US 3726764 A
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Description (OCR text may contain errors)
April 10, 1973 F. K. WHITE 3,726,764
' MICROBIOLOGICAL CHAMBER APPARATUS Filed Aug. 6, 1971 FIGURE 4.
INVENTOR FRED K. WHITE S 6M ATTORNEY United States Patent O 3,726,764 MICROBIOLOGICAL CHAMBER APPARATUS Fred K. White, Glen Ellyn, Ill., assignor to Miles Laboratories, Inc., Elkhart, Ind.
Filed Aug. 6, 1971, Ser. No. 169,628 Int. Cl. C121 1/02 US. Cl. 195-127 4 Claims ABSTRACT OF THE DISCLOSURE Microbiological chamber apparatus having a closed top Wall, a bottom wall and sidewalls and having at least one sealable sidewall access port, which apparatus is useful, for example, to produce a tissue culture directly on a base, such as a microscope slide, for subsequent examination and storage, comprises in combination a base which forms the bottom of said chamber and a receptacle removably adhesively attached in liquid-impermeable contact to the base, such receptacle forming the other walls of the chamber. In one use of this apparatus, a liquid tissue culture medium is placed through the access port into the receptacle chamber in contact with the base, the medium is incubated to allow the tissue culture to grow and to attach such growth to the base. The liquid medium is then removed from the chamber, and the receptacle is removed from the base. The tissue culture growth on the base can then be treated as desired and microscopically examined. This apparatus is also useful as an anaerobic chamber or as a blood culture chamber for microbiological assays.
BACKGROUND AND PRIOR ART It is frequently desirable in medical laboratory practice as well as in biological research to grow various bacteria, cells or tissues in particular media and then to examine the resulting growth. A particularly major use of this technique is in virology laboratories where host cells are grown and then used to detect viral activity.
In general, the prior art tissue cultures have been obtained in the following manner. A known measured amount of liquid growth medium containing a suspension of the cells to be grown was placed in a sterilized glass test tube. The tube was then sealed with a non-toxic stopper and placed on its side in an appropriate rack and oriented so that the area of cell attachment and growth was always immersed in a nutritional fluid environment. The tube was then incubated until a complete cell monolayer was grown. The tube was then examined microscopically to'check for growth and then again to check for any cytopathologic changes induced by any experimental treatment during such growth.
In order to obtain a permanent stained slide of any such cytopathologic changes which occurred during the above growth, a second cell culture must be prepared. A cleaned glass coverslip was placed in a specially modified glass tube and a portion of liquid growth medium containing a suspension of the cells to be grown was placed in the tube in contact with the coverslip. The tube was then sealed and incubated as described above to grow a cell monolayer on the coverslip. The same experimental treatment was repeated to effect the same cytopathologic changes. When the desired cell growth and changes were accomplished, the coverslip was removed from the tube, rinsed, fixed and stained. The stained coverslip was then mounted on a microscope slide for subsequent examination and storage.
This prior art technique required many manipulative steps and complex apparatus in order to produce stained tissue cultures on microscope slides.
In addition to the production of tissue cultures, there are other microbiological procedures which are carried out in chambers using complex and cumbersome prior art apparatus. In such procedures it was difficult using prior art apparatus to add materials to the contents of a reaction chamber or to withdraw materials during the reaction.
It is an object of the present invention to provide improved microbiological chamber apparatus.
SUMMARY OF THE INVENTION In accordance with the present invention, microbiological chamber apparatus useful for carrying out biological reactions or growth therein, such as growing tissue cultures, is provided which comprises in combination a base member and a cooperable receptacle having sidewalls, a top wall and an open bottom opposite from said top wall surrounded by a bottom surface, said bottom surface being removably mated in liquid-impermeable contact with said base, whereby said base member forms the bottom wall, said sidewalls of said receptacle form the sidewalls, and said top wall of said receptacle forms the closed top wall of said microbiological chamber apparatus, said receptacle having at least one scalable sidewall access port.
DESCRIPTION OF THE DRAWINGS FIG. 1 is a partially-exploded perspective view with a portion removed of one embodiment of the present in- 'vention;
FIG. 2 is an enlarged plan view of the underside of the receptacle portion of FIG. 1;
FIG. 3 is a vertical cross-section view taken along line 3-3 of FIG. 1 showing a liquid growth medium contained within the apparatus; and
FIG. 4 is a side elevation view of another embodiment of the apparatus of the present invention.
DESCRIPTION OF THE INVENTION Referring to FIG. 1, the microbiological chamber apparatus 10 of the present invention comprises in part a base member 12 having a planar upper surface 13 and opposing end faces 15 and 17 which are normal to surface 13. A box-like receptacle 14 is located near end face 17 and mates against surface 13 of base member 12 so as to leave a substantial portion 19 of surface 13 exposed near end face 15. This free surface area 19 can be etched so as to render it capable of receiving appropriate identification markings, or an appropriate label can be atfixed to this free surface area.
Base member 12 is preferably a glass microscope slide when the microbiological reaction or growth chamber apparatus 10 is intended to be used for tissue culture. Such microscope slide is preferably fabricated from soda glass which has been cleaned to remove oils, greases, surfactants, abrasives or other materials inhibitory to biological or bacterial growth. Upper surface 13 of base member 12 must be reasonably fiat to form a mating surface for receptacle 14. Glass microscope slides useful in the present invention are marketed by the Erie Scientific Co. under the designations 'ESCO No. 2955-1 or No. 3A. It is understood that other materials, such as sintered alumina, organoplastics and the like, can also be used for the base member 12 for biological reaction chamber apparatus of the present invention, providing such materials are appropriately cleaned and do not inhibit the desired biological reaction or growth.
Receptacle 14 is formed with a top wall 30, opposing longitudinal sidewalls 16 and 18 and opposing transverse sidewalls 20 and 22 defining an open bottom 21 therewithin at the base thereof. At its base, receptacle 14 is formed with a peripheral outwardly directed flange 24 surrounding the bottom opening 21. Flange 24 has a bottom surface 40 shown in FIG. 2, as well as end faces 26 and 28. The opposing sidewalls of receptacle 14, as shown in FIG. 1, are preferably inclined inwardly toward each other at an angle of about from the vertical. One wall of recptacle 14, for example wall 20, is formed with an access port from which conduit 32 extends. Conduit 32 has external threads 34 which are capable of threadably mating with internal threads (not shown) of removable cap 36 for sealing the access port. Other suitable convenient means can also be used to removably seal the outer end of conduit 32.
Receptacle 14 also has an injection port formed by conduit 38 located in peripheral flange 24 near end face 28. The internal passage 39 of conduit 38 extends through flange 24. As shown in FIG. 2, the bottom surface 40 of flange 24 is formed with a rectangular groove 42 which is in general alignment with the sidewalls 16, 18, 20 and 22 of the receptacle. The internal passage 39 of conduit 38 communicates with groove 42 as shown.
Receptacle 14 is preferably formed from transparent organoplastic materials. Useful organoplastic materials are polystyrene, polypropylene, celluloid, polymethacrylate, polymethylmethacrylate and the like.
In the fabrication of the microbiological chamber apparatus of the present invention, the bottom surface 40 of receptacle 14 is mated against surface 13 of base member 12 near end face 17 and clamped thereto by suitable clamping means (not shown), the mating surfaces having been previously cleaned. An adhesive gasket material in liquid or slurry form is then injected through passage 39 of conduit 38 to fill groove 42 with such material. The gasket material is then allowed to solidify slowly at ambient temperature, or more rapidly under heating con ditions, to form an adhesive gasket 44 (shown in FIGS. 2 and 3) which provides a liquid-impermeable seal between the receptacle 14 and the base 12 at its points of contact therewith. When the injected gasket material 44 has solidified, the clamping means can be removed, since the receptacle 14 is adhered to the base member 12 by means of the adhesive gasket 44. When desired at a subsequent time, the receptacle can be easily removed from the base by pulling it away from the base.
A preferred gasket material is an organopolysiloxane elastomer. An especially useful gasket material is an organopolysiloxane elastomer composition marketed by the General Electric Co. under the designation 'RTV 630. This material is further described in US. Pat. No. 3,436,- 366. It is further preferred to remove entrapped air from the above RTV 630 material before it is used to form the gasket. This is conveniently accomplished by placing the material in a desiccator and evacuating it to a vacuum of about 10-15 in. of mercury for about 20-30 min.
Other useful gasket materials are microcrystalline waxes and various synthetic organic elastomers. The principal criteria for a useful gasket are that it provide desired removably adhesive characteristics between the receptacle 14 and the base 12, provide a liquid-impermeable seal, be non-toxic to the biological material subsequently employed in the apparatus, and not act as a source of growth for undesirable microorganisms.
While it is preferable to form the gasket in the above described manner, it is also suitable to coat bottom surface 40 of flange 24 with the suitable gasket material and then press the coated receptacle into contact with surface 13 of base 12 to achieve the desired adhesive and sealing conditions.
After fabrication of the microbiological chamber apparatus, it is then sterilized in any well-known manner, and the access port conduit 32 is then sealed with a sterile cap 36 or other means. The apparatus can then be stored until ready for use.
The previously sterilized microbiological chamber apparatus of the present invention is employed in the following manner to grow tissue cultures, for example. The cap 36, or other sealing means, is removed from conduit 32. The desired liquid tissue culture medium 46 (FIG. 3) containing a suspension of cells to be grown is then placed into the receptacle. As shown in FIG. 3, the adhesive gasket 44 forms a liquid-impermeable seal between the receptacle 14 and the base 12 to prevent any leakage from the apparatus. Conduit 32 is then resealed by re placing cap 36, for example, in threaded engagement with threads 34. Appropriate identification markings are then entered on the free surface area 19 of base 12 to describe the contents of the receptacle chamber. The apparatus is then placed in a suitable incubator and is incubated under well-known conditions to carry out the tissue culture growth. If desired, suitable treatment may be carried out on the cells and medium during this growth to achieve cytopathologic changes in the cells. Various reactants can be introduced through conduit 32, and samples can be withdrawn through conduit 32. As shown in FIG. 3, at the conclusion of the growth period a mass of tissue cells 48 is attached to base 12. The tissue culture medium can then be removed from the chamber by aspiration, for example, through conduit 32, and the receptacle 14 is then removed from the base 12. The mass of tissue cells attached to the base is then rinsed and fixed on the base, and the alfixed tissue culture is then treated with an appropriate stain to stain the culture.
The stained culture can then be microscopically examined, and the base 12 with the culture attached can be stored for further use. This provides a permanent record of the results of the tissue culture growth which can be produced through use of the apparatus of the present invention in a far simpler and more eflicient manner than through the use of the techniques employed in the prior art.
A further apparatus embodiment of the present invention is shown in FIG. 4 wherein the receptacle 14' is provided with two access ports. Elements in FIG. 4 corresponding to similar elements in FIG. 1 have the same identification numbers primed. A conduit 32' projects from transverse sidewall 20' and is sealed by removable cap 36. A conduit 50 projects from and communicates through opposing transverse sidewall 22'. Conduit 50 is sealed by removable cap 52. In this form of the apparatus the injection port conduit 38' is conveniently located on flange 24' adjacent to longitudinal sidewall 16'. If desired a second injection port (not shown) can be located on flange 24' adjacent to the opposite sidewall (not shown). As shown in FIG. 4, the conduits 32 and 50 are substantially normal to the inwardly inclined sidewalls 20 and 22 and thus are both tilted upward with respect to the flange 24'. This assists in transferring material through these access ports.
This embodiment of the apparatus has the advantage of providing an access port at each end of the receptacle so that materials can be introduced or withdrawn from a specific location with minimal contamination to the entire contents of the receptacle chamber.
While access ports are shown in specific walls of the receptacles illustrated, it is understood that access ports may be positioned in other locations on the receptacle as suitable or desired. Moreover, though the receptacles described above all have rectangular-shaped cross-sections, it is understood that apparatus of the present invention can employ receptacles having other cross-sectional shapes, such as circular, for example.
What is claimed is:
1. Microbiological chamber apparatus comprising in combination a flat base member and a cooperable receptacle having sidewalls with at least one scalable sidewall access port, a top wall and an open bottom opposite from said top wall surrounded by an annular flange extending outward from said sidewalls having a bottom surface coplanar with said open bottom, said bottom surface being formed with a groove means therein containing an adhesive gasket, said bottom surface being removably mated through said adhesive gasket in liquid impermeable contact with said flat base member.
2. Apparatus according to claim 1 wherein the cooperable receptacle contains an injection port which communicates with said groove means and through which gasket material is injected to form the adhesive gasket in good groove means.
3. Apparatus according to claim 1 wherein the opposing sidewalls of the receptacle are inclined toward each other.
4. Apparatus according to claim 3 wherein the opposing sidewalls are each inclined inward at an angle of about 10 from the vertical.
References Cited UNITED STATES PATENTS Bazil 195-142 Orst 88-40 Henderson 195-139 Newby 206-1 Lamal 350-95 Andelin 195-139 A. LOUIS MONACELL, Primary Examiner US. Cl. X.R.