U.S. patent number 3,768,914 [Application Number 05/290,170] was granted by the patent office on 1973-10-30 for electron microscopy tissue grid staining and storing rack and method.
Invention is credited to Thomas D. Kinney, John E. P. Pickett.
United States Patent |
3,768,914 |
Kinney , et al. |
October 30, 1973 |
ELECTRON MICROSCOPY TISSUE GRID STAINING AND STORING RACK AND
METHOD
Abstract
An apparatus and method for staining, washing and storing a
plurality of grid mounted electron microscopy tissue specimens are
based upon placing the grids having the mounted tissue in a rack
which isolates and provides an identifying file indicia and fluid
passages for staining and washing each of a plurality of grids. The
same rack also receives a protective cover for grid storage thereby
eliminating much individual grid handling and the requirement for
separate staining and storage apparatus.
Inventors: |
Kinney; Thomas D. (Durham,
NC), Pickett; John E. P. (Durham, NC) |
Family
ID: |
23114831 |
Appl.
No.: |
05/290,170 |
Filed: |
September 18, 1972 |
Current U.S.
Class: |
356/244;
359/398 |
Current CPC
Class: |
G01N
1/31 (20130101); B01L 3/5085 (20130101); G01N
2001/315 (20130101) |
Current International
Class: |
G01N
1/31 (20060101); G01N 1/30 (20060101); G01n
021/16 () |
Field of
Search: |
;356/244,246,36,38
;250/49.5B ;350/92,95 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Sikes; William L.
Claims
What is claimed is:
1. An assembly of cooperative laboratory components adapted to
facilitate staining, washing and storage of grid mounted electron
microscopy tissue specimens comprising, in combination:
a. a generally rectangular base container having a central plate
portion providing a plurality of individual wells each adapted to
receive in one open end thereof an electron microscopy specimen
grid and to hold the grid by its peripheral edge whereby the
specimen itself remains out of contact with well surfaces;
b. a first cover adapted to be removably secured to said base
container, said base container and first cover being constructed
for operational cooperation such that when said first cover is so
secured said grids are retained in their respective wells by said
first cover and each well is provided with at least one fluid
exchange path having external communication for exchanging staining
and washing fluids;
c. a fluid container adapted to hold a selected fluid and to
slidably receive and maintain said base container and first cover
in a vertical position during exchanges of fluids with said grids;
and
d. a second cover means adapted to be secured to said base
container in place of said first cover and being adapted in
cooperation with said base container to enclose each said well to
provide an enclosed storage void for each said grid.
2. An assembly as claimed in claim 1 wherein said base container
includes on said plate portion opposite the side thereof having the
grid receiving ends of said wells an attached porous pad, said pad
serving to provide a porous closed end for each said well and
thereby establishing said fluid exchange path.
3. An assembly as claimed in claim 2 wherein said pad is fixedly
secured to said plate portion.
4. An assembly as claimed in claim 2 wherein said pad is slidably
secured to said base container adjacent said plate portion.
5. An assembly as claimed in claim 3 wherein said first cover is
provided with a plurality of holes of substantially smaller size
than and mating with respective said wells to facilitate passage of
fluids therethrough.
6. An assembly as claimed in claim 5 wherein said second cover
means has integral portions adapted during storage to cover both
said pad and the grid receiving side of said base container.
7. An assembly as claimed in claim 4 wherein said first cover is
imperforate and including a further imperforate cover, said pair of
imperforate covers being adapted to be slidably secured to said
base container on opposite sides of said plate portion during
storage of said grids.
8. An assembly as claimed in claim 1 wherein said first cover
comprises a perforate cover having holes mating with said wells,
wherein each said well on the end opposite the grid receiving end
thereof is closed and provided with a fluid passage adapted to
communicate with fluid sources externally thereof, and wherein said
second cover means comprises a pair of imperforate covers adapted
to be slidably secured to said base container during storage of
said grids.
9. An assembly as claimed in claim 1 wherein said first cover is
imperforate and said base container wells are closed at the ends
opposite said open grid receiving ends and in such closed ends are
provided with holes which provide for each respective well a said
fluid exchange path.
10. An assembly as claimed in claim 1 wherein said first cover is
provided with a plurality of holes of substantially smaller size
than and mating with respective said wells to facilitate passage of
fluids therethrough and wherein said base container wells are
closed at the ends opposite said open grid receiving ends and in
such closed ends are provided with holes which provide for each
respective well a said fluid exchange path.
11. The method of staining, washing and storing electron microscopy
grids having tissue specimens mounted thereon, comprising the
steps:
a. mounting a plurality of said grids in a base container having a
plurality of open ended grid receiving wells shaped to engage the
grid peripheries and hold the tissue specimens out of contact with
the well surfaces;
b. securing the grids in place by installing a cover on said base
container over the wells open ends;
c. treating the plurality of said grids simultaneously with
appropriate staining and washing fluids in sequence while confined
in said base container;
d. drying and selectively examining said grids as required; and
e. covering said wells and the grids contained therein for storage
by detachably securing imperforate covering means to said base
container.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to histologic processes and
apparatus and more specifically to electron microscopy grid mounted
tissue staining and storage.
2. Description of the Prior Art
Specimens are stained for electron microscopy to increase the
contrast of the section under the electronic beam on the electron
microscope. In electron microscopy it has been difficult to develop
staining procedures that are specific for particular substances,
but a general stain to increase contrast is very helpful,
particularly when the contrast is low. Most staining is done with
reagents containing metal ions to increase the electron density of
the tissue structures. Stains include lead citrate, uranyl acetate,
phosphotungstic acid, phosphomolybdic and ammonium molydate.
It has been the practice to mount electron microscopy tissue
specimens on small diameter, e.g., approximately three
thirty-seconds inch, copper grids, e.g., 200 mesh. After the
mounting procedure, each minute grid having a tissue specimen is
placed with a fine pair of forceps on a drop of stain and is
allowed to stay for a predetermined period, e.g., 5 minutes. Each
grid is then carried with the forceps through several distilled
water rinses. Through all of these individual operations on each
grid, provision must be made for maintaining the identity of each
grid. It is also often desirable that particular groups of grids
receive as near as possible the same staining exposure to
standardized results.
It would be desirable to stain electron microscopy sections on
grids in volume in order to speed up processing and also give
better and more uniform quality material for electron microscopy
study. However, prior art practices have over a long period of time
followed the individual grid staining and washing procedures
described above.
In addition to the mounting, storing, and washing procedures
mentioned above in which grid identification, freedom from
contamination and physical protection are most important for the
very delicate grids, it also becomes important that the grids be
suitably protected and identified when stored after examination so
that they may be retrieved if necessary for other examinations.
There is now sold under the trademark "LKB" what is called a
"Specimen Grid Box" for use in storing, by a file number, electron
microscopy grids having tissues mounted thereon. The manufacturer
is LKB-Produkter A B, Stockholm-Bromma 1, Sweden. This grid box
comprises a holder made of plastic having a transparent sliding
cover. The cover includes a pair of integral, inturned flanges and
receives a mating flanged grid storage container which has twenty
rows of diamond shaped holes or wells which are arranged in ten
columns. The rows each have five wells and the rows and columns are
marked lengthwise with letters and laterally with numbers so that
each well has an identification such as "A-5." This allows the
technician to record where a particular grid is stored. Each LKB
grid box also has a serial number on one end of the container such
as the number "Box 52134" which allows the box to be identified.
The diamond shaped well is specifically designed, dimension and
shapewise, so as to receive the grid periphery by opposed well
edges and to allow the grid to be stored without the delicately
mounted tissue touching the well surfaces.
It is significant to note that the LKB type grid box container is
designed to that the grid storage wells are open at one end but are
closed at the opposite end. It should also be noted that the
sliding cover is designed with a relatively close sliding fit over
the open end of the wells. Thus, it can be appreciated that while
the conventional LKB type grid box does serve as a useful storage
and filing device for electron microscopy grids, it has no utility
for staining or washing procedures.
From the foregoing, it can be seen that the prior art methods and
apparatus for the very delicate handling of electron microscopy
grids during storage, staining and washing procedures have required
individual grid handling in all steps and have required separate
apparatus for storage as distinct from staining and washing. Thus,
the prior art would benefit from an apparatus which is adapted to
serve both the purpose of being a file storage rack as well as a
staining and washing rack and from a method which substantially
reduces the amount and hazards of individual grid handling.
SUMMARY OF THE INVENTION
The invention is directed to a method and apparatus based upon
utilization of a container adapted to provide a plurality of
indicia identified wells or holes for staining and washing of
electron microscopy grids and to also provide means whereby the
grids so stained may be stored. In one embodiment, the grids are
stored in a container having a plurality of wells adapted on one
side with open ends to receive and provide identification for an
equal number of electron microscopy grids and on the other side to
provide small holes communicating with the wells for transfer of
staining and washing fluids. During staining, a cover having small
holes corresponding with the wells covers the grid receiving side
of the container. This container once so covered is fitted
vertically into a receptacle which receives a selected staining
fluid so that the grids may be stained and then washed in another
similar receptacle having a washing fluid. For storage, a pair of
imperforate covers are slidably received by the container on both
sides of the wells which secures and covers all the grids for
indefinite storage.
In a second embodiment, the container which is used in the first
embodiment is fitted with a porous pad which provides a porous
closure for one end of the wells and is fitted with an imperforate
cover on the other, open end, side of the wells for staining and
washing procedures. For storage purposes, the porous pad is removed
and replaced by an imperforate cover so that both ends of the wells
are covered by imperforate covers during storage.
A third embodiment utilizes a modification of the LKB grid storage
box in which the conventional grid box container is provided on one
side with a porous pad which provides porous ends for the wells so
that when the grid box is mounted in its conventional storage cover
and the grid box is placed in a suitable container of staining or
washing fluid, a fluid procedure may be effected. Another cover is
provided to cover the wells during storage.
A fourth embodiment is based upon providing the normally closed end
of each of the wells in a LKB type grid storage box with an
aperture which, with the conventional storage cover installed,
allows staining or washing fluids to penetrate each well during
fluid treating. Another cover such as used for the third embodiment
is provided to cover the wells during storage.
In all embodiments what is important to recognize is that the grids
remain in the same base container during staining, washing, and
storage. It is further important to recognize that unlike the prior
art approach of individually fluid treating each grid, the grids
are processed in volume.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is an exploded view of the first embodiment base grid
container and perforated cover components used during staining and
washing.
FIG. 2 is a perspective view of a fluid container used to hold
rinse and wash solutions and which mates with the base
container.
FIG. 3 is a perspective view of the first embodiment components as
they appear assembled for staining and washing.
FIG. 4 is a top view of the FIG. 2 container with the FIG. 3
assembly installed.
FIG. 5 is a longitudinal section view taken in the direction 5--5
of FIG. 3.
FIG. 6 is a cross section view taken in the direction 6--6 of FIG.
3.
FIG. 7 is an enlarged cross sectional portion of FIG. 5 showing the
typical disposition of several grids.
FIG. 8 is an exploded view of the first embodiment components used
for storage.
FIG. 9 is a cross section view taken through the assembled
components of FIG. 8.
FIG. 10 is an exploded view of the components used in the second
embodiment for staining and washing as well as storage.
FIG. 11 is a perspective view of the second embodiment components
assembled for staining and washing procedures.
FIG. 12 is a side view of the FIG. 11 assembly installed in a fluid
container of the FIG. 2 type.
FIG. 13 is a top view of the assembly of FIG. 12.
FIG. 14 is a cross section view taken in the direction 14--14 of
FIG. 11.
FIG. 15 is a longitudinal section view taken in the direction
15--15 of FIG. 11.
FIG. 16 is an enlarged cross sectional portion of FIG. 15 showing
the typical disposition of several grids.
FIG. 17 is an exploded view of the components used in the third
embodiment for staining and washing as well as storage.
FIG. 18 is a longitudinal section view of the third embodiment
components assembled. for staining and washing.
FIG. 19 is a cross section view of the third embodiment components
assembled for staining and washing.
FIG. 20 is an enlarged cross sectional portion of FIG. 18 showing
the typical disposition of several grids.
FIG. 21 is a plan view of a fourth embodiment showing a modified
base grid container without a cover.
FIG. 22 is a cross section view of the base container taken in the
direction 22--22 of FIG. 21 and having a cross section ef the cover
added to show its relation.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The preferred embodiment is shown in FIGS. 1 through 9, a second
embodiment in FIGS. 10 through 16, a third embodiment IN FIGS. 17
through 20, and a fourth embodiment in FIGS. 21 and 22. Since the
third and fourth embodiments more closely follow prior art
practices these will be first described and then a description will
be given for comparison of the second and finally of the first and
preferred embodiment.
Referring further to FIGS. 17 through 20, the third embodiment
represents a simple but significant modification of the LKB type
grid storage box. The conventional LKB type storage box includes a
solid cover 30 which normally does not contain the holes 31 shown
in FIG. 17. These holes are of the smallest diameter which allows
fluid flow. Cover 30 normally slides over a container 32 having the
diamond shaped wells 33 and the vertical and horizontal indicia, 1,
2, 3, 4, 5, A, B, C, etc., shown in FIG. 17. Container 32 does not
normally have the slot 41 which receives the porous pad 40. Thus,
cover 30 and container 32 represent the conventional LKB grid
storage box except for the presence of the holes 31 in cover 30 and
except for the presence of the slot 41 and porous pad 40 in
container 32. Also, the supplementary storage cover 34, shown in
FIG. 17, is not provided with the conventional LKB storage box
since cover 30, without holes 31, serves the same purpose during
storage when installed on container 32 in its conventional
form.
Slot 41 is cut out of the normally solid side of container 32. As
best seen in FIGS. 18 through 20, the formation of slot 41 to
receive pad 40 causes what appear in those figures as the bottom
ends of the grid receiving walls to be opened and covered by pad
40. A variety of porous materials may be used for pad 40. A
preferred material for pad 40 which provides both filtering and
fluid passage is identified as Labpor porous polyethelene plastic
sheets one-sixteenths inch thick, 120 micron porosity,
hydrophically treated, made by Porex Company of Fairborn, Georgia.
Pad 40 is suitably rigidly secured to container 32. When using this
third embodiment, the grids having mounted tissue are placed in
their respective wells 33, cover 30 having holes 31 is installed
and the assembled grid box is placed in a stain solution container
such as indicated in FIG. 2 and which contains the appropriate
stain. Holes 31 and pad 40 provide fluid passages. Here it should
be mentioned that successful staining and rinsing has been achieved
without holes 31 in cover 30 and with only the presence of pad 40
as a means of fluid passage. This alternate third embodiment does
appear, however, to be more time consuming with such experience as
has been gained to date. Under normal procedures, after staining,
each grid is handled individually with forceps and is passed
through several rinses of distilled water. Such procedures subject
the very delicate and small, e.g., three-thirty-seconds inch, grids
to damage and contamination at every step. With the present
invetion, however, the delicate and small grids remain in container
32 and may be washed through several separate rinses and dried
while remaining in the grid box, thus eliminating much of the
hazard previously experienced.
Once the particular sections on the grids have been stained,
rinsed, and individually examined under an electron microscope as
required, the grids may then be returned to the same grid box and
solid cover 34 installed which now becomes a permanent storage by
box and file designation. Thus, each grid, i.e., each tissue
section, may be assigned a box number, e.g., F-1235 shown in FIG.
17 and a file number in the box, e.g., A-3, which it may retain for
future reference for the original tissue mounting, the staining,
examination and subsequent storage. Thus, box F-1235 may be
referenced with regard to a staining technique whereas before each
individual grid had to be so referenced and the staining techniques
could not be uniform for a group of related grids.
From what has been described, it can be seen that the presence of
fluid passages into the wells in container 32 allows the
conventional LKB type grid storage box to be used for both staining
and washing of large numbers of grids and with solid cover 34
installed allows the grids to be safely stored in the same
container 32 in which they were stained. This simple modification
in both structure and procedure thus produces significant
advantages.
The fourth embodiment shown in FIGS. 21 and 22, like the third
embodiment, basically constitutes a modification of the LKB type
grid storage box. In particular, the modified LKB grid box
container 32' has holes 50 through the normally closed base ends of
each grid receiving well 33' in the container 32'. Thus, container
32'may be identical to the container portion of the conventional
LKB grid box except for the fact that the normally closed ends of
the grid receiving wells 33' are apertured with holes 50. The
diameter of holes 50 should be of the minimum size necessary to
provide fluid flow. Additionally, for purposes of the fourth
embodiment, it is desirable to provide shallow longitudinal slots
51 on the underside of the cover 30' and which are aligned
lengthwise with the rows of grid receiving wells 33' to provide
additional fluid passages. Slots 51 may, for example, be about one
thirty-second inch wide and one thirty-second inch deep.
In using the fourth embodiment, the delicate and small size grids
with the tissue mounted thereon are individually installed as
previously explained in the respective apertured wells 33' in the
container 32'. The slotted cover 30' is installed and then the
assembled grid box is placed vertically in a fluid container such
as shown in FIG. 2 and having the desired staining solution. During
staining, the staining fluid reaches and stains the tissue sections
by flowing through the holes 50 and the slots 51. After the grids
have been in the staining solution for the required time they are
retained in the grid box and passed through other containers
containing distilled water as required to complete washing. While
still remaining in the grid box, the grids are allowed to dry and
are then available for examination and eventual return to the grid
box for storage. A cover such as cover 34 in FIG. 17 is installed
and adapts the grid box for permanent storage under a suitable box
number and file indicia as previously explained.
With the above description in mind, reference is next made to the
second embodiment shown in FIGS. 10 through 16. The second
embodiment, like the third and fourth embodiments is directed to
providing a grid box which can serve both as a staining and washing
rack as well as a storage rack. Additionally, the grid box of the
second embodiment is adapted to be mated with a container such as
shown in FIG. 2 which serves in place of a beaker to hold stain and
wash solutions and positions the grid box vertically during
staining and washing.
In FIG. 10, the "grid box", per se, numbered 60, has a plurality of
diamond shaped wells which extend completely through the central
plate portion 61. Diamond shaped wells such as used in the LKB box
are preferred, as previously mentioned, since they tend to support
the respective grids on the grid peripheries and prevent the
mounted tissue from touching the sides of the wells. Wells of other
shapes may, of course be used. Integral with plate portion 61 grid
box 60 includes on the well grid receiving side a pair of slotted
flanges 62, 63 which slidably receive an imperforate cover 65 and
engage stop protrusions 66, 67. The fit between cover 65 and the
face of plate portion 61 should preferably allow at least some
fluid passage. In one embodiment, a polyethelene plastic sheet
1-3/8 inch wide by 2 inches long by one-sixteenth inch thick has
been employed for cover 65. On an opposite side slotted flanges 70,
71 slidably receive a replaceable and cleanable porous pad 72 which
provides a bottom surface. A Labpor one-tenth inch thick sheet, 120
micron porosity, hydrophically treated sheet as made by Porex
Company, Fairborn, Georgia, of a length and width similar to that
of cover 65 has been employed successfully as pad 72. Unlike pad 40
in FIG. 17 which is intended to be permanently secured, pad 72 is
removably secured and, thus, can be cleaned.
With pad 72 in place, the grids positioned and identified by
appropriate indicia 75, e.g., numbers 1-25, and cover 65 installed,
the grid box as shown in FIGS. 12-13 is next placed in a mating
open top container 80 (like the container of FIG. 2) having
sidewalls 81, 82, end walls 83, 84 and bottom wall 85. End walls
83, 84 are provided with slots 86, 87 adapted to slidably receive
the flange portions 88, 89 (FIG. 11) of grid box 60 and during
staining and washing to maintain the grid box in a vertical
position. A tab portion 90 is formed integral with box 60 and
provides a convenient means for installing and removing the grid
box assembly.
After the grids have been appropriately stained, washed and dried
while remaining in grid box 60 and after the required individual
examination under an electron microscope, pad 72 is removed, solid
cover 65 is installed and a similar solid cover 91 (FIG. 10) is
substituted for pad 72. In this configuration, the grid box
assembly becomes a storage assembly but at no time must any one of
the individual and very delicate grids be removed from box 60
except when being examined. A suitable file box number, e.g.,
Z-1234, may be used as shown in FIGS. 10 and 11.
The first and preferred embodiment is shown in FIGS. 1 through 9
and its construction and use will be immediately understood from
what has already been explained. The grid box assembly used for
staining and washing comprises the grid box 100, the perforated
cover 101 and the fluid container 102. Grid box 100 is generally
similar to the previously explained grid box 60 except that a
porous pad, like pad 40 or pad 72, is not employed and the
individual wells 103 instead of being of a uniform diamond shape
size throughout the thickness of the grid box, as in FIG. 16, have
closed ends which in turn are provided with small holes 105 like
holes 50 (FIG. 22) as best shown in FIG. 7. Thus, with the grids
installed as in FIG. 7 and with the perforated cover 101 installed,
the cover holes 106 and the well holes 105 insure a fluid exchange
path for both staining and washing fluids when installed in the
container of FIG. 2 and as depicted in FIG. 4.
Once the grids have been stained and washed as a group and then
individually examined as required, storage is effected by
installing a pair of solid, relatively snug fitting, covers 110,
111 as shown in FIGS. 8 and 9. With such covers installed, the grid
box staining-washing assembly becomes a storage assembly for
essentially indefinate storage. A suitable file box number, e.g.,
M-4621, as shown in FIGS. 1, 3 and 8, may be used.
In all embodiments, it can be seen that individual handling of the
delicate and small size grids during staining and washing has been
eliminated. Thus, this extremely crucial, tedious and time
consuming procedure has been abolished. Each grid box embodiment,
furthermore, mates and is preferably used with a fluid receptacle
that holds the box vertically in a uniform position during staining
and washing and with minimum opportunity for damage to the grids.
More uniform results are now assured with substantially less risk
to the sometimes critically important minute tissue sections.
* * * * *