US 3227522 A
Description (OCR text may contain errors)
Jan. 4, 1966 A. B. sALlsBURY, JR., ETAL 3,227,522
SSAYING APPARATUS Filed Sept. 19, 1961 2 Sheets-Sheet 1 @www Jan. 4, 1966 A, B. sALlsBuRY, JR., ETAL 3,227,522
ASSAYING APPARATUS Filed Sept. 19. 1961 2 Sheets-Sheet 2 fr-cey United States Patent O 3,227,522 ASSAYNG APPARAUS Alvin B. Salisbury, Jr., Fairborn, Guy A. Baiser, Dayton,
and eClifford I. Clt, Xenia, Ohio, assiguors to Laboratories, line., Fairborn, Ohio Filed Sept. 19, 1951, Ser. No, 139,211? 6 Ciaims. (Ci. 23-253) This invention relates generally to apparatus for assaying the reactions of different agents, for example, chemical agents, to each other and, more particularly, to apparatus for so-called agar diffusion assays in which a fiat surface of an agar is impregnated with one agent and the effect of one or more testing agents on the first agent is obtained by bringing the other agents into contact with spaced portions of the surface so that they diffuse into the agar and produce a visible reaction with the tirst agent. Such reactions may be visiele as changes of color or as the presence or absence or" growth where microorganisms constitute the rst agent and the other agents are growth inhibiting or encouraging substances.
A general object of the invention is to provide novel apparatus of the above character which, compared to similar prior apparatus, insures a more uniform and controlled distribution of each testing agent on the agar surface and thereby achieves more accurate and reliable results.
Another object is to support each testing agent on a projecting post which is constructed in a novel manner to insure Contact of a controlled quantity of the agent with the agar surface and to avoid reduction of that quantity or shifting of the agent on the agar due to air bubbles trapped on the surface.
A further detailed object is to proportion the parts of the projection in a novel manner to obtain the desired conv-ex contour while avoiding cracks in the agar.
The Vinvention also resides in the novel and simple manner of forming and connecting a plurality of projections for use with standard commercially available assay apparatus of the Petri dish type including an enclosing lid and inverted during incubation to avoid moisture collecting on the lid and dropping onto the agar.
Other objects and advantages of the invention will become apparent from the following detailed description taken in connection with the accompanying drawings in which FiG. l is a plan view of the test agents supporting plate embodying the novel features of the present invention.
FIG. 2 is a side elevation of the plate.
FlG. 3 is a fragmentary sectional view taken along the line 3 3 of FIG. 1 and showing an agent supporting projection.
FG. 4 is a perspective view of the plate and position on agar in a Petri dish bottom.
FlG. 5 is a plan view oft -e parts in FEG. 4.
FlG. 6 is a sectional view of plate in contact with agar in a complete Petri dish inverted for incubation.
FIG. 7 is a view similar to FIG. 5 with the plate removed.
FIGS. 8 and 9 are fragmentary sectional views taken along line 8 8 f FiG. 5 and showing successive positions of a projection with respect to the agar after different lengths of time of contact between the two.
FIGS. 10 and 11 are views `similar to FGS. 8 and 9 and showing -a modified proiection configurations.
FG. l2 is a side elevational view of a stack of plates some parts of which have been `broken away and shown in section.
FIGS. 13 and 14 are plan views of modified plates of different size with different number of agents supporting projections.
The present invention is especially suited for agar diffusion assays in which an agar medium 16 having a at exposed surface 17 is impregnated with an agent to be ested and quantities of one or more other agents are placed on the surface in spaced relation for observation of the effect of the other agents on the first agent. Such assays may be conducted for various purposes, one of the more common being biological assays to determine the effects on microorganisms of growth inhibiting or encouraging agents. Another is spot test analysis of solutions containing diiferent trace metals, complexes or ions. These are placed on the agar and suitable indica-tors forming the other test agents are placed at spaced points on the agar to produce chemical reactions such as visible color changes.
ln the case of microbiological assays, the agar usually is contained in a Petri dish having a bottom 18 with upstanding sides. The agar, as is well known, is a gelatinous material partially filling the bottom of the dish and containing moisture. The exposed agar surface 1'7 is streaked with bacterial inoculum to be tested and a quantity of one or mor-e growth affecting agents is placed on the surface at a point spaced from other agents. The dish bottom then is covered with a lid 19 and the assembly is incubated, the agents diffusing into the agar and discouraging or encouraging the growth of the bacteria depending on the type of agent used. Examples of inhibiting agents are antibiotics, sulfonamides and other chemotherapeutic substances. Some growth encouraging agents are carbohydrates, proteins, fats, vitamins, horins-nes, etc.
The visual effects of inhibitors on the growth of bacteria is illustrated in FIGS. 5, 6 and 7. Referring to these figures, different inhibitors carried by supports 26 are brought into contact with spaced portions of the agar surface 17 and diffuse into tbc agar as indicated by the dotted spaces 21. These spaces represent zones where the growth of the bacteria has been inhibited, the sizes of the zones varying with the effectiveness of the different agents. The absence of zones of inhibition around two of the supports indicates that the agents on these supports had no inhibiting effect on the bacteria. ri`he reason 'for inverting the bottom 18 and cover 19 of the Petri dish as shown in FIG. 6 is to prevent moisture accumulation on the inside cover surface as well as evaporation and loss of water from the agar during incubation. Handling of a plurality of agents is facilitated bythe provision of a body ember 22 which connects the supports and maintains them in a desired spaced relation. I
To obtain accurate and reliable test results, the present invention Vcontemplates a novel construction of each agent support 2i) insuring uniform and .complete diffusion of the agent into the agar 16 and avoiding shifting of the support with respect to the agar. The support thus is formed as a projection which terminates in a continuous smooth surface 23 of convex exterior contour covered by a test agent in the form of a thin layer. As the support is moved toward the exposed agar surface 17, this configuration results in contact of the agent and the agar first at a central portion of the convex surface and then progressively to the peripheral portions, any air bubbles thus are advanced ahead of the progressing contact line and do not become entrapped between the support and the agar so as to prevent contact of the agar surface by all of the agent or to space the end surface of the protection from the agar surface. The projection thus is able to sink partially into the agar as shown in FEGS. 9 and 11 and prevent sidewise shifting relative to the agar.
The preferred test agent supporting surface 23 as shown in FIGS. 1 to 9 and 12 to 14 is of a rounded or spherical convex contour with a circular periphery 24 (FIG. 3).
Its height, that is, the lateral or transverse spacing from the outermost center portion to its periphery, is correlated with its diameter to provide the desired convexity to insure contact of all of the surface with the agar while avoiding a sharp point liable to penetrate the agar sufficiently to produce cracks resulting in errors in the assay. A suitable height, indicated at A in FIG. 3, found to be satisfactory for a surface of 6.6 millimeters to 13 millimeters diameter is .001 to .010 of an inch or .0254 to .1524 of a millimeter. Convexities with heights outside of this range were found to be subject to entrapment of air bubbles or cracking of the agar, heights of .003 to .006 of an inch being the most satisfactory for the prevention of air entrapment. A suitable length of each projection of this approxhnate size is from two to five millimeters.
Another convex contour of agent supporting surface 23 found to be satisfactory is a conical surface as shown in a modification of FIGS. and 11. In this construction as in the preferred rounded surface, the height or transverse spacing of the center of the surface and the periphery is important to insure full contact of the agent with the agar and to avoid air bubbles without cracking the agar. With both configurations, moisture collects between the center and periphery of the surface when the surface first contacts the exposed surface 17 of the agar. With the lapse of time, the agar surface beneath the convex surface assumes a complementary concave contour resulting in a pit or recess and tending further to reduce shifting of the projection relative to the agar. Such formation of the recess beneath each projection is believed to be due to the weight of the projection and its connecting body and also to what may be termed interfacial tension. As the pit is formed, the moisture collected between the projection and the agar assumes a more uniform thickness and forms a film 26 (FIGS. 9 and l1) which, due to capillary action or interfacial tension, resists movement of the projection away from the agar.
In another of its aspects, the present invention contemplates forming a plurality of the projecting supports 2li and their connecting body 22 in a novel manner enabling the capillary action or interfacial tension of the film 26 between the agent surface 23 and the exposed agar surface 17 to retain the projections in position with respect to the agar when the covered Petri dish is inverted for incubation as shown in FIG. 6. The invention further contemplates inexpensive manufacture of the projections and body and simplification and accuracy in their use. To these ends, the body is formed as a thin sheet or plate and the projections and supports are formed as integral parts therefo, the weight of the plate being less than the sum of the tension forces due to the film 25 on the projections. Spaced portions of the sheet thus are depressed laterally to form recesses on one side and the projections on the other.
To facilitate insertion and withdrawal of the plate 22 between the upstanding walls of the Petri dish bottom 13, the plate has the same peripheral shape as the dish but is smaller thereby leaving a space between the two when the plate is inserted in the dish. This space is distributed around the plate, that is, the plate is centered in the dish, by locating lugs 27 engageable with the dish wall and projecting radially and outwardly from angularly spaced ports of the periphery of the plate. If the plate is removed from the dish and is desired to be reinserted in its original position, the proper orientation is insured by the provision of another projection 28 which is rectangular and therefore provides a recess 29 on the agar (FIG. 7) easily distinguishable from the circular recesses 25 of the agent carrying projection 20.
A material found to be suitable for the plate 22 is a thermoplastic resin such as a polyvinyl chloride sheet .093 to .004 of an inch thick, which is known to be nonabsorbent. If desired, one side may be roughened to provide a pleasing appearance while leaving the plate translucent to permit observation of the zone 21 of inhibi- CII tion through the plate. A plate of this material and thickness weighs less than the force resulting from the capillary action or surface tension of the moisture between the agar lo and the projection 20 when inverted as shown in FIG. 6. Shipping and storing of a plurality of plates is facilitated by making each projection between its end and the plate of tapered contour, the projections in this instance being conical with circular cross sections. The projections of one plate thus may nest in the hollow projections of the next plate for stacking the plates in spaced relation and thereby keeping the agents on the projections of one plate out of contact with the projections of the next plate as shown in FIG. l2.
Various common commercial methods of forming the plate with its projections may be used, for example, stamping or vacuum forming using male or female dies with a sheet of the plastic material heated during the shaping and then cooled in a well known manner.
The diameter of the plate 22 and the number and spacing of the projections 2li depend on the size of Petri dish i8 intended to be used and the desired available size of ti e zones 21 of effectiveness. For example, seven projections 2@ of a diameter of 6.6 to 10 millimeters may be arranged in a uniform pattern as shown in FlG. l with their centers spaced apart 3 centimeters from each other on a plate having a diameter of 73 millimeters and intended for use in a Petri dish of 9() or 100 millimeters. Smaller or larger numbers may be arranged in regular patterns on smaller or larger plates as shown in i3 and i4, the projections on the larger plate of PlG. i4 having a smaller center to center spacing. Preferably suitable indicia Si@ such as names (FIG. l) or numbers (PEG. 14) are provided on the plate adjacent the respective projections and are visible from the upper side of the plate to identify the different agents. The latter are placed on the projections in solutions which dry leaving each agent in a dry state in a layer covering the end 23 of the projection.
The contour of the ends 23 of the projections is especially important in the use of the plate 22 when it is dropped freely on an inoculated surface 17 of agar 16 in a Petri dish i3. Air bubbles thus are removed from between the agar and each projection so that the latter seats firmly and remains in the same position on the agar and, also, so that the agent on the projection diffuses uniformly from the entire area of the projection end. After a short time, on the order of two to five minutes, the recess 23S forms in the agar beneath each projection and the film Z5 assumes a uniform thickness. The formation of the recess helps prevent shifting of the plate on the agar and, in conjunction with the uniform diffusion of the agent provides controlled test conditions contributing to accurate results.
With the cover i9 on and the dish 1S inverted as shown in FiG. 6, the projections 2i? are retained against the agar 16 due to the tension forces of the film 26 and the lighter weight of the plate. The dish being transparent and the plate translucent, the growth of bacteria or other reaction may be easily observed. If it is desircd to remove and replace the plate in its original position, its proper location is insured by the orienting lug Z8 and its recess 29.
lt will be apparent that the plate with its projections 20 may be made inexpensively and thus are disposable after a single use. They are easy to use by personnel with little training and a large number may be stored in a relatively small space. By virtue of the continuous convex contour of the end 23 of each projection, there results controiled positioning of the projection on the agar i6 and control of the amount of agent diffused into the agar so as to achieve accurate test results. By utilizing the light weight plate 22 to connect a plurality of the projections, a corresponding number of agents may be tested simultaneously while they are maintained at accurate spacing even during inversion of the Petri dish. The ease and accuracy of the assay thus are enhanced still further.
While the invention is susceptible of various modifications and alternative constructions, the preferred embodiments are shown in the drawings and are described herein in detail. It is to be understood, however, that such disclosure is not intended to limit the invention but, that the aim is to cover all modifications and alternative constructions falling within the spirit and scope of the invention as expressed in the appended claims.
We claim as our invention:
1. In apparatus for assaying the effect of a rst agent on a second agent upon diffusion of the rst agent into an agar-like medium having a generally ilat exposed surface impregnated with the second agent, a supporting body and an elongated projection extending outwardly from said body and providing an outwardly facing continuous end surface to receive a layer of said rst agent for diffusion into said medium and reaction with said second agent, said end surface of said projection being formed of non-absorbent material and having a convex contour to engage said medium surface rst at a central portion of the end and then progressively to the peripheral portion of the end surface to avoid the trapping of air between the projection and the medium surface and to insure uniform distribution of the rst agent throughout the area covered by the end surface of the projection.
2. In apparatus for use in assaying the effects of a plurality of rst agents of different compositions upon a second agent upon diffusion of the first agents into an agar-like medium having a generally at exposed surface impregnated with the second agent, said apparatus comprising an integral at disk of thin sheet material having a plurality of spaced portions offset laterally from the plane of the sheet to form hollow projections extending from one side of the sheet and terminating in continuous outer end surfaces adapted to receive and support said rst agents in layers on the ends and insure contact of the agents with said medium surface over the entire area of each end, said end surfaces being formed of non-absorbent material and each having a convex contour to engage said medium surface rst at a central portion of the end surface and then progressively outwardly to the peripheral portion of the end surface to avoid the trapping of air between the projection and the medium surface and to insure uniform distribution of the first agent throughout the area covered by the end surface of the projection.
3. The apparatus of claim 2 in which said disk is formed of a light-weight material such that, when the disk is disposed horizontally beneath said medium with said projections engaging the medium, the interfacial tension between the medium and the projections is sucient to overcome the force of gravity and maintain the contact.
4. The apparatus of claim 2 in which in egral projections extend radially and outwardly from the periphery of the disk at angularly spaced points to engage the upstanding wall of a container for said medium and space' the disk inwardly from the wall.
5. The combination of claim 2 in which said hollow projections are conical to permit nesting of the projections of one disk in the projections of a similar disk with the disks spaced apart.
6. The apparatus of claim 2 in which said end surface is circular and has a diameter of approximately 6.6 to 10 millimeters and a height of .003 to .006 of an inch.
References Cited bythe Examiner UNITED STATES PATENTS 2,956,931 10/ 1960 Goldberg 195-103.5 2,986,497 5/1961 Pagano et al 195-103.5 2,998,353 8/1961 Ryan l95-103.5 3,010,880 11/1961 Littman et al. l95-103.5
MORRIS O. WOLK, Primary Examiner.
ANTHONY SCIAMANNA, Examiner'.