FI60316C - APPARATUS AND EQUIPMENT FOR THE CONSTRUCTION OF ANTIBIOTICS - Google Patents

Description

@ SU OM I —FI N LAN D Patent Publication — patentkrift 6031 6 IT © © Kv.lk7lnt.CI? G 01 N 21/03 φ © Patent application - Patentansökning 762861 ® @ Application date - Ansöknlngsdag O7.lO.76 @ Start date —Giltlghetsdag 13.08.73 ® Published to the public - Blivlt offentllg 07.10.76 @ Date of publication and publication.

Ansökan utlagd och utl.skriften publicerad 31.08.8l

National Board of Patents and Registration © Patent granted - Patent meddelat 10.12.81

Patents and registration Λ

Claim claimed - Begärd priority 18.08.72 USA (US) 2819 ^ 6 (73) Warner-Lambert Company, 201 Tabor Road, Morris Plains, New Jersey O795O, USA (US) (72) Julius Praglin, East Lyme, Conn., David Kenneth Longhenry, East Lyme,

Conn., Alan Clarkston Curtiss, Old Lyme, Conn., James Edward McKie,

Jr., Ledyard, Conn., USA (US) (7 * 0 Oy Kolster Ab (5 * 0 Method and apparatus for determining the susceptibility of antibiotics - Förfarande och apparatur för bestämning av antibioters känslighet (62) Separated from application 2537/73 (patent 53591 ) -Avdelad frän trap 2537/73 (patent 53591)

The invention relates to a container for use in dispensing a solution into a plurality of identical samples to which different reagents are added, each of which must be tested for reaction with the solution, comprising filling blocks in which the dispensed solution is evenly distributed and transferred to respective test blocks.

Hospital clinical laboratories have had problems identifying the antibiotic to which the disease-causing bacterium isolated from the patient is most sensitive. In the Kirby-Bauer method described in the article entitled "Disc Suspeptibility Testing" in "Hospital Practice", February 1970, Vol. 5, No. 2, pages 91-100, a zone of inhibition is measured around an antibiotic plate or vortex in a gel containing bacteria. This takes about one day and requires quite a bit of handling, labor, and exposure to the disease-causing bacterium. A highly automated particle counting system is described in "Applied Microbiology", December 1971, pages 98Ο-986. It gives results in a couple of three hours, but is extremely complex and expensive and thus kills bacteria, thus preventing replication. Light scattering laser beam photometers have also been used to study changes in the scattering curve obtained with a rotating detector to determine the antibiotic susceptibility of bacteria. It is an object of the present invention to provide a relatively simple and inexpensive compartment container which is suitable for antibiotic susceptibility testing and is not only based on accepted and validated principles of antibiotic susceptibility testing, but is also fast, efficient, economical and simple to use.

According to the invention there is provided a compartmentalized container for use in dispensing a solution into a plurality of identical samples to which various reagents are added, each of which must be tested for reaction, the container comprising a longitudinal series of compartments having a longitudinal axis. are capable of holding fluids with the filling and test blocks of all compartments substantially in line, to keep the test block support block set test blocks down to allow fluid to flow into and remain in the compartment set test blocks, to fill the block to all the filling blocks to cause the solution to flow from the distribution space to the filling blocks between all the filling blocks of the connecting openings in order to make the solution evenly distributed among the filling blocks, each filling block being connected to allow fluid to flow between them so that rotating the container about its axis from the down position to the down position allows the amounts of solution evenly distributed in the charge blocks to flow from the upper block to the test block walls.

The compartmentalized container of the present invention is very useful as part of an apparatus for carrying out the method described in application No. 2537/72 for determining the relative efficacy of various antibiotics on bacteria.

The invention is mainly characterized in that each filling block and the associated test block are located in their own longitudinal longitudinal compartment set compartment, that the container is provided with a distribution space which receives the solution to be dispensed and through the connecting openings between the filling blocks, that each filling block is connected to an associated test block so that the solution flows from the filling blocks to the test blocks when the container is rotated about the longitudinal axis until it rests on the test block support to place in the test block.

Using this compartmentalized container, hereinafter referred to as a cuvette, the efficacy of various reagents, such as antibiotics, can be determined by adding antibiotics 3,60316 simultaneously to several identical samples of the bacterial suspension. The preparation of samples, the addition of antibiotics to them and their analysis are greatly facilitated when they are placed in a compartmentalized container, i.e. a cuvette, and then the antibiotic plates are simultaneously transferred from the plate dispenser to the samples. In preparation for this insertion of plates, the interconnected filling and test blocks are turned sideways to fill them with equal amounts of bacterial suspension as it moves from the distribution space to the blocks. When the cuvette is then turned to a position where the interconnected blocks are in an upright position, the sample solution flows from the confluent filling blocks to separate test blocks. Antibiotic plates can be placed in the cuvette even before the inoculum, i. subsistence wear, if required by practical needs. The test blocks are preferably light transmissive and shaped to allow rapid photometric analysis, e.g. by passing them quickly past the light source and the photometric sensor, preferably a sensor of the light odor detection type. If the determined growth index is too low, the cuvette can be re-incubated and re-read before discarding.

For a better understanding of the present invention, it will now be described in more detail with reference to the accompanying drawings, in which like parts are designated by like reference numerals and in which Figure 1 is a partially schematic diagram of an antibiotic susceptibility test apparatus including a cuvette according to the present invention; Figure 2 is a plan view of the cuvette of the apparatus shown in Figure 1; Figure 3 is a front view of the cuvette shown in Figure 2; Fig. 11 is a plan view of the cuvette shown in Figs. 2 and 3; Figure 5 is a rear view of the cuvette shown in Figures 3 and 11; Figure 6 is an end view of the left end of the cuvette shown in Figures 2 and 3; Figure 7 is an end view of the right end of the cuvette shown in Figures 2 and 3; Fig. 8 is a cross-sectional view taken along line 8-8 of Fig. 3; and Figures 9-13 are partially schematic views of the sequential filling steps of the cuvette shown in Figures 2-8 as it is filled from the feed tube.

Figure 1 shows an apparatus for determining the relative effectiveness of a variety of reagents, such as antibiotics (e.g., twelve), in inhibiting bacterial growth, comprising a disposable plastic cuvette 12 in which sensitivity tests are performed, a plate 16 in a dispenser 14, an incubator shaker 30 and shaking the cuvettes and an automatic light scattering photometer analyzer 62 to expose bacterial growth and write the results on a preformed form or tape 22, as will be explained in more detail later.

The clinical isolate obtained prior to the assay described in detail herein is placed in a petri dish 20 where it is allowed to incubate overnight.

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The bacteriologist then picks up several morphologically similar growth colonies from the dish using loop 24, after which they are vortexed into saline in tube 13. Using the photometer conditioning method, the suspension in the tube is brought to the normalized bacterial concentration, which is checked in analyzer 62 by inserting the tube into the opening in cover 74 and viewing the reading on meter 68. Two milliliters of moth suspension is added to 18 milliliters The test tube 7Θ is screwed into a plastic cuvette 12 and a simple treatment causes the contents of the test tube to be evenly distributed in the cuvette triplicate in the test compartment Sc and S1_12. 17 in the growth medium 28 by means of plastic tubes 29 at the top of the cuvette. The thirteenth chamber SQ is a control chamber.

The cuvette 12 is now incubated for three hours in an incubator-trot 30 which is constructed to receive 30 cuvettes. After three hours, cuvette 12 is injected into analyzer 62 and growth in each chamber SQ and evaluated.

By comparing the control chamber SQ, the relative inhibitory effect of each antibiotic in the chambers S.j_12 is calculated and written as described in more detail below.

The lush growth medium has the following composition in grams / liter and has a pH of 7 »0.

Ingredient concentration peptone "C" 15.0 peptone "S" 5.0 dextrose 5.5 sodium chloride 4.0 sodium sulphite 0.2 1-cystine 0.7

The detailed structure of the four components of the apparatus 10 is explained as follows: A. Cuvette 12

Measuring the effect of antimicrobial agents on the growth of microorganisms (bacteria) in the resin requires a chamber (cell) containing the inoculated resin. Measuring growth in an elastic medium by light scattering requires that this chamber be both transmissive to the light emitted and geometrically compatible with the light scattering photometer. The effect of many antimicrobial agents on the growth of a microorganism can be conveniently and quickly viewed by a system of these optical chambers directly in queue form into a single unit. The cuvette 12 also comfortably allowed to transfer an equal amount of inoculated 6 u 31 6 medium to each chamber S. The cuvette 12 is also able to easily receive an antimicrobial impregnated round paper sheet in all its chambers, but cannot receive such an antimicrobial sheet in one of its control chambers. In addition, the cuvette 12 is waterproof, optically polished, optically reproducible, inexpensive, relatively small, stackable, and possibly disposable.

Cuvette 12 is shown in Figures 2-6. It is an optically clear and inert plastic such as polystyrene and is manufactured by injection molding into two-part optical! using polished steel molds. After injection molding, the two parts are sealed together with either solvent or ultrasonic energy to make a cuvette. Ultrasonic sealing is performed because it then avoids damage to the optical surface due to excessive dissolving. The cuvette 12 has one control chamber S in a straight line, i.e. in series, and two test chambers 12 for antimicrobial agents. * The only other material in addition to the polystyrene used in the cuvette 12 shown is a flexible polymer such as a styrene-butadiene polymer 34 . The seal 32 and closure 34 are placed in the cuvette 12 prior to final packaging.

Cuvette 12 comprises a Soisi part: (1) Inoculum orifice (P) (Figures 2-7)

Threaded orifice receiving the inoculated medium 18-43 of the 18-143 spherical tube 78 · A seal 32 placed in the bottom of the orifice forms a watertight seal between the cuvette and the inoculum tube.

(2) Distribution space (r) (Figures 2-7)

It receives the inuculated medium from the inoculum tube when the cuvette is inverted by hand.

(3) Filling blocks 15 connected to their tolls The row of filling blocks 15 extended from end to end of the cuvette in the direction of its longitudinal axis (except for the division space R). They are connected to the dispensing space by the main dispensing opening 31 and receive the inoculated medium from the dispenser when the cuvette is turned by hand to cause equal volumes of solution to flow. to the blocks through the manifolds 33, which is aided by the return flow of air through the air holes 35. The area of the distribution openings 33 increases away from the distribution space R.

(4) Test blocks 17 (Figures 7 .1a 8)

Thirteen separate test blocks 17 disintegrating in the compartments Sc1 (S2, S2 "si2 ^ va * oa) receive an equal volume of inoculated medium from the interconnected filling blocks 15 when the cuvette is manually rotated 90 ° about its longitudinal axis to count the filling blocks. filled with inoculated medium, they are insulated from each other by 6 6 L »ό 1 6 partitions 3β · Distribution openings 33 and ll holes 35 located at the top of the partition 36 and well above the level of the medium are the only connections between the compartments. 13 «as described above.

(5) Cavities of antimicrobial plates (29) (Figures 7 .1a 8)

Twelve apertured hollow rods 29 extend down to the test blocks of the twelve compartments. Each hollow rod, known as a plate holder, receives a round antimicrobial paper plate 16 (6.5 mm in diameter) through twelve plate openings 26 in the upper surface of the cuvette 12. The plate falls into the plate holder 29 and rests on the bottom of the holder 73 · Two elution openings E are formed in the wall of the plate holder near the plate 16, allowing the antimicrobial (i.e., antibacterial) agent to elute into the inoculated medium in the test block. Twelve bumps 40 (called plugs) of the closure 34 are inserted into the plate openings 26 to form a watertight seal in each plate holder. The closure 34 is interposed between two parallel rails 34a between which the openings 26 in the upper surface of the cuvette 12 are located.

(6) Bracket B (Figures 2, 1a 4-8)

At the rear of the cuvette 12 is a longitudinal L-shaped bracket B with which the cuvette can be attached to the holders 42 of the incubator shaker 30 and to the spreader of the photometer carriage. Thus, the cuvette holder B allows its correct accumulation for both the incubation-shaking period and the photometer wiping period.

The incubator shaker, shown in Figure 1, receives thirty cuvettes in three interchangeable shelves 54. Each sleeve 54 has ten holders 42 for holding the cuvettes by means of their own holder B.

The cuvettes are rotated or mixed in an incubator maintained at 36-0.5 ° C with an on-no temperature controller. The incubator has a safety thermostat 60 to provide a backup temperature control system in the event of a break in the main thermostat. the lacubator-shaker 30 has a speed control knob 63 with which its circulating frequency of the shaking movement and a meter 65a for changing the rotational frequency and a temperature setting knob 65 for changing the temperature can be changed.

A. Preparation of vacloin wear

The standard type of inoculum is a suspension of pure bacteria in 0.90 wt.% Sodium chloride solution with 1-3 x 10 'viable cells per milliliter. This normal type of saline solution wear in an optically acceptable, i. in a clean, scratch-free 16 x 125 un round bottom silicon I tube, gives a scattering signal at 35 angles - log S between 2.2 (1x 10 cells / 7 cells / ml) and 1.9 (3 x 10 'cells / ml) when set the photometer.

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The photometer conditioning meter 68 has a center range (covering 40 i ° of the entire range of the meter), which is the "right inoculum range". Its two limits correspond to the limits of the acceptable decomposition range. The left area of the meter (covering $ 30 of the entire area of the meter) means "below" and / or "more microorganisms." The area on the right (covering the remaining 30 # full area of the meter) indicates "over" and / or "dilute with saline".

Standard inoculum is prepared by transferring the growth colony or colonies of a particular bacterium 16-24 h agar agar to a standard x 16 mm saline inoculum tube 13 containing 6 x 0 ml of 0.45 membrane filtered sterile 0.90 saline. A microbiological loop 24 is used for this purpose and a standard flame is used for sterilization.

Although the solution of how many growth colonies must be placed in an aqueous solution tube to obtain a suitable concentration range is ultimately a matter of practice and depends on colony consistency and size, approximate guidelines for colony diameter and number of colonies can be developed to facilitate rapid inoculum production.

Once the colonies have been looped into the saline tube 13 (careful rubbing of the loop against the inside of the tube just below the concave portion helps to remove the particularly pasty colonies from the loop), the tube 13 is flamed, covered with a clearing cap, held in a vortex for 13 seconds and placed in the photometer cover. A white, vertical mark on the top of the tube helps to position it (the white mark is aligned with a similar line on the lid of the photometer). The conditioning button 79 is then pressed and it is noted where the pointer of the meter 66 is located. If the pointer is within the normal inoculum range, the saline inoculum is ready to be introduced into the cuvette. If the pointer is in the "below" range, the tube 13 is removed from the photometer and growth colony or colonies are added.

If the pointer is in the "over" range, then sterile, filtered 0.90 wt% saline is gradually added to the tube until the inoculum is diluted to the normal range.

B. Antibiotics exposed to standardized inoculum (l) Ryvetin nanostructured with antimicrobial plates

Once the desired group of antimicrobial agents (antibiotics) has been selected, the plate dispenser 14 is loaded by inverting it and inserting the charged cartridges 39 (mouth up) into the holes 19 of the dispenser 14. Care must be taken that the plates 16 of each cartridge 39 are properly packed (i.e., 90 ° at an angle to the longitudinal axis of the cartridge) and that the distance between the top plate and the mouth of the tube is no longer than 3 mm. Once the lime cartridges have been charged, the dispenser 14 is turned back to its normal vertical position. The closure 34 is removed 6 O 31 6 from the cuvette 12. With the dispenser 14 standing on a bench (or suitable table), the cuvette 12 is placed in the dispenser guide and pushed to the stop 45. The dispenser lever arm 51 is then depressed and then released, with one plate 16 entering each plate opening 26 of the cuvette 12. The cuvette 12 is removed from the dispenser and the closure 54 is firmly in place to close the cuvette.

(2) Filling the cuvette with maintenance nine wear11a (Figures 9-15)

Following the standard procedure, 2.0 ml of euum solution wear is transferred from tube 13 to a 20 x 12 mm tube 78 (flat-bottomed, silicate, 18-145 screw cap) with 18.0 ml of sterile, 0.45 μm membrane-filtered lush growth medium (described above) using a sterile pipette. . The usual microbiological method of flaming the mouths of the tubes is used, and after insertion of the ino-wear, the tube is closed with a screw cap. This tube 7Θ is then gently inverted several times to mix the contents, the lid is removed and immediately thereafter the tube is screwed vertically into the inoculation opening P of the cuvette until it presses firmly against the seal 52. The first step and subsequent lies in filling the cuvette with eLoader consumption are shown in Figures 9 and 10-13, respectively. The cuvette 12 is now carefully rotated 180 ° so that the contents of the inoculum tube 78 flow completely into the cuvette distribution space R (Figure 10). The cuvette 12 is set to rest on its end space 112 on a horizontal surface 114 · In this position, the longitudinal axis of the cuvette is perpendicular to the horizontal surface, as shown in Figure 11. The cuvette 12 is then rotated 90 ° so that the inoculum flows from the manifold to the interconnected dispensing blocks 15, as shown in Figure 12. This inversion is most easily accomplished by gripping the non-dividing end of the cuvette and lowering it onto a horizontal surface so that the back side 9 of the cuvette (where the cuvette holder B is) rests on the horizontal surface 114 (as shown in Figure 13), the back side 9 providing support for holding blocks. And to cause the media wear to be divided into filling blocks. Drainage into the filling blocks is complete after 8 seconds, after which a final inversion is performed. This only involves rotating the cuvette 90 ° about its longitudinal axis to a vertical position (i.e., the position where it was loaded with antimicron plates) to rest on its lower walls 116 and the leg 118 below its Distribution R, the edges 116 and leg 118 forming a test block support to hold the test block assembly so that are down to allow the medium to run into and remain in these plots. It is very important that the cuvette remains horizontal during this last turn, this is ensured by performing the turn in such a way that both ends of the cuvette remain in contact during the horizontal surface of the turn. Examining a correctly filled cuvette, it can be seen that the height of the medium wear is the same for all

Claims (16)

6031 6 9 sa in the test blocks. The plate 16 in each cavity holder 29 should be just below the surface of the medium. In some cases, the plates 16 are not located on the same level, however, this does not cause problems as long as the plate is in contact with the medium. C. Injection of filled cuvette, 1a shaking As soon as the medium wear is divided into a cuvette loaded with antimicrobial plates, this is placed in an incubator shaker at 5 °. It is recommended that If, for example, 10 bacterial isolates are to be tested per hour, ten standard inoculations are first prepared, after which the cuvettes are filled and placed on one shelf of the incubator-shaker. They are then simultaneously incubated and shaken for a constant 3 hours at 36 ° C. During the incubation period, the incubator shaker may be momentarily stopped to insert a second or third cuvette shelf. After a three-hour incubation and geculation step, the cuvette shelves are removed and taken to a photometer for reading. The reading process is repeated for each chamber, i.e. the compartment, until a reading has been obtained from all the chambers and the result has been listed. 1. A container intended for use in dividing a solution into several identical samples, to which are added different reagents, each of which must be tested for reaction with the solution, and which contains filling blocks in which the solution to be distributed is evenly distributed and transferred to corresponding test blocks. each filling block (15) and the associated test block (17) are located in their own longitudinal longitudinal compartment set (S 1 -S 2), the container (12) is provided with a distribution space (R) which receives the solution to be dispensed and the container rests on the filling block support ( 9) on the basis that the filling blocks are located below the distribution space, place the solution in the filling blocks evenly distributed through the connecting openings (33) between the filling blocks, each filling block connected to the associated test block until the solution flows around the filling blocks to the test blocks;on a test block support (116, 118), wherein the test blocks are located below the filling blocks, and that there is an opening (26) in the wall of each compartment above the test block for placing the reagent in the test block. ίο 60 31 6 Container according to claim 1, characterized in that the distribution space (R) has a connection (P) for a supply pipe (78) containing a predetermined amount of solution, both the supply pipe and the distribution space being able to receive an amount of solution that can be dispensed and accommodated separately. to the tray of the longitudinal tray set (S ^ ~ S ^). Container according to Claim 1 or 2, characterized in that each compartment (S 2 -S) is approximately in the shape of a pistol housing with its stern and barrel parts, the filling blocks (15) forming the rear parts and the test blocks (17) being barrel parts. A container according to claim 3, characterized in that said wall of each compartment comprises an aperture cavity rod (29) inserted inside the respective compartment and that the tips of the cavity rods have openings (E) through which the contents of the cavity rods elute into the samples in the test blocks (17). A container according to claim k, characterized in that each hollow rod (29) is a substantially closed tube with slits adjacent said tip. Container according to Claim h or 5, characterized in that a removable stopper (3 ** a) is arranged in each opening (26) which leads to a corresponding cavity rod. A container according to claim 6, characterized in that the stoppers comprise a flexible strip (3 * 0) with a longitudinal series of individual stoppers connected to the strip to facilitate their insertion and removal. Container according to claim 1, characterized in that the distribution space (R) is delimited by one of said compartments and is connected to it by a filling opening (31) and that the container also has a partition (36) between each of two adjacent compartments, the connecting openings (33) at the bottom of the filling opening when the filling blocks are below the distribution space, as a result of which the solution flows and is evenly distributed in all the filling blocks. Container according to Claim 8, characterized in that one air hole (35) is formed in each partition wall in order to facilitate the even distribution of the solution in the filling blocks. A container according to claim 9, characterized in that it has a top wall forming a surface remote from the blocks, that the openings (26) are in this top wall and each of them forms said top wall. the open end of the hollow pin connected to the upward wall, the tip of each hollow pin being inserted into the corresponding compartment to be immersed in the root. to the solution in the test block of the tray. 11 60 31 6 Container according to claim 10, characterized in that the tips of the hollow needles are substantially closed so that each tip has a slot. Container according to Claim 2, characterized in that the connection (P) for the supply pipe is arranged substantially parallel to the longitudinal set of compartments, so that the container and the stationary supply pipe are substantially parallel to save space and facilitate pouring of the supply pipe into the distribution space (R). A container according to claim 1, characterized in that it comprises a top part and a bottom part, wherein the top part has an opening top wall for said compartments and the partition space and the top wall are sealed to the bottom part to cover the compartments and the partition space. la. Container according to Claim 13, characterized in that the distribution space has a connection (P) for the supply pipe and that this connection is evenly distributed between the upper and lower parts in order to facilitate its manufacture. A container according to claim 13 or 1U, characterized in that the apertured top wall comprises a series of perforated cavity pins (? 9) »connected to the top and intended to be inserted into the trays when the top and bottom are sealed together. Container according to one of Claims 1 to 15, characterized in that a bracket (B) is attached to it for facilitating its attachment for handling. Container according to claim 16, characterized in that the bracket (B) comprises a strip connected to the second longitudinal side of the set of compartments and the lower edge of which is placed at a distance from said longitudinal side. from the side to provide a mounting gap.