Luminescence test and exposure apparatus

Abstract: Apparatus for performing and measuring chemical reactions includes a reaction test apparatus having reaction wells wherein reactants are controllably mixed, and exposure apparatus which receives and positions the reaction test apparatus adjacent a photographic film. Each of the reaction wells includes at least two reaction chambers, arranged in a side by side fashion. All but the final reaction chamber have upwardly sloping sides, so that liquid placed in one reaction chamber can flow to the next reaction chamber when the apparatus is tilted. In a preferred embodiment, the reaction wells are supported in a plate that is structurally integral with the wells but separates the wells from each other. The test plate is retained in the exposure apparatus, and liquid is controllably flowed from one reaction chamber to the next by tilting the exposure apparatus. The apparatus of the invention is particularly suited for measuring reactions that produce luminescence of short duration, as the reactants can be conveniently mixed in darkness, while the film is being exposed.

Description: This invention relates to a reaction test apparatus for assessing reactions by the production of luminescence according to claims 1 and 2, to an apparatus for conducting luminescence reactions tests according to claim 19 and to a method for assessing reactions according to claim 22. In several types of chemical and medical test procedures, a liquid such as a body fluid must be reacted with individual reactants in a sequence of related but separate chemical reactions, and then the final product analyzed. Traditionally, such procedures have been performed by placing the fluid into a reaction tube or the like, adding the remaining reactants for the first reaction, and permitting the first reaction to proceed to completion. The further reactants for the second reaction are added, and the second reaction is permitted to proceed to completion. This stepwise operation can be repeated as many times as necessary, until a final reaction product is obtained and analyzed. Certain types of testing procedures do not lend themselves to such wet chemical techniques where the test must be performed in a field location. One example is a test procedure wherein the final reaction product is analyzed by a luminescence reaction. In this type of procedure, the amount of a final product is indicated by the amount of light produced in a light producing reaction, one of whose reactants is the final product of one or more prior reactions. An example of such a testing procedure is the detection of harmful bacteria in the urine of persons by luminescence, termed a bacteriuria test. The luminescent test for bacteriuria can be performed by conventional wet chemical procedures such as previously described, but in this form may be subject to errors of several types because light production starts almost immediately after the final reagents are mixed together where the test is positive. There is therefore a need for an apparatus and test protocol for performing qualitative or semi-quantitative tests for bacteriuria by the luminescent analysis procedure. The apparatus must permit controlled sequential chemical reactions, and specifically must permit removal of non-bacterial ATP prior to the release of bacterial ATP for luminescent detection. The apparatus should be sufficiently simple that it can be used by technicians with little training, should serve to minimize errors due to chemical deterioration or technician error, and should be inexpensive so that tests can be performed economically. The present invention fulfils this need, and further provides related advantages. In the document DE-A-1 220 083 a reaction test apparatus comprising some of the features of the preamble of the present invention's independent claims is described. In this reaction test apparatus the individual wells of the reaction test apparatus are not optically separated, so that from a well, in which a strong reaction occurs, light may be transmitted to the adjacent wells. In the EP 0 106 662 a non-fluorescent vessel for holding test samples in fluorescent assays is disclosed. The vessel for holding test samples to be measured is formed from a material having a native fluorescence. It is provided with dispersing particles for a barrier which blocks penetration or at least reduces the extent of penetration of an exciting light into the vessel's fluorescable material to prevent fluorescent excitation of the vessel or at least reduces the extent to which the vessel is fluorescently excited when exposed to an exciting light during the fluorometric measurement. The GB 2 062 674 A discloses a tray for identifying microorganisms, the tray comprises several test wells with two reaction chambers arrenged in line one after the other. SUMMARY OF THE INVENTION It is therefore an object of the invention to provide a reaction test apparatus and a method of the above mentioned kind in which the problem of a poor recording of produced light in the final reaction chamber is solved. According to the invention this problem is solved by a reaction test apparatus according to claims 1, 2 and 22. Furthermore, the present invention is embodied in an apparatus according to claim 19 being specifically adapted for the reaction test apparatus of claim 1 or 2. The luminescence is recorded on photographic film for further analysis. The direct contact of the bottom of the final reaction chamber and the film material guarantees an exact recording of the light emitted from the reaction. The apparatus thus provides excellent flexibility of use, and is adapted for use either in a busy laboratory or at remote sites. Practice of the invention involves two separate but interrelated pieces of apparatus. The chemical reactions are performed within the reaction test apparatus of claim 1 or 2, which in turn is contained within the exposure apparatus of claim 19 which controls operation of the reaction test apparatus and records light output. The invention provides a reaction test apparatus in which two or more chemical reactions may be performed sequentially. The final reaction results in production of light by luminescence, depending upon the test results. Reaction wells with multiple chambers are provided having pour plates in which chambers have sloping interior walls are used, so that the act of tilting the pour plate moves reactants to the next stage of the process. The inventive features ensure that care is taken to minimize the likelihood that light can stray from one test well to another, which could lead to erroneous results. The invention also provides an exposure apparatus in which the reaction test apparatus is placed, to record on photographic film the luminescence produced by each test well. In the preferred bioluminescent bacteriuria analysis, a urine specimen is added to the first reaction chamber containing reactants to release non-bacterial ATP into solution and to eliminate the non-bacterial ATP from solution. This release and elimination reaction typically requires about 10 to 60 minutes to complete. The urine treated to completion in the first reaction chamber, containing only ATP within bacteria, then is flowed to the final reaction chamber by tilting the apparatus so that the liquid flows along the surface and into the final reaction chamber. It is necessary that the elimination of non-bacterial ATP be completed before beginning the reaction in the final reaction chamber, or erroneous results will be obtained. In the final reaction chamber the bacterial ATP is released and reacted with light-producing reagents which react with free ATP to create luminescence in an amount related to the prescence of bacterial ATP in the original sample, a reaction typically requiring only about 30 seconds. An important feature of the preferred embodiment of the present invention is the provision of certain key reactants in a solid form. The apyrase enzyme and somatic release reagent packaged in the first reaction cup are provided in a solid form, as are the light producing reactants, bacterial release reagent, bovine serum albumin, and buffering agent in the final reaction cup. These reactants normally deteriorate in effectiveness when stored in a liquid or liquid solution form for extended periods of time. Providing these reactants in a solid form has the important advantage of prolonging their lives in the absence of refrigeration or other means for reducing deterioration, and also eliminates the possibility of spilling. It will be appreciated that the present invention provides a highly flexible system for conducting sequential chemical reactions and then measuring the results of the reactions with a light producing reaction. The apparatus is readily provided in a kit form, with the exposure apparatus being reusable indefinitely. The test can be performed by relatively inexperienced personnel, as all reactants except the test specimen are prepackaged into the reaction test apparatus. The resulting exposed film provides a permanent record of the test results, which can be evaluated either by the technician or a doctor. The testing procedure yields qualitative or semi-quantitative results particularly useful in a screening of specimens, and can be economically used in central or remote laboratory facilities with equal ease and identical results. Complete screening of specimens is often accomplished within less than an hour, so that the results are available before the patient leaves the doctor's office. Other features and advantages of the invention will be apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a first embodiment of a reaction test apparatus, which does not form a part of the invention; Figure 2 is an exploded perspective view of the reaction test apparatus of Figure 1; Figure 3 is a side sectional view of the reaction test apparatus of Figure 1, taken along line 3-3; Figure 4 is a fragmented side sectional view like that of Figure 3, except illustrating an alternate configuration of the lower reaction cup, which does not form a part of the invention, too; Figure 5 is a fragmented side sectional view of a reaction test apparatus having three reaction cups in each test well; this test apparatus being no part of the invention; Figure 6 is a perspective view of the exposure apparatus used in conjunction with the reaction test apparatus of Figure 1, opened to illustrate the interior structure; Figure 7 is a side sectional view of the exposure apparatus of Figure 6, taken generally along line 7-7; Figure 8 is an end sectional view of the exposure apparatus of Figure 6; Figure 9 is a perspective view of the exposure apparatus of Figure 6, with the top cover broken away to illustrate the internal mechanism; Figure 10 is a perspective view of a detail of the locking mechanism of the exposure apparatus; Figur 11 is a perspective view of an embodiment of the reaction test apparatus; Figure 12 is a side elevational view of the reaction test apparatus of Figure 1; Figure 13 is a perspective view of a variant of the reaction test apparatus of Figure 1; Figure 14 is side elevational view of another variant of the reaction test apparatus of Figure 1; Figure 15 is a side elevational view of a portion of the pour plate support and two adjacent wells; and Figure 16 is a perspective view of an exposure apparatus used in conjunction with the reaction test apparatus of Figures 11 to 14, opened to illustrate the interior structure. Figures 1 to 10 show embodiments which do not form a part of the invention but which are helpful for understanding the invention, embodiments of which are shown in figures 11 to 16. One form of a reaction test apparatus 20 is illustrated in Figures 1-5, and a cooperative exposure apparatus 22 is illustrated in Figures 6-10. The cooperative relationship between the reaction test apparatus 20 and the exposure apparatus 22 is illustrated in Figures 7 and 8. As shown therein, the reaction test apparatus 20 is operated by placing it within the exposure apparatus 22, closing the exposure apparatus 22, and operating the externally extending controls of the exposure apparatus 22 to effect and record chemical reactions occurring within the reaction test apparatus 20. Figure 1 illustrates generally the reaction test apparatus 20, while Figure 2 illustrates the apparatus 20 in exploded view and Figure 3 illustrates the interrelationship of the elements within the apparatus 20. The reaction test apparatus 20 includes an upper plate 24 and a lower plate 26 which include a plurality, preferably 12, of test wells 28. The upper plate 24 and the lower plate 26 are preferably each one-piece molded plastic structures, although it would be possible to use an alternate structure wherein the test wells 28 were fabricated as separated elements and pressed into bores in the plates 24 and 26. As illustrated in Figures 2 and 3, each test well 28 includes an upper reaction cup 30 and a lower reaction cup 32. One upper reaction cup 30 for each test well 28 is molded into the upper plate 24, and one lower reaction cup 32 for each test well 28 is molded into the lower plate 26. The shape of the upper plate 24 is defined, and the upper reaction cups 30 are held in position, by an upper web 34 that forms the skeleton of the structure of the upper plate 24. Similarly, the shape of the lower plate 26 is defined, and the lower reaction cups 32 are held in position, by a lower web 36 that forms the skeleton of the structure of the lower plate 26. Each cup 30 and 32 has an open top and sides that taper inwardly from the top to the bottom of the cup. The inward taper permits the upper reaction cups 30 to be nested within the lower reaction cups 32 during assembly of the reaction test apparatus 20. The two plates 24 and 26 are dimensioned to be otherwise conformable. The reaction test apparatus 20 can therefore be assembled in the manner illustrated in Figure 2, by slipping each upper cup 30 downwardly into the corresponding lower cup 32, and then joining the upper plate 24 to the lower plate 26, either permanently or releasably. Preferably, the plates 24 and 26 are releasably joined, as an aid in assembly of the apparatus 20. The joining mechanism includes a pair of molded catches 38, one on each end of the lower plate 26. Each catch 38 is a tab having a tapered upper end extending upwardly from the outside end of the lower web 36, and is dimensioned to be slightly flexible. The upper plate 24 is releasably secured to the lower plate 26 by slipping it downwardly into secure contact with the lower plate 26, so that the catch 38 on each end captures and snaps closed over the respective end of the upper web 34. The upper plate 24 can be disassembled from the lower plate 26, if desired, by manually pulling each catch 38 outwardly and lifting the upper plate 24 upwardly. As more fully illustrated in Figure 3, the upper reaction cup 30 has an orifice 40 in the bottom thereof. The orifice 40 is closed by a removable plug 42, preferably a small amount of chemically inert silicone laboratory grease. An increased internal pressure within the upper reaction cup 30, as controllably applied by the exposure apparatus 22, causes the plug 42 to be ejected. Ejection of the plug 42 opens the orifice 40, so that any liquid therein falls downwardly into the lower reaction cup 32, which is positioned directly below the upper reaction cup 30 in their nesting relationship. In some instances, surface tension of the liquid in the upper reaction cup 30 is sufficient to prevent it from flowing through the orifice 40, as illustrated at numeral 42a of Figure 3. In those instances, no plug 42 is provided. (In Figure 3, the two leftmost upper reaction cups 30 are illustrated with the plugs 42 in place, while the two rightmost upper reaction cups 30 are illustrated without plugs.) The lower reaction cup 32, also illustrated in Figure 3, has no orifice therein, but instead has a flat bottom 44 adapted to be placed in close contact with the surface of a photographic film. In another embodiment illustrated in Figure 4, the lower wall of the lower reaction cup 32 is further tapered into a flat bottomed tubular section 46, to achieve direction of the light produced by a chemical reaction within the cup 32 into a smaller area as defined by the size of the bottom of the tubular section 46, and thereby increase its intensity within that area. In typical use of the reaction test apparatus 20, chemical reactants are placed into the reaction cups 30 and 32 prior to shipment to customer laboratories. To retain the chemical reactants in the cups 30 and 32, and to prevent their contamination, a foil cover 48 is preferably sealed over the top of the upper plate 24. In use, the foil cover 48 is removed or punctured when a particular test well 28 is used, and this physical disruption of the foil cover 48 serves as an indicator of which test wells 28 have been used. In another embodiment illustrated in Figure 5, a reaction test apparatus 50 contains an upper plate 24 and a lower plate 26, which are preferably identical to those previously described, and in addition a top plate 52. The top plate includes a plurality of top reaction cups 54, supported by a top web 56. The top reaction cups 54 are conformably arranged to nest with the upper reaction cups 30 in the upper plate 24. With the use of this embodiment in mind, the upper plate 24 is fabricated with an upwardly projecting lip 58. The top web 56 is dimensioned to fit snugly within the bounds of the lip 58 and be retained therein. As illustrated in Figure 5, the top plate 52, upper plate 24, and lower plate 26 are conformably nested and retained together in the manner described. The top reaction cup 54 has an orifice 60 in the bottom thereof similar to that in the bottom of the upper reaction cup 30, so that removal of a plug 62 from the orifice 60 allows liquid to flow out of the top reaction cup 54 into the upper reaction cup 30. The addition of the top reaction cup 54 permits another chemical reaction or procedure to be performed prior to the reaction in the upper reaction cup 30, and prior to the reaction in the lower reaction cup 32. As is apparent, the principle of multiple reaction cups can be extended to four, five, or more vertically aligned reaction cups in a single reaction test apparatus. The reaction test apparatus 20 or 50 is preferably used in conjunction with the exposure apparatus 22 illustrated in Figures 6-10. As illustrated in Figure 6, the exposure apparatus 22 includes a lower housing 64 and an upper housing 66 which is hingedly joined along one side to the lower housing 64. The hinged arrangement permits the upper housing 66 to be closed with the lower housing 64 to form a light tight container, and to be opened to insert a reaction test apparatus therein in the manner to be described. The sectional view of Figure 7 shows the apparatus 22 with the upper housing 66 in the closed position and a reaction test apparatus 20 in place therein. The upwardly facing portion of the lower housing 66 is a fixed shutter 68 having a plurality of openings 70 therethrough. The positions of the openings 70 are selected to correspond to the positions of the test wells 28 of the reaction test apparatus 20, so that each test well 28 is positioned directly above one of the openings 70. A collar 72 surrounds and projects upwardly from each opening 70. The collars 72 are cylindrical and have inner diameters sufficiently large that the test wells 28 fit slidably within the collars 72. The cooperative engagement between the test wells 28 of the reaction test apparatus 20 and the collars 72 of the exposure apparatus 22 permits the reaction test apparatus 20 to be comfortably engaged into the exposure apparatus 22. A biasing arrangement on the upper surface of the fixed shutter 68 resists the downward movement of the reaction test apparatus 20 when it is placed into contact on the top of the fixed shutter 68. The biasing arrangement preferably includes a pair of biasing springs 73, on opposing sides of the upper surface of the fixed shutter 68. The biasing springs 73 are preferably leaf springs, as illustrated, or may be coil springs or any other suitable type. The biasing springs 73 cooperate with other portions of the mechanism in translation of the reaction test apparatus 20 within the exposure apparatus 22, in a manner to be described subsequently. A movable shutter 74 is slidably disposed below the fixed shutter 68, and can be moved relative to the fixed shutter with an external shutter handle 76. In the preferred embodiment, the movable shutter 74 has a plurality of openings 78 therethrough. The openings 78 have the same pattern and spacing as the openings 70 in the fixed shutter 68, but each of the openings 78 is physically offset from its corresponding opening 70 in the direction of the movement of the movable shutter 74. Consequently, when the movable shutter 74 is closed, the movable shutter 74 blocks the openings 70, and there is no continuous light path through the two shutters 68 and 74. When the movable shutter 74 is opened by grasping the handle 76 and pulling the movable shutter 74 outwardly, at the full extension the openings 78 line up with the openings 70, and there is a line of sight path through the two shutters 68 and 74 at each of the openings 70 and 78. This preferred approach is for a short throw design for the movable shutter 74. Alternatively, the movable shutter can be made a solid piece, requiring that it be withdrawn fully to create a line of sight path. Within the lower housing 64 below the movable shutter 74 is a piece of photographic film 80, which is exposed to the test wells 28 when the movable shutter 74 is opened to align the openings 70 and 78. Preferably, the film 80 is of the instant developing type, and there is provided an instant processing film pack 82 to accomplish the developing of the film 80. The preferred type of film 80 has an ASA rating of 20,000, which is available in packs of eight instantly processing prints from Polaroid Corporation as its Type 612 film. The film pack 82 is self contained, and need only be inserted into the conforming lower housing 64 with the film 80 below the movable shutter 74. The upper housing 66 includes a mechanism for applying pressure to the tops of the test wells 28 when the exposure apparatus 22 is closed. An arched rubber diaphragm 84 is positioned above the location of each of the collars 72, as determined when the apparatus 22 is closed, but separated from the collars so that the diaphragms 84 contact and cover the tops of the test wells 28 when the apparatus 22 is closed with a reaction test apparatus 20 in place. The rubber diaphragms 84 are mounted on a diaphragm support 86, which has a pressure bar 88 portion thereof extending through the top of the upper housing 66 through a light-tight seal. The pressure bar 88 is supported on the diaphragm support 86 by rods 130. Coil springs 134 and 136 position the diaphragm support 86 so that the diaphragms 84 are loosely in contact with the tops of the upper reaction cups 30, until an external downward force is applied to the pressure bar 88. The upper spring 134 reacts between the pressure bar 88 and a portion of the housing 66, indicated by numeral 132. The lower spring 136 reacts between the portion of the housing 132 and the diaphragm support 86. The lower spring 136 normally has a higher spring constant than the upper spring 134, to restore the position of the pressure bar 88 after operation is complete. A downward force applied to the pressure bar 88 against the resistance of the spring 134 brings the diaphragms 84 into close, sealing contact with the tops of the upper reaction cups 30, and continued downward force compresses the air inside the diaphragms 84 and the upper reaction cups 30. A sufficient downward force builds up sufficient pressure within the upper reaction cups 30 to pop out the grease plug 42 from the bottom of the orifice 40, so that liquid in the upper reaction cups 30 can flow through the orifice 40 and into the lower reaction cups 32. Where there is no grease plug 42 because surface tension of the liquid is sufficient to prevent liquid from otherwise flowing through the orifice 40, the downward pressure is sufficient to overcome the surface tension and force the liquid of the test specimen downward through the orifice 40. The diaphragms 84 provide a structurally simple, reliable method for moving the liquid reactants from the upper reaction cup 30 into the lower reaction cup 32. The luminescent chemical reactions that produce the light recorded on the film 46 produce relatively faint light, and several techniques are utilized to use that light as effectively as possible. The tubular sectioning 46 of the lower reaction cup 32, discussed previously, is one such technique. A very fast film, such as the ASA 20,000 film previously discussed, is typically used. Another technique to utilize the light most fully is to contact the bottom 44 of the lower reaction cup 32 directly to the film 80 during exposure. This can be accomplished by translating the reaction test apparatus 20 downwardly to bring the bottom 44 of the lower reaction into contact with the film 80, after the movable shutter 74 is opened. An externally operable translation mechanism is provided for this purpose. The translation mechanism includes a rod 92 mounted horizontally through the center of the upper housing 66 and fixed at each end in a bearing surface, so that the rod 92 can be rotated. The rod 92 extends outside of the upper housing 66 through a light-tight baffle, and ends in a translation control lever 94. Inside the upper housing 66, the rod has a cam lobe 96 on each end thereof, positioned over the top of the reaction test apparatus 20. Operation of the lever 94 rotates the cam lobe 96 into contact with the top of the reaction control apparatus 20, and continued rotation forces the reaction control apparatus 20 downwardly toward contact with the top of the fixed shutter 68, against the biasing force of the biasing springs 73. At full operation of the cam lobe 96, the reaction test apparatus 20 is forced downwardly sufficiently far that the bottoms 44 of the lower reaction cups 32 directly contact the film 80. This downward movement of the reaction test apparatus 20 is done only when the movable shutter 74 is opened, so that the lower reaction cups 32 can be contacted to the film 80. It is in this position that the exposure pressure bar 88 is operated and the exposure of the film 80 is completed. After exposure is complete, the lever 94 is operated in the reverse direction to rotate the cam lobe 96 to release the pressure on the reaction control apparatus 20, so that the reaction control apparatus 20 moves upwardly under the influence of the biasing spring 73. With the apparatus 20 in its upward position, the movable shutter 74 can be closed. As is apparent, the movable shutter 74, lever 94, and pressure bar 88 must be operated in the correct sequence for the exposure of the film to be properly completed. To reduce the chances for operator error, an interlock system has been incorporated into the exposure apparatus 22 to prevent an incorrect sequence of operations. The interlock system is best seen by reference to Figures 7-10. The shutter handle 76 projects from the side of the lower housing 64 in a position to contact a side of the translation control lever 94 in the manner illustrated in Figure 9. The control lever 94 cannot be rotated to translate the reaction test apparatus 20 downwardly unless the shutter handle is moved to the outward position to align the openings 70 and 78. The shutter handle 76 is attached to the shutter 74, which cannot be moved unless the upper housing 66 is closed to the lower housing 68 to form a light-tight seal therewith, due to an interlock between the two parts. When the shutter handle 76 is operated to open the shutter 74, a notch 110 in the handle 76 engages a tab 112 on the upper housing 66 to lock the upper housing 66 closed, thereby preventing its inadvertent opening at any time at which the film is exposed. The slide interlock, illustrated in Figure 10 in a reverse angle view, is effected by a slide lock 116 mounted in the lower housing and biased upwardly by a spring 120. In the locked position illustrated in Figure 10, a locking tab 118 on the slide lock 116 engages a conforming recess in a locking rail 114 that projects upwardly from one side of the movable shutter 74, preventing movement of the shutter 74. An extension 122 extends upwardly from the top of the slide lock 116, through an opening in the housing 64. The wall of the upper housing 66 depresses the extension 122 when the upper housing 66 is closed, thereby depressing the slide lock 116 against the force of the spring 120 to unlock the shutter 74. As the shutter handle 76 is moved to open the shutter 74 after being so unlocked, the upper housing 66 is locked closed by the engagement between the shutter handle 76 and the tab 112. After the film exposure is complete, the housing 66 can be opened only when the shutter 74 is returned to its closed position by operation of the handle 76, where it is again locked closed by the slide lock 116. This same movement of the handle 76 unlocks the upper housing 66 so that it may be opened, by disengagement of the tab 112. Mounted on the rod 92 is a pressure bar locking cam 98. The cam 98 is a part-circle cam that contacts the bottom side of the diaphragm support 86 when the translation control lever 94 is in the position that the reaction test apparatus 20 is in its upward position. Thus, in this position, the pressure bar 88 is locked into place and cannot be inadvertently operated. When the translation control lever 94 is operated to force the reaction test apparatus 20 downwardly, the locking cam 98 is rotated away from the diaphragm support 86, and the pressure bar 88 (connected to the diaphragm support 86) can be operated. After exposure of the film 80, the translation control lever 94 is operated to raise the reaction test apparatus 20, again locking the diaphragm support 86 in place. With the translation control lever 94 in this position, the shutter handle 76 can again be operated to close the shutter 74 and unlock the upper housing 66, so that the reaction test apparatus 20 can be opened. This interlock mechanism prevents an incorrect sequence of operations of the external control apparatus. In the most preferred use, the present invention is used for analysis of the bacteriuria content of urine samples. This test depends upon the reaction of bacterial ATP with luminescent reagents, to produce light that is measured on the film 80. There are two sources of ATP in a urine sample, bacterial and non-bacterial sources. The objective of the preferred bacteriuria test is to measure bacterial sources only. In the preferred test, the ATP in non-bacterial sources is first removed so that it cannot adversely affect the test results, and then the ATP in bacterial sources is released and reacted with the luminescent reagents. In this test procedure, the upper reaction cup 30 contains a release reactant to lyse somatic cells, releasing non-bacterial ATP therefrom. The release reactant does not affect the bacterial ATP, which remains bound to the bacteria. The release reactant is Triton X100 detergent (polyoxyethylene ether). The upper reaction cup 30 also contains an enzyme elimination reactant (preferably apyrase) to dephosphorolate the non-bacterial ATP to adenosene monophosphate, also known as AMP, which is not measured by the subsequent luminescent assay. The preferred elimination reactant is apyrase enzyme. To prepare the reagent used in the upper reaction cup 30, about 0.16 units of solid apyrase enzyme and about 100 microliters of 0.2 percent aqueous solution of Triton X100 detergent liquid are mixed with 0.5 milligrams of bovine serum albumin. This mixture is added to the upper reaction cup 30 and freeze dried. Upon completion of the reaction between the non-bacterial ATP in the urine specimen with the reactants in the upper reaction cup 30, the reacted urine specimen is passed through the orifice 40 into the lower reaction cup 32 for the second reaction. In the lower reaction cup 32, the bacterial ATP is released, and reacted with luminescent reagents. The result is light produced in proportion to the bacterial ATP, with the amount of light measured indicating the amount of bacterial ATP present in the urine specimen. To prepare the solid material for the lower reaction cup, the following ingredients are mixed together: about 100 microliters of a 0.005 percent to 0.6 percent concentration aqueous solution of a chemical bacterial releasing agent (such as polyoxyethylene ether, hexachlorophene, chlorohexadine, or dimethylsulfoxide), about 10 to 100 micrograms of firefly luciferase enzyme, about 15 micrograms of solid D-luciferin, about 10 microliters of a 10 millimolar magnesium chloride luminescent cofactor, about 0.5 milligrams of bovine serum albumin, and a N-2-Hydroxyethylpiperadine-N'-2-ethanesulfonic acid buffer to bring the pH of the solution to 7.75. The solution is added to the cup 32 and freeze dried. Urine specimens are then tested in the manner discussed previously. The urine sample is added to the upper reaction cup 30 and reacted therein for a period of time sufficient to eliminate all non-bacterial ATP, which usually requires about 1-10 minutes. The reaction test apparatus 20 is placed into the exposure apparatus 22, the apparatus 22 is closed to form a light-tight enclosure, and the shutter handle 76 is operated to lock the apparatus 22 closed and open the movable shutter 74. With the shutter handle 76 moved to the position with the shutter 74 opened, the interlocked translation control lever 94 is operated to force the reaction test apparatus 20 downwardly to place the bottom 44 of the lower reaction cup 32 in contact with the film 80. At this point, the urine sample is still in the upper reaction cup 30, and there has been no light output. Rotation of the control lever 94 unlocks the diaphragm support 86 and pressure bar 88 in the manner previously described, and the pressure bar 88 can then be operated to press the rubber diaphragms 84 against the tops of the upper reaction cups 30. The pressure thereby created in the upper reaction cup 30 ejects the grease plug 42, so that the liquid in the upper reaction cups 30 flows into the lower reaction cups 32. The liquid mixes with the release reagent and luminescent reagent in the cup 32, producing light in an amount related to the amount of bacterial ATP released. The light is recorded on the film 80. The light producing reaction begins within about 2 seconds or less after operation of the pressure bar 88, and is completed after no more than 30 seconds. After a sufficiently long time that the light-producing reaction is essentially complete, typically about 30 seconds, the translation control lever 94 is operated to release the downward pressure on the reaction test apparatus 20 so that it moves upwardly to its uppermost travel under the urging of the biasing spring 73. The shutter handle 76 is closed to end the exposure and unlock the upper housing 66. The instant film pack 82 is operated to develop the film 80, either before or after the shutter handle 76 is operated. After the film is developed, typically 60 seconds for Type 612 ASA 20,000 speed instant film, the test results on the film are ready for viewing. The reaction test apparatus 20 (if there are any remaining unused test wells) and exposure apparatus 22 are ready for further testing. The presence of any previously unused test wells 28 of a particular reaction test apparatus 20 is immediately apparent upon visual search for undisrupted portions of the foil cover 48, as the foil cover 48 above used test wells 28 is torn or disrupted during insertion of the specimen into the test well 28. The embodiment of the reaction test apparatus described to this point operates by application of a positive pressure to force liquid from one superimposed cup downwardly into another cup. The structure of the exposure apparatus is dictated in part by the need to provide the positive pressure by external application. Liquid flow from one portion of the reaction test apparatus to another portion can also be accomplished solely by gravity-induced flow, and in another embodiment the reaction test apparatus is structured for this operation. The exposure apparatus has a structure permitting this mode of transfer. Figure 11 illustrates the inventive form of a reaction test apparatus 200 wherein the flow of liquid is accomplished completely by gravity feed, without the application of a positive pressure. A plurality of test wells 202, preferably 18 wells arranged in three rows of six wells per row, are formed in a pour plate 204. The structure of each well 202 is seen more clearly in Figure 12. The test well 202 includes a first reaction chamber 206 extending downwardly from the pour plate 204. The first reaction chamber 206 has on one side an upwardly sloping interior wall 208 that slopes upwardly and outwardly from the bottom of the chamber 206. A final reaction chamber 210 is positioned adjacent the first reaction chamber 206, near the top end 212 of the upwardly sloping interior wall 208. The final reaction chamber 210 is illustrated as a generally cylindrical cup with an open top and a closed, flat bottom 214. In use, a liquid specimen is placed into the first reaction chamber 206 and reacted with a first reactant. When sufficient time has passed for the first reaction to be as complete as required, then one end of the pour plate 204 (in the illustrations of Figures 11 and 12 the right end) is displaced upwardly so that the pour plate 204 is rotated (counterclockwise in Figures 11 and 12). The reacted liquid specimen in the first reaction chamber 206 flows along the sloping interior wall 208 and thence into the final reaction chamber 210, where it reacts with a second reactant. In the preferred luminescence reaction described previously, light is emitted and measured through the flat bottom 214 of the final reaction chamber 210. A foil seal 215 covers the top of the test apparatus. The seal 215 is shown in Figures 12 and 14, but is removed in Figures 11 and 13 for clarity. The transmission of light between adjacent wells must be reduced as much as possible, because such stray light from a well in which a strong reaction occurs can result in false readings in adjacent wells that perhaps do not contain as strong a light emitting reaction. Several techniques are used in the reaction test apparatus of the invention to prevent light from passing between adjacent wells. To prevent light produced in one of the final test chambers 206 from being mistakenly recorded as light produced from another of the chambers 206, the pour plate 204 provides a support 216 between each of the chambers 206, which supports but also separates the adjacent wells. Transmission of light directly from one well to an adjacent well is therefore greatly reduced, particularly where the exposure apparatus places an opaque wall in the separating region created by the support 216. This approach is to be contrasted with that used for test apparatus that is not used for luminescence testing, where for cost reasons the adjacent wells are not separated. However, there remains the possibility that light will be piped through the material of the supporting pour plate 204 from one well to another. An effective approach to reducing the light transmission through the plastic itself, which is operable in the reaction test apparatus of the invention as well as other types of reaction test apparatus used in luminescence measurements systems, is to provide grooves 280 in a web 282 portion of the pour plate 204 that extends between the test wells 28 in the manner illustrated in Figure 15. A number of opposing but offset grooves 280 extending inwardly into the web 282 diffract light traveling within the plastic to prevent it from being transmitted through the plastic from one well to the adjacent well. When the light beams from a well strike the sloping walls of the grooves, they are diffracted away and out of the support structure, and therefore do not reach the adjacent test well. Another aid to preventing erroneous measurements due to light transmitted through the plastic from one well to another is to include a dispersion 284 of opaque dispersing particles within the plastic. The plastic is made thin in the region of the bottom of the flat bottoms 214 of the wells 202, and therefore the dispersing particles have little influence. However, when interposed in the longer light transmission path between adjacent wells, the particles aid in reducing light transmission between wells. A preferred approach is to mix into the plastic, prior to molding, on the order of 5 percent by weight of carbon particles. Figure 15 also illustrates the previously discussed approach of providing the support 216 that separates the adjacent wells. The web 282 is part of the support 216 that spaces the wells 202 apart from each other. Light cannot easily travel directly, in a straight line, between the adjacent wells 202 due to their spacing. Moreover, in practice there is ordinarily provided an opaque wall 290 in the exposure apparatus 244, which fits into the separating space between the wells 202. The wall 290 ensures that no light passes directly through the space between the wells. In the embodiment of Figures 11 and 12, there is a single individual first reaction chamber for every final reaction chamber. In a variant test apparatus 218, illustrated in Figure 13, there is a single first reaction chamber 220, which receives the liquid specimen. There are a plurality of individual final reaction chambers 222 that each receive a portion of the reacted liquid specimen upon tilting and rotating of the pour plate 204. The structure of the test wells 202 in this embodiment is otherwise essentially the same as previously described. This type of apparatus is useful where the liquid sample is to be uniformly reacted in the first reaction chamber, and then reacted with individual final reactions in the final reaction chamber. In yet another embodiment, illustrated in Figure 14, each test well 202 includes three reaction chambers rather than two, as in the previously described embodiments. The test well 202 of an apparatus 224 includes a first reaction chamber 226 having a first upwardly sloping interior wall 228. Adjacent a top end 230 of the wall 228 is a second reaction chamber 232, positioned so that liquid flowing along the wall 228 can pour into the chamber 232. The second reaction chamber 232 has a second upwardly sloping interior wall 234 that is preferably oppositely disposed across the chamber 232 from the top end 230 of the wall 228. A final reaction chamber 236 is positioned adjacent a top end 238 of the wall 234. The final reaction chamber 236 is generally cylindrical with a flat bottom or a shaped bottom to concentrate light onto an underlying photographic film, in the manner previously described. There is a wall in the pour plate 204 between each of the final reaction chambers 236, and the grooves and dispersion of particles are preferably provided to prevent leakage of light between adjacent test wells. All of the embodiments of Figures 11-14 utilize only gravity to move fluid from one reaction chamber to another. An exposure apparatus 244, illustrated in Figure 15, suitable for conducting the photographic recording of the test results using these embodiments is generally similar in most respects to the apparatus 22 previously described and used in conjunction with the pressure-activated reaction test apparatus 20. However, the rubber diaphragms, pressure bar, and related structure are omitted from the exposure apparatus 244. As shown in Figure 15, the apparatus 244 includes a lower housing 246 having an internal structure identical to the lower housing 64. A shutter handle 248, extension 250, film pack 252, and bias spring 254 have the same placement and function as the corresponding elements in the lower housing 64. A fixed shutter 256 is placed and oriented in a manner similar to the fixed shutter 68, except that it has a plurality of conforming recesses 258 therein to receive the downwardly projecting portions of the test wells 202 of the pour plate 204 that cooperates with the apparatus 244 in measuring liquid specimens. The lower housing 246 has a housing extension 260 extending from the end of the housing 246 nearest the final chambers of the last row of test wells. The housing extension is a solid piece having an upwardly inclined lower surface 262 that has an angle with the horizontal preselected to aid in the transfer of liquid from one chamber to another in the test wells. In use, the apparatus 244 (with the cover closed and reaction test apparatus in place) is placed upon a flat support surface and then rotated by pushing the housing extension 260 downwardly. The extension 260 is pushed downwardly until the lower surface 262 touches the support surface, and at that point further rotation is prevented. The apparatus 244 is constructed such that the angle of the lower surface 262 to the horizontal is greater than the angle of any of the upwardly sloping interior walls of the pour plate with the horizontal, so that the rotation necessary for liquid transfer is achieved. An upper housing 264 is similar to the upper housing 66, except that it has no pressure control mechanism. There is a translation control mechanism that operates in a manner like that of the apparatus 22. A rod 266 extends across the interior of the housing 66, and can be turned by an external translation control lever 268. Rotation of the lever 268 and the rod 266 causes two cam lobes 270, one on either end of the rod 266, to rotate. These lobes 270 push downwardly against the reaction test apparatus that is inside of the apparatus 244, causing it to move downwardly against the biasing springs 254 and into contact with the film within the lower housing 246. The interlock system preventing inadvertent exposure of the film operates like that described for the exposure apparatus 22. The exposure apparatus 244 therefore permits a user to conduct tests using a pour plate type reaction test apparatus, which achieves fluid flow within the reaction test apparatus by rotation of the pour plate and a gravity feed and does not require a positive pressure transfer. Although a particular embodiment of the invention has been described in detail for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims: A reaction test apparatus for assessing reactions by the production of luminescence, comprising: at least two test wells (202), each test well (202) including a first reaction chamber (226) having a first upwardly sloping interior wall (228); a second reaction chamber (232) disposed adjacent the top (230) of the first upwardly sloping interior wall (228) of the first reaction chamber (226), whereby tilting of the apparatus (224; 218) causes liquid in the first reaction chamber (226) to flow from the first reaction chamber (226) to the second reaction chamber (232) along the first upwardly sloping interior wall (228), and further having a second upwardly sloping interior wall (234) oppositely disposed to the location of the first reaction chamber (226); a substantially planar support (204) for the test wells (202) including a web section (282) supporting but separating the at least two test wells (202), said web section (282) extending between adjacent test wells (202) and positioning these test wells (202) in the web section (282), characterized in that a final reaction chamber (236) is disposed adjacent the top (238) of the second upwardly sloping interior wall (234) of the second reaction chamber (232), whereby tilting of the apparatus (224) causes liquid in the second reaction chamber (232) to flow from the second reaction chamber (232) to the final reaction chamber (236) along the second upwardly sloping interior wall (234), the final reaction chambers (236) being arranged with respect to the web section (282) so that the bottoms of the final reaction chambers (236) of each of the at least two test wells (202) are coplanar in a plane that lies parallel to the plane of the substantially planar support (204), and further the bottom (214) being transparent so that light produced in the final reaction chamber (236) may be monitored from beneath the final reaction chamber (236), that bottom (214) forming the lower extremity of the apparatus being designed to be pressed onto a photographic film material. A reaction test apparatus for assessing reactions by the production of luminescence, comprising: at least two adjacent test wells (202), each test well including a first reaction chamber (206; 220) having a first upwardly sloping interior wall (208); a final reaction chamber (210; 222) disposed adjacent the top (212) of the first upwardly sloping interior wall (208) of the first reaction chamber (206; 220), whereby tilting of the apparatus (200; 218) causes liquid in the first reaction chamber (206; 220) to flow from the first reaction chamber (206; 220) to the final reaction chamber (210; 222) along the first upwardly sloping interior wall (208); a substantially planar support (204) for the test wells (202) including a web section (282) supporting but separating the at least two wells (202), said web section (282) extending between adjacent test wells (202) and positioning the test wells (202) in the web section (282) so that the bottoms of the final reaction chambers (210; 222) of each of the at least two test wells (202) are coplanar in a plane that lies parallel to the substantially planar support (204), characterized in that the bottom (214) of the final reaction chamber (210; 222) of each of the at least two test wells (202) form the lower extremity of the apparatus and are designed to be pressed onto a photographic film material (80). Apparatus according to claim 1 or 2, wherein the apparatus (200; 218; 224) includes a plurality of substantially identical test wells (202), each of which test wells has the structure set forth in claim 1 or 2. Apparatus according to any of the preceding claims, wherein the test wells (202), the support (204) and/or the web section (282) are made of a plastic. Apparatus according to claim 4, wherein the plastic has a dispersion of light diffracting particles (284) dispersed through the plastic, said plastic being made thin in the region of the bottom (214) of the test wells (202). Apparatus according to any of the preceding claims, wherein said web section (282) has grooves (280) therein to reduce the transmission of light through the support (204) from one test well (202) to the adjacent test well (202). Apparatus according to any of the preceding claims, further including means (280, 284) for preventing light from passing from one of the at least two test wells (202) to the other of the test wells (202) through the web section (282). Apparatus according to claim 7, wherein the means for preventing includes at least one groove (280) in the web section (282) to reduce the transmission of light through the web section (282). Apparatus according to claim 7, wherein the means for preventing includes a distribution of light diffracting particles (284) dispersed through the web section (282) to reduce the transmission of light through the web section (282). Apparatus according to any of the preceding claims, wherein the final reaction chamber (210; 222; 236) of each of the two test wells (202) is supplied from the same first reaction chamber (206; 220; 226). Apparatus according to any of the preceding claims, wherein the first reaction chamber (226; 206; 220) includes a first chemical reactant capable of reacting with other chemicals to release adenosine triphosphate. Apparatus according to claim 11, wherein the first chemical reactant is in a solid, dry form. Apparatus according to any of the preceding claims, wherein the final reaction chamber (210; 222; 236) contains a chemical selected from the group consisting of luciferin, luciferase, and combinations thereof. Apparatus according to claim 13, wherein the chemical in the final reaction chamber (210; 222; 236) is in a solid, dry form. Apparatus according to any of the preceding claims, characterized by a luminescence exposure apparatus (244) including means for sensing a light output from a reaction chamber (236; 206; 220) of the apparatus (224; 200; 218). Apparatus according to claim 15, wherein the means for sensing comprises a solid, dry chemical reactant in at least one of the reaction chambers (206; 210; 220; 222; 232; 236). Apparatus according to any of the preceding claims, wherein there is a solid, dry chemical reactant in the first reaction chamber (206; 220; 226). Apparatus according to any of the preceding claims, wherein there is a solid, dry chemical reactant in the final reaction chamber (210; 222; 236). Apparatus according to any of the preceding claims for conducting luminescence reactions tests on specimens contained within the test well (202) comprising: a lower housing (246) including a flat, upwardly facing internal surface for receiving a piece of film (80) thereupon, a fixed shutter (68) covering the upwardly facing surface and having at least two openings (70) therethrough positioned to correspond to the locations of the test wells (202) in the reaction test apparatus (200, 218, 224), biasing means (73) on the fixed shutter (68) for resisting the downward movement of the reaction test apparatus (200, 218, 224), when the reaction test apparatus (200, 218, 224) is placed upon the fixed shutter (68), and a movable shutter (74) between the internal interior wall and the fixed shutter (68); and an upper housing (66) including translation means (92) for controllably urging the reaction test apparatus (200, 218, 224) downwardly against the resistance of the biasing means (73), thereby contacting the bottom (214) of the test well (202) in the reaction test apparatus with the piece of film (80) in the lower housing (246). Apparatus of claim 19, further including an extension (260) on one end of the apparatus (244), the extension (260) having an inclined surface (262) on the bottom thereof to limit the angular rotation of the exposure apparatus (244) to a predetermined amount when the reaction test apparatus is placed onto a flat surface and rotated toward the inclined surface. Apparatus of claim 19 or 20, further including locking means which include a movable shutter lock, the movable shutter lock preventing the opening of the movable shutter (74) when the reaction test apparatus is not in contact with the film (80) in the lower housing (246). Method for assessing reactions by production of luminescence by use of a reaction test apparatus according to one of the preceding claims, characterized in the steps of: adding the test sample to the first reaction chamber (206; 220; 226), placing the apparatus into an exposure apparatus (244) which is then closed to form a light-tight enclosure, placing the bottom (214) of the final reaction chamber (210; 222; 236) in the exposure apparatus (244) in contact with a photographic film material (80), passing the test sample to the final reaction chamber (210; 222; 236) where a release reagent and a luminescent reagent react with the sample producing light in an amount related to the amount the bacterial ATP release and the light produced is recorded on the film material (80).

Inventors: SMITH JERRY W, WANNLUND JON C, SMITH, JERRY W., WANNLUND, JON C.
Applicants: DIFCO LAB, RMS LAB INC, DIFCO LABORATORIES, RMS LABORATORIES, INC.
Application year: 1989
Main classification: G01N21
IPC codes: G01N21, C12M1, B01L3