US 3826717 A
Description (OCR text may contain errors)
July 30, 1974 v. E. GILBERT HTM, 3,826,717
QUANTITATIVE NTIBIOTIC TEST CONTAINER 3 Sheets-Sheet l` Filed Feb. 26, 1973 v v n. QN
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QGGG NCQOG @CDOGO @W600 mGQQQ@ [@GQQQQQ @SQQQQ N3000 @SGGQ@ @0000 wm@ cm@ fgcmCQGm@ @Q0 Qklll 30, 1974 v E. @LEER-r ET AL 3,826,717
QUANTITATIVE ANTIBIOTIC TEST CONTAINER Filed Feb. 26, 1973 3 Shouts-Ghent 2 July 3G, 1974 v. E. GILBERT ET AL 3,826,717
QUANTITATIVE NTIBIOTIC TEST CONTAINER 25 Shouts-Shut 3 Filed Fob. 26, 1973 `FIG. l0.
United States Patent O 3,826,717 QUANTITATIVE ANTIBIOTIC TEST CONTAINER Verne E. Gilbert, Oak Ridge, Term., and Arnold B.
Silverman, Pittsburgh, Pa., assignors to Verne E. Gilbert, Oak Ridge, Tenn.
Filed Feb. 26, 1973, Ser. No. 335,520 Int. Cl. C12k 1/04 U.S. Cl. 19E- 103.5 R 26 Claims ABSTRACT F THE DISCLOSURE A quantitative antibiotic test container having a plate member provided with a plurality of integrally formed upwardly open test wells and an elongated upward-ly open specimen receiving trough. The Wells are preferably d1sposed in a number of generally parallel rows which are oriented generally transversely with respect to the trough. The Wells are filled with a solid nutrient media, such as Trypticase Soy agar, and a predetermined concentration of an antibiotic, with the upper surface of the mixture being disposed generally at the well opening. A specimen distributor having a handle portion and an elongated head portion is provided with a specimen receiving section so c'onlfigurated as to be at least partially receivable lwithin the specimen receiving trough. A cover member overlying the plate member. The plate member preferably has elongated barrier elements to resist flow of fluids between adjacent rows. An antibiotic is provided in a given row with a number of predetermined concentrations in successive wells with higher concentration in a given row preferably being in the well more remote from the trough. One or more standard wells having solid nutrient media but no antibiotic may be provided.
vA method of effecting antibiotic sensitivity testing employing the container described.
BACKGROUND OF THE yINVENTIN Field of the Invention This invention relates to a pre-poured miniaturized multiple antibiotic sensitivity test container which provides multiple concentrations of a number of antibiotics and, more specifically, it relates to such a container which has means for simultaneously inoculating a large number of test wells with the organism to be tested and the method of using such a container.
Description of the Prior Art As a part of conventional medical practice in the treatment of patients with a wide range of infections, it is essential to obtain samples of the mcroorganism which is believed to be causing the illness and to culture the same in efforts to identify and determine the sensitivity of the organism to a particular antibiotic. As prompt, accurate and quantitative antibiotic sensitivity results are of obvious urgency, laboratories must have effective means for accomplishing these desired objectives.
In the sophisticated laboratories at a substantial number of university medical centers antibiotic sensitivity testing is accomplished by means of the accurate but tedious and cumbersome tube dilution or agar dilution antibiotic sensitivity tests. In this type of testing, the organism is challenged with multiple concentrations of many antibiotics. These exacting procedures give lthe physician very accurate results and allow the physician very accurate results and allow the physician to choose antibiotics based upon the degree of sensitivity of the organism to various antibiotics. These systems, however, require the individual inoculation of organisms into numerous tubes or nutrient agar filled Petri dishes or plates r, lCe
containing multiple concentrations of different antibiotics. In addition to the inefficiency of such time consuming systems, substantial space for storage of the large number of containers both prior to use and during use is required. Also, the time beting consumed is generally that of skilled or semi-skilled laboratory personnel. Furthermore, such laboratory personnel have been kept extremely busy as a result of the ever increasing number of new antibiotics with respect to which the microorganisms need to be tested. As a result, these time consuming procedures may contri-bute to delay in getting proper medication to the patient.
lIn most hospitals and doctors ofiices there h'as not been an effective means of accomplishing this quantitative type of antibiotic sensitivity tests. Such locations frequently use the antibiotic medicated disc system .for testing organisms. Suc'h tests are undesirable as they provide an unacceptable degree of reliability and they do not give the physician a choice of effective antibiotics based on the degree of sensitivity. Ideally, a physician would like to use an antibiotic to which the organism is exquisitely sensitive, as opposed to one which demonstrated only moderate sensitivity. It has been found that on the basis of a professional study the reliability of antibiotic medicated disc tests were only 36 to 90% as accurate as tube dilution sensitivity tests. See Turck, Lindemeyer, and Petersdorf, Ann-als of Internal Medicine, Volume 58, page 56 (|1963) and Chitwood, Tube Dilution Antimicrobial Susceptibility Testing: Efiicacy of va Micro-Technique Applicable to Diagnostic Laboratories, Applied Microbiology, page 707, May 1969. Among the errors which result from such disc systems is the fact that the test m-ay indicate that a given organism is sensitive to a particular antibiotic when in fact the organism may be quite resistant to one or more given antibiotics or the test may indicate that the organism is resistant when, in fact, it is sensitive. The life threatening implications are obvious.
'In some uses of the disc type systems, a dye agent is employed in order to attempt to obtain a color change responsive to certain interactions between the organism and the medication. In addition to other weaknesses inherent in disc type systems, such as discussed above, a further area of difficulty results from the fact that no all bacteria will produce the desired color change. Such a system iS shown in U.S. Pat. 3,-l07,204.
It has been suggested that miniaturized unitary test containers be provided with a number of compartments so that mutual testing could be undertaken. The use of such a system wherein the individual hospital laboratory prepares the individual compartments with antibiotics -is contemplated in the Chitwood article noted above. Additional disclosures of compartmentalized individual contamers are found in U.S. Pats. 3,272,719, 3,308,039, 3,632,478 and 3,649,464.
Another difficulty of the prior systems is in connection with the method of inoculating the compartmentalized specimen test containers. These prior approaches in volve the cumbersome and' inefficient approach of requiring individual inoculation of each compartment. Such an approach is not only time consuming, but in addition the organisms represent a potential health hazard for laboratory personnel.
There remains, therefore, a substantial need for au antibiotic sensitivity testing system which provides quick quantitative results and is readily employed in a simple and inexpensive way in all hospitals, medical laboratories and doctors offices. In addition, there remains a need for such a system wherein a multiplicity of tests employing many concentartions of numerous antibiotics may be preformed in a single container with the specimens being inoculated into the various container compartments in a rapid and eicient manner not involving individual sequential inoculation. superimposed' upon these needs is the great need to accomplish these objectives in an economical fashion and with the reduction in the amount of laboratory time required to perform such tests effectively.
SUMMARY OF THE INVENTION The antibiotic test container of this invention has met the above-described need. It comprises a plate member which may preferably be formed of transparent material such as plastic. The plate member has a plurality of integrally formed upwardly open wells which contain a solid nutrient media. The wells are positioned in rows with each row containing a single antibiotic and different wells Within the row having different concentrations. In addition it is preferred to provide at least one well in the plate member which has solid nutrient media, but no antibiotic. Such a well serves as a standard to confirm the fact that the test organism has grown in the container and provides for visual comparison with the inhibited growth of the organism on the wells containing antibiotic.
The plate member also has an elongated upwardly open specimen receiving trough. A specimen distributor having a handle portion and an elongated head portion provided with a specimen receiving sector is also provided. The organism to be tested will generally be grown for a short period in a tube of ordinary broth and then be introduced into the trough, transferred to the specimen distributor and, by means of the distributor, substantial- 1y simultaneously inoculated into many rows of wells. A suitable cover member is provided.
The plate member preferably has the wells disposed in substantially parallel rows and barrier members provided between adjacent rows to resist ow of uid therebetween.
It is an object of this invention to provide a sterile antibiotic sensitivity testing container which provides a plurality of wells containing a pre-poured moist nutrient solid agar, and a large number of antibiotics in a wide range of concentratioins and is adapted to provide rapid quantitative results and the method of employing the same.
It is another object of this invention to provide such a container which is factory lled and sealed and is adapted to test for a wide range of types of bacteria including aerobic and anaerobic varieties, as well as fungi.
|It is another object of this invention to provide such a container wherein means for initially holding a test organism in liquid broth are provided and means for simultaneously and effectively inoculating a large number of wells are also provided.
It is another object of this invention to provide for positioning of the various concentrations of antibiotics in the wells in such a fashion that undesired mixing will be resisted during inoculation of the organisms.
It is another object of this invention to provide a sealed container design which will effect retention of moisture in the solid nutrient agar during both storage of the containers and incubation of the same during testing.
It is a further object of this invention to provide such containers wherein only a minmum amount of laboratory time is required and' wherein extensive use in smaller laboratories and doctors offices may be initiated in order to provide prompt and accurate quantitative test results.
It is another object of this invention to provide such a system which is adapted to have extremely high sensitivity such that a single antibiotic-resistant colony of an organism may readily be detected.
`It is yet another object of this invention to provide for economical adoption of such a container as a result of sterile factory prepackaging and economic materials and design such that the container once used may be discarded.
It is yet another object of this invention to provide a simple quality control system which confirms the stability of the antibiotics in the test container.
These and other objects of the invention will be more fully understood from the following description of the invention, on reference to the illustrations appended hereto.
BRI-EF DESCRIPTION OF THE DRAWINGS FIG. 1 is a partially schematic top plan view showing one embodiment of the antibiotic test container of this invention.
FIG. 2 is a cross sectional illustration of the plate member of FIG. 1 taken through 2 2.
FIG. 3 is a fragmentary cross sectional illustration of a portion of a plate member of FIG. 1 taken through 3 3.
FIG. 4 is a top plan view of a form of specimen distributor of this invention.
FIG. 5 is a cross sectional illustration of the specimen distributor of FIG. 4 taken through 5 5.
FIG. 6 is a bottom plan view of a cover element of this invention.
FIG. 7 is a cross sectional illustration of a container of this invention.
FIG. 8 is a top plan view of a modified form of specimen distributor of this invention.
FIG. 9 is a cross sectional illustration of the specimen distributor of FIG. 8, taken through 9 9.
FIG. 10 is a fragmentary cross sectional illustration showing a portion of the specimen distributor of FIG. 8 in combination with a portion of a plate member.
FIG. 1l is a fragmentary cross sectional illustration of a modified form of cover and specimen distributor member contemplated by this invention.
FIG. l2 is a fragmentary cross sectional illustration of a modified form of cover and plate of this invention.
FIG. 13 is a fragmentary, partially schematic plan view of a plate of this invention after completion of a sensitivity test.
DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now more specically to FIGS. 1 and 2, the embodiment of the invention there illustrated will be considered in greater detail. As is shown in FIG. 1, a plate member 2 is, in the form shown, of generally rectangular peripheral configuration. The plate member is preferably formed as a unitary article, as by molding. It is provided with a plurality of integrally formed upwardly open wells, with the wells in each row bearing the same number with the wells designated by the even numbers between 4 and 30. In the form illustrated, the plate member 2 has fourteen rows of wells 4-30 and five lines of wells 4-30 which are designated by the letters 8, A, B, C and D. The wells are downwardly directed and of generally cylindrical coniiguration. Between adjacent rows of wells are barrier elements designated by the even numbers between 40 and 66. These barrier elements 40-66, in the form shown, are upwardly directed hollow ribs which are preferably continuous and have a length at least equal to the length of the adjacent rows of Wells 4-30. A cross section of rib 66 is shown in FIG. 3.
Referring now to the upper portion of FIG. 1, there is shown a specimen receiving trough 70 which is oriented generally transversely with respect to the rows of wells and, in the form shown, is generally perpendicularly oriented with respect thereto. It is noted that the trough 70 is, in the form shown, slightly longer than the distance between row 1 and row 14. The reason for such as arrangement will be discussed in greater detail below.
Referring now to FIGS. 1 and 2, it is seen that the plate member 2 has an upwardly projecting hollow wall 74 which is peripherally substantially continuous around the plate. The height of hollow wall 74 is preferably suthcient to resist undesired spillage of the organism containing broth from specimen receiving trough 70 or the well containing portions of the plate member 2 onto the user or surrounding environment. The wall preferably has a height of about 1'1/2 to 3 times the average well depth. In the form shown, hollow wall 74 terminates in an outwardly directed foot 76. The trough 70 is shown as extending continuously between sectors of wall 74 at opposed ends of plate member 2. Depending upon the depth of the wells 4-30, the plate may rest upon the ange foot 76 or the wells between or both.
Referring again to FIGS. l and 2, it is noted that each of the wells is filled (preferably by automatic filling means in a sterile environment) with a solid nutrient vgrowth media such as trypticase soy agar, for example, in such fashion that the upper surface 80 of the media will be disposed generally at the opening of the well 4. This will generally position the surface 80 substantially coplanar with the upper surface 82 of adjacent portions of the plate member 2. It will be appreciated that while for purposes of convenience of description herein, a plate member 2 having fourteen rows each containing five wells is being shown, it will be understood that different numbers of rows containings different numbers of wells may be provided, depending upon the number of antiboitics used, and
fabricating costs of making several types of plates as contrasted with a single versatile plate. Also, for a particular test, only those rows of wells which are to be employed in the test need be filled.
Considering now, by way of example, line S of wells, it is contemplated that this line will represnt a standard line and that the Wells of each row disposed in this line will be prepackaged solely with the solid nutrient media but no antibiotic. When the organism to be tested is inoculated, in a manner to be described below, into the wells of row S, and incubation is effected, growth of the organisms in the line S wells will confirm the fact that the organism has grown in the test system and will provide a basis for visual comparison with the other wells. In the absence of such a standard, one could obtain ersoneous indication that the organism was sensitive to all of the antibiotics tested in all concentrations tested. It is preferred to provide a standard well within each row so that it may be used for easy visual comparison with the growth inhibiting effects of the antibiotic containing wells, If desired, one may provide a lesser number of standard wells, only a single standard for the entire plate or no such standard wells. It is preferred, however, that at least one standard well be employed, with the preference remaining the use of a standard Well in each row.
Considering now the antibiotic concentrations, row 1 will have no antibiotic in the wells in the S line, but will contain different concentrations of the same antibiotic in the Wells of lines A through D. Each of the wells in lines A through D of line 1 will contain a different concentration, with the preference being that the well of line A will have the lowest concentration and that the concentration be progressively increased through D which will contain the highest concentration. By arranging the concentration of antibiotics in a row in this preferred way, substantially simultaneous inoculation of the entire plate with a rapid single stroke is readily accomplished without undesired carryover of antibiotic from one Well in a row to a next succeeding well in the row. As it is contemplated that inoculation will be effected sequentially from the line A wells through the line D wells, the lower concentration inthe firstl inoculated wells minimizes the risk of such carryover. For example, a specimen distributor which passes over a well having a 1 microgram concentration of an antibiotic will not add significant amounts of antibiotic to a next succeeding well having a 5 microgram concentration of the antibiotic.
It has been found that in order to obtain effective, accurate results it is preferred that the wells have a certain size. The wells should have a depth of about 3 to 10 millimeters (preferably 5 to 7 millimeters) and an internal diameter of about 5 to l0 millimeters (preferably about 6 to 8 millimeters) with each of the antibiotic containing wells being substantially of the same size. In the event that the wells are smaller in depth or diameter, it has been found that the nutrient agar may dry out and shrink from the sides of the `well thereby interfering with organism growth and its visual inspection. In the event that the depth of the well is larger, difficulties may be encountered in making proper visual examination of the surface growth after incubation as one needs to see through the agar in order to make an accurate evaluation. With respect to diameter, the diameter should be suflicient to permit easy visual observation of the organism growing on the nutrient surface. As one of the objectives of this invention is t0 provide a miniaturized system, it is desired that the diameter of the wells not be excessively great as this does not contribute to the accuracy of the tests but does needlessly increase the size of the container.
While for purposes of illustration there have been shown the preferred cylindrical wells, other configurations such as wells having a rectangular configuration in plan, for example, may be employed, if desired, and the term diameter in such context shall be interpreted as referring to the maximum transverse direction between opposed walls of the wells.
Referring now to FIGS. 4 and 5, one preferred form of specimen distributor will be considered. The specimen distributor 86 has a head portion 88 and a handle portion 90. It is seen that the handle portiony has a base web a and an upstanding reinforcing n 90b. The head portion 88 is preferably substantially rigidly secured to handle portion 90, and has an upper web 92 and a specimen receiving portion 94. The handle 90 and web 92 may conveniently be formed of any inert, relatively strong material including plastics, glass, metal or combinations thereof. The specimen receiving portion `94 is preferably composed of a material which will absorb a liquid specimen. For this purpose, natural and artificial sponges or natural or synthetic fabric or fibrous materials such as cotton may be employed effectively. The specimen receiving portion is preferably of smaller dimension than the recess defined in the specimen receiving trough 70, such that the specimen receiving portion 94 may be at least partially received within the trough 70. The specimen receiving portion 86 should have sufficient length as to extend the full distance between the first filled row of wells and the row of wells most remote therefrom.
In employing the plate member 2 shown in FIG. l in combination with a specimen distributor of the type shown in FIG. 4, the container may be prepackaged in such fashion that the specimen distributor 86 has its specimen receiving portion 94 positioned within specimen receiving trough 70 and the handle portion 90 resting upon the generally fiat portion 96 of the plate member 2. In such a situation it may be desirable to establish the relative sizes of the trough 70 and specimen receiving portion 94 such that the volume of specimen desired to be introduced to the trough 70 may be received therein while the specimen receiving portion 94 is disposed, at least partially, in the trough 70. In the event that this form of prepackaging is not contemplated and the specimen distributor 86 will be supplied separately, substantially hat portion 96 of the plate member 2 may be eliminated and row 7 may be positioned adjacent to each other with a single barrier element 52, 54 positioned therebetween.
By the method described below quantitative antibiotic sensitivity test results are made available within only about 18 to 24 hours for aerobic organisms and about eighteen to forty-eight hours for anaerobic organisms.
In inoculating the rows 4-30, in general the organism isolated from patient material will first be grown in a tube of ordinary broth media of the type generally available in laboratories for a period of about 2-5 hours. The organism in broth will be introduced into specimen receiving trough 70 by pipets or other suitable means. The test specimen will be absorbed into specimen receiving portion 94 of specimen distributor l86. The specimen distributor will then be lifted from the base of the trough and moved downwardly along the upper surface of plate member 2. As is shown in FIG. 2, the flat region 82 between trough 70 and first well 4 and the flat region between the last well 4 and end wall 74 facilitate efficient inoculation by the distributor 86. It will be appreciated that when the specimen distributor 86 is over line S of wells 4-30, it will by means of a number of inoculation zones overyling the wells f430 simultaneously transfer the test specimen to the upper surface of the solid nutrient material in each filled well of line S. Continued movement of the specimen distributor 86 will result in simultaneous inoculation of all wells of line A, line B, line C and line D sequentially. It will, therefore, be apparent that where all fourteen rows of plate member 2 are filled the specimen distributor 86 would simultaneously inoculate each line of the fourteen rows. The full inoculation of all ve lines may be effected substantially simultaneously.
During the movement of the specimen distributor 86 the increasing antibiotic concentrations which have been established in a direction moving away from trough 70 substantially prevent meaningful pickup of antibiotic from one well of a given row and deposition of the same in a succeeding well. Also, barrier elements y40-66 resist contamination of an antibiotic of one row with an antibiotic of another row.
The specimen receiving portion 94 is preferably cornposed of a resiliently compressible material such that it will move over the barrier elements 40-66 without damage. If desired, as an alternative, the specimen receiving portion 94 may be notched or segmented so as to minimize interference between the barrier elements 40-66 and the specimen receiving portion 94, but this is not believed to be essential. Also, while the barrier elements have been illustrated as being upstanding continuous hollow ribs, it will be appreciated that they might be provided as solid ribs, downwardly directed solid or hollow grooves, discontinuous elements or in the form of other suitable means of resisting flow between adjacent rows. Finally, undesired lateral movement of the specimen distributor 86 is prevented by the upstanding peripheral walls 74 disposed at opposite ends of the plate. The distributor 86 should preferably have a length just slightly less than the distance between the plate member end walls. This serves to prevent specimen distributor carryover of antibiotics from one row to another. The trough 70, wells 4-30 and movement of the specimen distributor 86 all are within the recess defined by peripheral wall 74. As a result, the risks of organism spillage and contamination of laboratory and personnel are substantially reduced.
Referring now to FIGS. 6 and 7, a form of cover construction adapted for use with the plate member 2 will be considered. The cover element 98 in the form shown has a substantially flat top panel portion 100 and an integrally formed downwardly directed peripheral flange 102 which preferably is continuous about the periphery of the cover element. As for certain stages of storage of the test container and for certain types of tests, improved sealing greater than that which will vbe obtained by the friction t of the cover 98 and plate member 2 may be desired, a compressible gasket element 104 is provided about the periphery of the top panel 100. This gasket 104 may be composed of any suitable resiliently compressible materials, such as a plastic or rubber or combinations thereof, for example. Gasket 104 may conveniently be provided in the form of a tape which establishes a substantially continuous sealing gasket about the periphery of top panel 100. It may be secured to the top panel by self-adhesion, through heat sealing or independent adhesive means. Where it is contemplated that the container will be employed for anaerobic tests it is preferable to have the sealing member 104 secured to the top panel 100 so as to be manually removable from the cover element 98. In order to facilitate such removal, a gripping tab 106 may be provided, After removal of the sealing member the container may then be introduced into an anaerobic chamber, such as a Brewer jar.
As is shown in FIG. 7, the cover element 98, is frictionally secured to the plate member 2 by ange 102 being received within the upwardly open channel in the plate member 2. The gasket member 104 is in peripherally continuous compressive sealing position between the cover top panel 100 and the upper portion of the plate hollow peripheral wall. While it is generally convenient to secure the gasket to the cover top panel 100 or flange 102, the gasket may be secured to the plate, if desired.
While, in general, it is contemplated that a relatively tight friction t or snap fit will be effected between the cover 98 and plate member 2 and that this will provide a reasonably tight seal, a gasket member may be employed to enhance sealing. This enhanced seal will serve to resist undesired escape of moisture from the nutrient media in the Wells during storage and incubation, If desired, as an alternate or supplemental sealing means a strippable plastic, rubber or metal foil tape may be sealed around the periphery of the container where the cover flange 102 meets the plate flange as is shown in FIG. 7. Also, if desired, a strippable plastic film or metal foil web may be secured to the plate in well covering position to help preserve sterility and resist moisture loss. Such a film or web would be removed and discarded prior to use of the plate.
In order to preserve the desired sterility of the assembly after manufacture of the container, and factory lling, during shipping and storage thereof prior to use, it is preferred that the entire assembly be placed in a sealed plastic or metal foil pouch or one composed of a laminate of plastic and foil. This will serve to guarantee sterility of the package prior to use. In addition, it will generally be desirable to store the factory created test container at a reduced temperature so as to preserve the viability of the antibiotics contained therein. A temperature of about 4 C. or lower should be effective for most antibiotics, but this can be altered in accordance with the needs of the group of antibiotics used.
Also shown in FIG. 7 is the preferred cover structure which provides top panel 100 in a position in spaced overlying relationship with respect to the underlying wells 4, 6, 8, 10, 30. The cover provides for entrapment of air within the sealed container to facilitate aerobic organism tests in a sealed sterile environment.
Referring now to FIGS. 8 and 9, an alternate form of specimen distributor 116 will be considered. In this ernbodiment a handle portion 118 is integrally formed with a head portion 120. In order to rigidify the members, integrally formed downwardly directed ribs 122, 124 are provided. The specimen receiving portion 126, in the form shown, is composed of an absorbent sponge material.
Referring now to FIG. 10, there is shown a section of the plate member, disposed at a position corresponding to flat section 96 shown in FIG. 1, which is designed to receive handle 118. It is noted that the plate member is provided with a depressed channel shaped sector which provides an upwardly open recess 132 which receives the handle 118. A downwardly directed hollow rib 134 provides a recess for receipt of handle rib 122.
Referring now to FIG. 11, an alternate form of cover and specimen distributor element will be considered. In the form shown, the specimen receiving trough is spaced from the nearest well 142. The plate member 144 is secured to the cover member 146 as a result of snap fit engagement between cover ange 148 and plate flange 150. A seal is provided by gasket 170. It is noted that the lower extermity of flange 148 terminates at a level above the level of the bottom wall 152 of trough 140 and the lower extremity 154 of flange 150. The lower edge of flange 148 must be suiciently high as to permit free inoculation by specimen distributor 160 in a fashion to be described below. In this embodiment of the invention the specimen distributor 160 has a specimen receiving portion 162 secured to an overlying web 164 and a connecting web or handle 166 which connects the same t0 cover panel sector 168. In this embodiment of the invention, one may inoculate the various wells by grasping the cover manually and moving the same along the upper surface of plate 144 with specimen receiving portion 162 contacting the upper surface of the solid nutrient media to effect inoculation as described above. For maximum strength, it is preferred that handle 166 have sufficient thickness to provide adequate rigidity and that it be substantially continuous longitudinally.
FIG. 12 lillustrates a modified form of sealing arrangement between cover element 176 and plate 182. The plate 182 has a specimen receiving trough 186 and weblike outer wall 188 as contrasted with the hollow outer walls shown in FIGS. 7 and l1. The cover element has a top panel 178 and a depending flange 180 which is in peripherally frictional engagement with wall 188. A gasket 190 of reduced width as compared with gaskets 104 (FIG. 7) and 170 (FIG. l1) is provided.
FIG. 13 illustrates an example of how a portion of a plate of this invention may appear after incubation on reading the antibiotic sensitivities. In general, the cover will be removed to view the wells, but, if desired, to reduce the exposure of personnel, reading may be made through the cover. As the S line of wells in rows 1, 2 and 3 are darkened, this confirms growth of the organism on the solid nutrient media in the absence of any antibiotic. As the wells of lines A through D in row 1 are completely clear, the organism is exquisitely sensitive to all tested concentrations of the antibiotic. As the wells of lines A through D of row 2 are all clouded, the organism is resistant to all tested concentrations of the row 2 antibiotic. Moderate antibiotic sensitivity is exhibited in row 3 wherein the line A well is clouded, the lines C and D wells are clear and the line B well is partialy clouded.
While the plate member and cover of this invention may be made from a wide range of materials such as plastic or glass, for example, it is generally preferred to mold these elements from a strong, impermeable transparent plastic material. Among the materials particularly suited for such use are styrene, polycarbonate, polyamides, vinyls (such as polyvinyl chloride, for example), acrylics, as well as polymers and copolymers thereof. These materials not only possess the desired properties, but can also be economically manufactured into the desired shape by modern conventional plastic fabricating techniques and may be advantageously molded as a unit.
In order to provide a specific illustration of how the microbiological test container of this invention might be employed, two examples will be considered. These will demonstrate that accurate quantitative antibiotic sensitivities can readily be determined within twenty-four hours after the organism is isolated in the laboratory.
EXAMPLE 1 A test container for use in testing for Gram-negative organisms will be considered first. The test container has a plate member generally of the type shown in FIG. l, with twelve rows of wells being lled. The plate member is of generally rectangular configuration and has a length of 15.1 centimeters and a width of 6.7 centimeters. Wells in a given row are spaced three millimeters apart and wells in adjacent rows are spaced 7 millimeters apart with a separation being effected by upwardly directed ribs having a height of 2 millimeters and a width of 2 millimeters. The specimen receiving trough is 7 millimeters wide and 5 millimeters deep. Each well is of cylindrical shape and has a depth of 5 millimeters and a diameter of 7 millimeters. Each of the twelve rows has the wells filled with a nurtient media having an agar concentration of 1.5% so that the media will be solid and not shift during transport. (Other solidifying agents such as methyl cellulose may be used, if desired.) The media upper level is generally coplanar with the upper surface of adjacent plate portions. The S line of wells contains no antiboitic and serves as a standard to confirm the growth of organisms on the agar nutrient. Each row has a different antibiotic from the other rows with the antibiotic being arranged in increasing concentrations in wells of lines A through D so that substantially simultaneous inoculation can be achieved. The concentration of the antibiotic will be 1.0 micrograms per milliliter of agar for the A line, 5.0 micrograms per milliliter of agar for the B line, 10.0 micrograms per milliliter of agar for the C line and 20.0 micrograms per milliliter of agar for the D line, except that 50.0 micrograms per milliliter of agar will be employed in the D line for rows with carbenicillin and nalidixic acid. The rst row is provided with penicillin, sodium G in wells A through D. The secondl row is provided with ampicillin, trihydrate. The following materials are provided in each of the suceeding rows in the concentrations indicated above: third row-cethalothin, sodium; fourth row-carbenicillin, sodium; fifth :row--kanamycim sulfate; sixth row-streptomycin, sulfate; seventh rowgentimycin, sulfate; eighth row-tetracycline, hydrochloride; ninth row-chloramphenicol; 'tenth row-nitroferanfoin; eleventh rownalidixic acid; and twelth row-polymyxin B. A colony of the organism to be tested is then inoculated into a test tube with a liquid broth at 37 C. for about 2 to 6 hours and then the broth is placed into the specimen receiving trough by means of a pipet. The organism containing specimen is absorbed into the head of the specimen distributor. The specimen distributor is then lifted from the trough and first simultaneously inoculates the organisms into the agar of each filled row in the S line. Similar simultaneous disposition is effected sequentially through lines A, B, C and D. The cover is placed on the plate and the container is incubated at 37 C. for 18 to 24 hours. For convenience, the plates for each organism being tested may be stacked vertically to provide more use of space. The S line will exhibit growth by appearing clouded thus confirming growth of the organism in the absence of any antibiotic. Inspection of the agar wells of lines A, B, C and D will show whether the organism is sensitive to a particular concentration of antiboitic. The lowest concentration of each antibiotic.
which completely inhibits the growth of the organism (clear surface of the agar) will be the minimum inhibitory concentration to which the organism is sensitive. A clear appearance will provide such an indication. Lack of sensitivity will be shown by a clouded agar appearance or a single colony present on the agar. In this fashion the degree of sensitivity of the organism to the effective antibiotic is established. Thus, klebsiella may be sensitive to one microgram of cethalothin, twenty micrograms of kanamycin, ve micrograms of gentimycin and have additional sensitivities to other concentrations of other antibiotics. It will be appreciated that this information is substantially more valuable than that provided by techniques which express results simply as sensitive or resistant.
After completion of the test, the container may be discorded.
EXAMPLE 2 The test of Example 1 may be repeated employing some different antibiotics in a test for Gram-positive bacteria. -In this test seven rows of wells are employed. The S line of wells is filled with solely solid nutrient agar. The following antibiotics (concentrations: expressed in micrograms per milliliter of nutrient media agar) are provided in the following concentrations inwellsA through D: row l-penicillin, sodium G (0.1, 0.5, 1.0 and 5); row
2-crythromycin, ethylsuccinate (0.1, 0.5, 1.0 and 5); row 3-methicillin (2, 4, 8, 16); row 4-lincomycin, hydrochloride (0.1, 0.5, 1.0 and 5); row S-celhalothin, sodium (0.1, 0.5, 1.0 and 5 row 6--clindamycin, palmate (0.1, 0.5, 1.0 and 5); and row 7-cloxacillin (0.1, 0.5, 1.5 and 5). The test is completed in the identical fashion as in Example 1 with the results indicating in which antibiotics and in which concentrations sensitivity of the organism exists.
For patients with staphylococcae infections the use of the one microgram concentration of penicillin is nedded. If the staphylococus grows on the one microgram it indicates that the organism makes penicillinase, and that penicillinase resistant antibiotics must be used.
The microbiological test container of this invention is adapted for use with both aerobic and anaerobic bacterial. In aerobic testing, the cover is sealed after inoculation of the plate so as to trap air and prevent the loss of moisture from the wells during incubation. When it is desired to perform antibiotic testing with anaerobic bacteria, the gasket sealing strip need not be provided or may be removed from the cover so that air over the plate can be evacuated when the plates are placed in an anaerobic jar (such as a Brewer anaerobic jar) which has been activated. In order to prevent excessive loss of moisture from plate member wells, a moisture containing sponge or other source of moisture should be placed in the air tight anaerobic jar to keep the water vapor at a sufficiently high level.
A quality control system test may be used to establish the stability of the antibiotics in the wells of the microbiological test containers. It may be used to test the effectiveness of a single container, of a .group ready for used, if desired. A Gram-negative organism which is known to be sensitive to the lowest concentration of each antibiotic is applied to the wells of antibiotics of the plate selected for testing the quality of a Gram-negative bacteria test container. After several hours of growth in broth, this test organism will be simultaneously inoculated on al1 the agar wells by the distributor member. If incubation produces growth of this bacteria on even the lowest concentration of any antibiotic it will indicate that the antibiotic has deteriorated and that the test needs to be repeated on a new set of plates. A Gram-positive organism can be used when testing the plate used for this type of bacteria.
It will be appreciated that the microbiological test container and method of this invention are uniquely suited to be employed in tests for a wide range of Gram-positive and Gram-negative material including both aerobic and anaerobic bacteria. Examples of the usual aerobic Gramnegative bacteria which may be tested by this invention are escherichiae, klebsiella, enterobacter, citrobacter, Proteus, pseudonomas, salmonella, Shigella, hemophilus, neisseria and other forms of aerobic Gram-negative bacteria. Growth factors such as X and V may be added to the media for growth of fastidious organisms such as hemophilus, for example. The test container is also suitable for all forms of Gram-positive bacteria such as streptococci, staphylocci and pneumoncocci, for example. The container may advantageously be employed to test for all forms of anaerobic bacteria such as bacteriodes, anaerobic streptococci, clostridia and fusobacterium, for example. While specific reference has been made herein to use of the container of this invention in testing bacteria, it will be appreciated that the invention may be employed to test fungi against antifungal agents such as amphotercin B and 5- fluorocytosine, for example.
While for purposes of simplicity of disclosure the specic plate member shown herein and the cover member have been illustrated as being composed essentially of a web of plastic which has been formed to provide the various wells and troughs, it will be appreciated that other forms of creating the desired wells and troughs may be provided. For example, one may provide a block of generally solid section within which the various wells and tr'oughs have been formed. Also, while certain forms of cooperation between the cover member and plate to establish a friction fit closed container have been shown, other forms of cover-plate closure relationships may be employed, if desired.
While the specific structure disclosed in detail herein has a generally rectangular periphery in plan and such geometry is preferred, the invention is not so limited and other external peripheral shapes may be employed while retaining the benefits of this invention.
While for simplicity of description herein reference has been made to the use of agar or methyl cellulose in the solid nutrient media, it will be appreciated that other suitable nutrient media and growth factors may be employed as well as a wide range of types of agar material to solidify the media. While for purposes of simplicity of reference there has been discussed visual examination of the container with the naked eye and such use will generally be effective, it will be appreciated that Aa magnifying glass or other instrument might also be employed in reaching a conclusion based upon the cultures taken with the present invention. One of the prime advantages of the present invention is that it permits observation on the surface of the agar wells of even a single antibiotic resistant colony which might be undetectable with many other systems.
It will therefore be appreciated that the present invention provides a factory filled sterile antibiotic testing container which is adapted to minimize laboratory time, storage space and to provide accurate quantitative results easily and rapidly in doctors offices, laboratories and other smaller medical facilities, as well as in major medical centers. All of this is accomplished while providing substantially simultaneous inoculation of a -multiplicity of agar wells and permitting the use of a large number of antibiotics with multiple concentrations of each. In addition, a standard nutrient test well or a series of the same is provided.
Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details may be made without departing from the invention as deened in the appended claims.
1. An antibiotic test container comprising a plate member having a plurality of integrally formed upwardly open test wells,
an elongated upwardly open specimen receiving trough,
said wells disposed in a number of generally parallel rows oriented generally transversely with respect to said elongated specimen receiving trough,
at least a major number of said wells in a number of said rows being filled with a mixture of a solid nutrient media and a predetermined concentration of an antibiotic with the upper surface of the mixture being disposed generally at the well opening,
a specimen distributor having a handle portion and an elongated head portion provided with a specimen receiving section having a number of inoculating zones to effect simultaneous inoculation of a number of said wells,
said specimen receiving section of said specimen distributor being so congurated as to be at least partially receivable within said specimen receiving trough, and
a cover member secured to said plate member, whereby upon removal of said cover member an organism containing broth may be introduced into said specimen receiving trough and be at least partially received by said specimen distributor section with movement of said specimen distributor over said plate member producing simultaneous inoculation of said organism on said nutrient-antibiotic mixture surfaces of a rst well of each said filled row and simultaneous inoculation of groups of the remaining Ifilled wells being effected sequentially to provide substantially simultaneous inoculation of all said lled wells.
2. The test container of claim 1 including elongated barrier elements disposed between adjacent rows of said filled wells for resisting ow of fluid between said rows.
3. The test container of claim 2 including said barrier elements being integrally formed ribs having a length not less than the length of the adjacent filled rows.
4. The test container of claim 1 including one said well in each said filled row being a standard well containing a solid nutrient media but no antibiotic, and
the remainder of said wells in said row being test wells having a specific antibiotic with a diiierent concentration of said antibiotic present in each said test well of said row.
5. The test container of claim 4 including said test wells of each said filled row having an antibiotic concentration generally related to the distance between said test well and said specimen receiving trough with the test well having the highest antibiotic Cil concentration being disposed farther from said specimen receiving trough than the other said wells of said row.
6. The test container of claim 2 including said wells being of generally cylindrical shape,
said wells having a depth of about 3 to 10 millimeters,
said wells having an internal diameter of about 5 to 10 millimeters.
7. The test container of claim 6 including said -wells having a depth of -about 5 to 7 millimeters,
said wells having an internal diameter of about 6 to 8 millimeters.
8. The test container of claim 6 including each said filled row having at least four said test wells.
9. The test container of claim 8 including each said filled row having a different antibiotic from the other said antibiotic containing rows.
10. The test container of claim 1 including said plate member having an integrally formed upwardly projecting peripheral wall which extends upwardly to a level spaced above the level of said well openings, whereby spillage of said organism containing broth is resisted.
11. The test container of claim 10 including said specimen receiving trough extending at least from the first said filled row to the filled row most remote from said first filled row, and
said specimen distributor head having a length not less than the distance between said first filled row and said remote filled row.
12. The test container of claim 1 including said cover member having a top panel and downwardly projecting flanges, and
said plate member having lateral surfaces cooperating with said cover flanges to close said container.
13. The `test container of claim 12 including a sealing member compressively secured between said cover member and said plate member when said members are in relatively closed position.
14. The test container of claim 13 including said sealing member being secured to the undersurface of said cover member top panel, and
said sealing member disposed in contact with the periphery of said plate member upper surface.
1S. The test container of claim 14 including said sealing member being readily manually removable from said cover member.
16. The test container of claim 12 including said cover member top panel being in spaced overlying relationship with respect to said -filled rows when said container is closed.
17. The test container of claim 6 including at least one said well of said plate member being a standard well containing solid nutrient media but no antibiotic.
18. The test container of claim 7 including said well rows oriented generally perpendicular with respect to said specimen receiving trough.
19. The test container of claim 10 including said upwardly projecting peripheral wall being substantially continuous, and
said peripheral wall having an overall height of about 11/2 to 3 times the average depth of said wells.
20. The test container of claim 19 including said peripheral wall being hollow and having a pair of spaced vertical wall elements and a connecting horizontal upper wall element.
21. The test container of claim 10 including said specimen distributor having a handle portion formed integrally with said cover member.
22. The test container of claim 10 including the first said well in each said filled row being in a line with the first said wells of the other said filled rows,
succeeding wells of each said filled row being in lines `with corresponding wells of the other said rows, and
said lines being disposed generally parallel with respect to said specimen receiving trough, whereby said specimen distributor will inoculate all filled wells within one said line simultaneously and will simultaneously inoculate the wells of each other said line in sequence.
23. A method of antibiotic sensitivity testing comprising providing a unitary plate member having an upwardly open, elongated specimen receiving trough and a plurality of upwardly open wells disposed in a number of lines with each said well being filled with a solid nutrient media and at least a major number of said wells having a specific concentration of a specific antibiotic,
providing a specimen distributor having a specimen receiving portion of a length not less than the length of said well lines,
introducing an organism containing broth into said specimen receiving trough,
introducing at least a part of said specimen receiving portion into said trough to permit at least a portion of said organism containing broth to be received by said portion,
substantially simultaneously inoculating said wells with said organism containing broth by positioning said specimen receiving portion in overlying relationship with respect to a first line of said wells and simultaneously effecting inoculation of said wells of said first line and subsequently effecting simultaneous inoculation of other lines of said wells, and
subsequently incubating said inoculated plate.
24. The method of antibiotic sensivity testing of claim 23 including 24 including providing said antibiotic concentrations within said rows with the well closest to said specimen receiving trough being devoid of said antibiotic and the antibiotic concentration of the other said wells within said rows being progressively greater from the antibiotic containing well within said row closest to said trough to the antibiotic Well farthest from said trough, and
preventing substantial loss of moisture by said solid nutrient media during said incubation period.
26. The method of antibiotic sensitivity testing of claim 25 including providing a cover member having a top panel and depending ange, and
after inoculating said wells but prior to said incubation, sealingly afiixing said cover member to said plate member with said cover top panel disposed in spaced overlying relationship with respect to said wells, whereby said sealed plate may be incubated without substantial loss of moisture and with the air entrapped between said plate member and said cover serving to support growth of aerobic organisms.
References Cited UNITED STATES PATENTS 3,234,107 2/1966 Kaufman etal. 195-139 0 A. LOUTS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner U.S. Cl. X.R. 195-139 TED STATES "A @FFME l mmmci'rr or MR EWMN Patent No., 3, 826 73.7 Dated July 30, 1974 Inventor) Verne E. Gilbert et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
Col. 2ly line 43 after "that" change "no" to n.o"c
ColIn 2, line 70 after "be" change "preformed" to mperzformeim-@-- Col. 3, line 43 after "of" change "eonc'entratioins" to concentratiozns L Colm, 5f, lime 29 after "of" change "antiboitics" to "antibiotics-- Colo 5 line 35 after "will" change "represnt" to --represent-- Col. 6 line '70 after "row 7" insert --and row 8 Col. 7 line 73 after "compressible" change "materials" to material Col. 9, line 2 after "lower" change "extermity" to -extremity- Col.D l0 line 4 after "a" change "nurtient" to u--nutrien-t Gol., ll@ line ll after "is" change "nedded" to -needed ColE 1l lines 16ml? after "anaerobic" change "bacterial" to --bacteriacor, im iin@ m after "as" chang@ "defiened" to undefined-m- Col@ 14g lime 54 after "and" change "subsequently" to .Signed amd sealed this 5th day of November 1974.
(SEAL) Atream MeCOY Mma GBSON JRn C DANN Artemio@ @ffimer Commiseioner of Patents FORM F30-1050 (1069) uscoMM-oc Goan-P69 U.S, GOVERNMENT PRINTING OFFICE z i919 0-'366-334,