylate resin(s) present therein—interfere and/or react with the organic solvent mixture used for washing the slide, resulting in the formation of a turbid layer on the slide and/or leading to other undesired effects which may detract from quality of the slide obtained and/or from the microscopic examination thereof. It has been found that these problems may in particular occur when the last washing step(s) has (have) been carried out with (a mixture of) saturated hydrocarbons, such as UltraClear.
Accordingly, it is an object of the invention to provide an improved mounting medium which can be used in conjunction with organic solvents, such as aromatic hydrocarbons and alcohols—i.e. as used in the preparation of the slides—and in particular with saturated hydrocarbons, without any of the problems indicated above.
It has now been found that such a mounting medium can be provided by the use of an acrylate resin or methacrylate resin as described below.
Thus, in a first aspect, the invention relates to a mounting medium for the preparation of slides, said mounting medium comprising a solution, in at least one organic solvent selected from the group consisting of saturated hydrocarbons optionally mixed with one or more alcohols, of at least one (meth)acrylate resin based on one or more monomers having formula I:
wherein is hydrogen or a methyl group and n has a value of 0-19.
In particular, the group —(CH
2
)
n
—CH
3
may comprise a linear “backbone” of between 1 and 10 carbon atoms, which may be substituted at any position(s) with one or more saturated hydrocarbon substituents such as methyl, ethyl, n-propyl, i-propyl, 1-butyl, 2-butyl, i-butyl, etc.; such that the total amount of carbon atoms in the group —(CH
2
)
n
—CH
3
is between 1 and 20.
Also, and/or alternatively, the group —(CH
2
)
n
—CH
3
may comprise/contain a saturated hydrocarbon ring and/or an aromatic hydrocarbon ring, which may for instance be connected to the (meth)acrylate group via a (further) saturated hydrocarbon residue, and/or which again may be substituted at any position(s) with one or more saturated hydrocarbon substituents such as methyl, ethyl, n-propyl, i-propyl, 1-butyl, 2-butyl, i-butyl, etc.; again as long as the total amount of carbon atoms in the group —(CH
2
)
n
—CH
3
is between 1 and 20.
Some particularly preferred examples of the group —(CH
2
)
n
—CH
3
include, but are not limited to:
methyl
ethyl
iso-propyl;
iso-butyl;
benzyl
iso-hexyl
iso-decyl;
For instance, the (meth) acrylate resin of Formula I may be iso-butyl methacrylate resin, methyl methacrylate, ethyl methacrylate, isopropyl methacrylate, iso-hexyl methacrylate, benzyl methacrylate, iso-decyl methacrylate, as well as the corresponding acrylate resins.
Generally, the mounting medium of the present invention will comprise a solution of one or more of such (meth)acrylate resins in an organic solvent of solvent mixture, such that the amount of (meth)acrylate resins makes up between 10 and 60 wt % of the total composition.
As the organic solvent, any suitable solvent or mixture of solvents may be used, as long as these solvents do not react with the (meth)acrylate resin and/or interfere with the intended action of the mounting medium. Preferably, an aprotic solvent or mixture of aprotic solvents is used, such as a (mixture of) apolair aprotic solvent(s). Some suitabl
Kieftenbeld Wilhelmus Hermanus Hendrikus Maria
Mallinckrodt Baker B.V.
Witz Jean C.
Young & Thompson
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