JPS6018400B2 - Test equipment and method for determining minimum inhibitory concentration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a test system, and more particularly to a novel test system in which the proceeding process or test is carried out quickly on a series of instruments in series in precise and automatic relation in several instruments. Regarding test systems.

Antibacterial agents (antibiotics) are generally used to treat infectious diseases.

For example, it is desirable to conduct tests to determine the resistance or susceptibility of infectious bacteria to certain antibiotics, allowing researchers to make informed choices about effective antibiotics. The most widely used technique is commonly referred to as the Kilby-Bawal method.・Take a sample from the patient to obtain infectious bacteria, identify it as gram enteric or negative,
Single machined using standard methods. A diluted medium of isolated bacteria is spread evenly over the surface of the nutrient agar. A paper disc impregnated with a standard known strength antibiotic is placed on the surface. an annular area surrounding a disc containing antibiotics to which bacteria are more or less susceptible (
Bacteria grow on the surface except for the "inhibition zone"). Measure each annular ring and compare it with standard data to determine whether the zone dimension indicates sensitivity or resistance;
Or determine whether it is neutral. However, bacteria are not just "susceptible" or "resistant."

Some bacteria, for example, are resistant to low concentrations of penicillin, but are sensitive above a certain threshold concentration. This test does not determine the above-mentioned cut-point concentrations and therefore does not allow researchers to determine the
They do not provide sufficient data to assess the efficacy in vivo, ie, to determine the required effective regimen at the level of antibiotic concentration that can be achieved in the actual breeding sites of the infectious disease. The Kilpie-Bowell method is relatively easy and inexpensive to perform in small numbers, and requires almost no special equipment.

Tests in which bacteria are cultured under different antibiotic concentrations provide quantitative results. Such a test is performed by preparing several tubes containing a known concentration of antibiotic, inoculating each tube with bacteria, and testing each tube for growth after incubation. Clearly, testing several concentrations of several antibiotics on one type of bacteria requires many test tubes and culture space, uses large amounts of materials, and is a laborious operation. This is completely impractical for high volume testing. Preparations of antibiotics of different concentrations can be prepared by the repeated dilution method, in which a fixed amount of a sample of known concentration is transferred to an equal volume of diluent to achieve a 2:1 dilution.

After mixing, a given amount of the mixture is transferred to an equal amount of diluent, resulting in a 4:1 dilution of the original sample. In theory, this method could be repeated many times to obtain a wide range of formulation dilutions. To carry out the small volume, one-time dilution method, a rhombus is available, using a plastic tray with many wells (see Astle US Pat. No. 3,536,449). This method provides a solution to many practical problems that arise with the dilution of soups in vitro. Practical repeated dilution methods have errors as large as 100% over the course of five or six dilutions, due to the accumulation of tolerances in transfer, distribution, and concentration.

Serial dilution methods are limited in practice to a range of concentrations of only 100:1. For this reason, the threshold effective concentration does not fall within this range, which significantly limits the range of effective data. This does not include the full range of drug concentrations that can be achieved in various infectious agents in vivo. Also, serial dilution devices are speed limited and therefore not suitable for large test volumes. Because of this speed constraint, the test tray is exposed to the risk of contamination during the unreasonable period during which the serial dilution method is performed. Currently, there is no accurate automated equipment available for simultaneously injecting large amounts of inoculum (medium containing test bacteria) into each well of a test tray.

It is therefore a primary object of the present invention to be able to determine the minimum effective concentration (commonly referred to as the minimum inhibitory concentration skin C) as accurately as the soup dilution method while providing the convenience and economy of using small amounts of test material. The objective is to provide a new, partially automated system that maintains the integrity of the test and increases the overall speed of the test in a practical manner for almost any number of tests.

Another object of the invention is to provide a novel, partially automatic system in which the entire sequence of tests is simplified and is not susceptible to errors in identifying test results.

Yet another object of the present invention is to enable test trays containing a large number of individual test materials to be transferred intact between different instruments in a series, ensuring that each well containing progressive test material has the same identity in successive instruments. The aim is to provide a new system that can be placed in any relative position possible. Important aspects of the present invention provide an antibiotic quality testing system in which the intervening instrumentation primarily involves the simultaneous application of different concentrations of antibiotics, mixtures, or other reagents to the wells of individual test trays. an inoculator, where the inoculum is injected into the individual wells of the test tray; a viewer, which illuminates the test tray to provide visual confirmation of the test result or progress; A particular object of the invention is to provide a new system in which the test tray is dispensed in only a single properly oriented position in each successive instrument.

Still another object of the invention is that the wells of the individual test trays are indexed and provided with means for forcing the test trays and instruments into mutually interacting forces, such as notches or protrusions, which join together. The present invention consists in providing a novel system of the above-mentioned type in which the trays are stationary only at properly associated oriented positions in each successive test instrument.

Other objectives will become apparent as the description proceeds with reference to the appended claims and the accompanying drawings.

The present invention is disclosed as shown in FIG. 1 as being used in a new antibiotic quality testing system in its preferred embodiment, but in this system, the mainly included instruments are a dispenser 10 in which the simultaneous distribution of substances takes place, an inoculator 11 in which the inoculum is transferred from said dispenser to the individual wells of the test tray, and the test tray is illuminated to check the result or progress of the test. and(
or) a viewer 13 that can record.

Preferably, a device 12 is provided for applying a sealing cover to the top of the well of the test tray after inoculation so that the tray can be handled or stored without risk until the time of observation. The specific components included in this system are described below.

Preferably, the test tray is a rectangular, generally flat, integral piece of synthetic plastic having a number of upwardly closing wells, each having a U-shaped bottom.

The advantage of the U-shaped bottom is that it concentrates the growing bacteria on the surface, making them easier to visually assess with a Puwa 13 or the like. In Figures 2 to 5, a rectangular tray 20 has a flat top surface 21 that is parallel to a narrow ridge 22 that projects outward over the entire circumference of the lower edge of the tray; An outwardly opening recess 23, preferably in the form of a rectangular notch, shown as being located approximately midway along the ridge, is formed.

A nine-needle well 24 of the same size enters the surface 21, with adjacent wells equally spaced apart. As shown, there are eight columns numbered A to 112 each having 12 wells numbered 1 to 112, so that each well is located in a recess 23 and in an identifiable position in the test tray. The test tray can be shaped with reference lines between each well, as shown at 21', to facilitate location identification.
The test tray is a transparent, one-piece structure that is a disposable titration plate disclosed in U.S. Pat. No. 3,356,462 to Cook et al., issued December 5, 1967. Preferably, the test tray is a plastic element, and although the patent is cited in detail in the text with respect to its composition and construction, the test tray may be provided with the stationary features of the present invention.

Figures 6 and 7 illustrate aspects of the invention incorporated into a dispenser.

The test tray 20 is mounted onto a drawer 25 whose front end is visible in a suitable recess 26 in the dispenser housing 27.

A suitable sliding guide (not shown) is provided for the movement of the drawer 25. The handle 28 is attached to the drawer section 25
The drawer portion 2 is of a length sufficient to place the tray thereon and can be slidably pulled out of the housing and then pushed back into the housing to place the tray in the operative position.
It is fixed at 5. FIG. 7 shows the tray 20 inserted into the drawer and the relative positions of the various parts when the drawer is fully pushed in.

The flat bottom tray has drawer flanges 30, 31 which are laterally inwardly curved between the side walls 34 and 35.
32, 33, with its front end in contact with the front wall 36 of the upright drawer, with the notch 23 facing towards the inside of the instrument. The flange 33 enters the notch 23 when in position when the tray is placed in the drawer. If the tray 20 is incorrectly placed in the drawer with the notch 23 forward, the rear of the tray will be raised upwardly from its proper position by the tab 33 and the drawer will be pushed into the displacer. When the dispensing device is opened, it contacts the front face of the dispensing neck or other fixing member, which prevents the drawer from closing fully and alerts the operator to his mistake. (See Figure 4A) Thus, the combination of tab and tray notch provides a control means to ensure proper orientation of the tray.

A suitable switch actuated by the drawer is connected to a signal circuit for indicating the status in some manner. The dispenser may be checked for other details, such as the individual passageways from the test tube feeder containing the material fed into the wells of each test tray as required. 1 of the title ``Method and Apparatus for''
973 King P., filed on July 11th. S. Citrine
No. 595,005, filed in May 2005.

In this way, tray 20 can be operatively positioned within the dispenser in only one oriented position. FIG. 8 is spaced from the top of the vertical front wall 48 from the vertical rear wall (not shown) to provide a mechanical space 51 (FIG. 9) housing the working parts described below. Inoculator device 45 with a metal plate casing 49 with a flat horizontal table 47 extending against the bottom of the vertical panel 46
shows.

It is desirable that the top surface of the table 47 is smooth, and the table 47 is connected to the tray holder 52 and the sterilization furnace cover 5.
Fix 3.

The tray holder 52 has two rectangular recesses 54 and 55 spaced apart in the longitudinal direction, and the tray holder is connected to the table 47.
Placement buttons 56 and 57 to place the item at the appropriate pointing position above.
is a flat stamped sheet metal element having an end port extending into the receptacle of the table 47.

Once the set button is set, its position will properly position any alternative configuration of tray holder. Since the flat tray holder is made of thin metal, it lies flat on the surface of the table, thus forming shallow rectangular upwardly closing pockets in recesses 54 and 55.

The two upwardly bent end tabs 58, 59 are specifically for removing the tray holder from the table.

In the configuration shown in FIG. 9, the pockets of the tray holder are each approximately the same size and adapted to receive spaced apart containers, such as trays 20 and 60.
As shown, vessels 20 and 60 are both of the microtitration type shown in FIGS.
It has 4. A tab 61 extends on one side of each recess 54 and 55 such that the notch 23 of each test tray mates with the tab 61 at a unique location of said test tray on the table. The test tray holder thus serves as a template for accurately setting the containers 20, 60 in the instrument relative to each other and to the furnace 53 which is fixedly mounted on the table.

The vertical panel 49 is formed with a gate 64 that extends horizontally, and horizontally spaced leg ports 65, 66, and 67 extend downwardly from the port 64.

Vertical inlets 66 and 67 are dimensioned to limit downward movement of inoculating head 68 to prevent unauthorized initiation of a furnace cycle by a control circuit (not shown). An inoculating head 68 extends horizontally through each of the entrances and is provided in J. D., cited in more detail below. F. Controlled along said closures as disclosed in pending U.S. Patent Application No. ``Inoculator'' filed April 26, 1970 by Howard et al. Post 6 for exercising
It is removably attached to the top of 9.

The furnace cover is provided with a nine-way closure 63 arranged to correspond to the position of the well of the tray 20.

The longitudinal rows of trays 20 are aligned with the longitudinal rows of the top dark holes of the furnace by the stationary action of holders 52. As shown in FIG. 8, the head 68 has a number of hanging inoculator pins 71 arranged in eight rows, each row consisting of IZ pins, and the arrangement of the pins corresponds to the arrangement of the openings 63 of the furnace. are doing.

Within the space 51, a support structure 72 is slidably mounted on the rod T3 or the like so as to move parallel to the longitudinal direction of the table 47. The support body 72 is a square post 69
It supports a depending guide structure which provides side guides 74 which can slidably move the parallel surfaces of the post 69 in vertical reciprocating motion. Post 69 is coupled to motor 75 by a suitable mechanism for vertically reciprocating the post in guide 74 as shown. Extending parallel to the rod 73 is a fixed rack bar 76 having a toothing 77 that meshes with a gear on the output shaft of a second motor 78 mounted on said support.

When the motor 78 is activated, the support 72 is moved to the table 47.
is moved horizontally in the longitudinal direction of the Control of motors 75 and 78 to effect linear movement of head 68 is preferably as disclosed in the aforementioned Howard et al. patent application. If a test is required, the tray 60 is also of the microtiter type, with the holder 52 positioned within the recess 55 and within the recess 54 so that the wells of the two trays are always placed in precise relative positions in discernible relationship. A tab 61 corresponding to the tab 61 is provided.

10 and 11 show a viewer with which test tray 20 can be inspected.

FIG. 10 shows opposing side wall parts 81 and 82 and a bottom part 83.
and a bottom wall portion 84.

On the front side there is a partial front wall portion 85 having a downward extension terminating downwardly to provide a forwardly closing space 86 extending across the lower portion of the housing. An inclined reflector member 87 is mounted within the space 86.

Above the reflector 87, a depending extension of the wall 85 is provided with a closure as shown at 88, and a tray carrier 89 is slidable on a guide as shown at 91 for supporting the transparent test tray 20. The transparent test tray 20 is mounted in a position inside the carrier (FIG. 10) with a light source, a target screen (not shown) on the top of the housing, and a reflector 87 where the bottom of the tray is viewed by an observer. is located between.

The guide 91 is a block of nylon or a material with good low friction support properties, and the sliding path of the carrier 89 is horizontal.

The preferred configuration of the carrier 89 is that the test plate 20 is mounted on the carrier in its normal viewing position.
This is shown in Figure 1.

As shown, a tab locking element 92, such as tab 33 in FIG.

The tab is located approximately in the middle of the rear edge of the opening 0 on the sliding surface and protrudes above the Sekiguchi green section. A slot and screw arrangement provides adjustment for proper alignment of the test tray with its associated target. Rectangular front part 95 of the tab
When fixed in place, the parallel side guides 96,9
The green notch 23 of the test tray 20 is placed laterally on the sliding surface between 7 and 7 and is pressed backwards so as to come into contact with the tab.
In this position the individual wells are precisely aligned with the closure 0 in the instrument and the optical system when the sliding surface is pushed fully rearward thereon as shown in FIG. The tray 20 has the cutout 23 on the front side, that is, on the 11th
If placed on the sliding surface 89 with the bottom side of the figure, the tray cannot be pushed forward to be properly positioned over the aperture 0 and with respect to the observation system, and this undesirable condition A device shall be provided within the instrument to prevent retraction of the sliding surfaces, or to be visually detected as in Figure 7, or to signal the condition to the operator in some manner. Become.

Emotion Carrier 89 is a F.A.F. patent application filed on October 3, 1973 under the name "Test Plate Reader". P. Tolosa (To
No. 619,183, pending US patent application Ser. FIG. 1 shows the sequence of the sealing device between the inoculator 11 and the viewer or reader 13. FIG.

The relative positions of the wells are not important in this device, so no orientation device is required. This sealing device is disclosed in the pending F.A. application filed Aug. 21, 1973, entitled "Combined Tape Disvenser and Tray Sealing Device." P. No. 606,454 to Tolosa et al. In the sequence of the system shown in FIG. 1, the tray 20 is thus the only one in each instrument.
These instruments can only be inserted in one correctly oriented position.
A device, such as a tab, is provided that is arranged to engage the notch in the tray only when the tray is properly positioned in the instrument.

In one embodiment, the notch 23 is an opaque area and the replacement tab is a direct replacement for the haze bulb and photovoltaic cell combination so that the tray is properly positioned only when the opaque portion blocks the light beam to the photovoltaic cell. This allows the wells of the test tray to be oriented in exactly the same relative position in each successive instrument, avoiding complicated record-keeping and the possibility of errors. The present invention, in its preferred embodiments, thus constitutes at least a statutory health protection factor. In a typical application of said system, a number of trays,
For example, a sufficient number of trays are allocated for approximately two weeks' worth of test doses, and these trays are frozen.

Each day, remove the number of trays required for that day's test, thaw, inoculate, and apply sealing cover tape. 4 to 4
Culture is carried out for a certain period of time, and the tray is inspected for growth using a viewer or the like.

Aspects of the present system provide the minimum inhibitory concentration (M
IC). In one aspect of the foregoing, the system can be used for simultaneous identification of minimum inhibitory concentration determinations.

This includes injecting an identifying agent into some of the wells of the tray instead of the antibiotic solution and seeing the results in the PUWA. In yet another embodiment of the system, when bacterial growth is detected after the above steps, further manipulation is performed to determine which concentration actually killed the bacteria.

In an extension of this test, samples from each well of the test tray showing growth are injected onto the surface of the nutrient agar using inoculator 11. This procedure removes bacteria free of antibiotics. Samples from wells where bacterial growth was inhibited but not completely killed will show bacterial growth on the agar medium. If the bacteria are killed by exposure to antibiotics, no growth will be observed on the agar tower surface;
In this way, the determination of the minimum concentration for sterilization is facilitated. This system has many advantages.

A single dispenser can be used to prepare trays for laboratories in different locations, in which case only one inoculator, tape sealer, and viewer are needed to complete the test. .

Because of the unpredictable delays between each testing process, it is advantageous to have a series of separate, interrelated instruments, such as in the present invention, rather than a single, integral device.
In each step, the identification of each well is clear and the trays are always placed correspondingly in several instruments. The present invention may be embodied in other forms without departing from its spirit or specific characteristics.

Therefore, the examples in the text should be considered in all respects as illustrative rather than restrictive, and the scope of the invention is expressed in the claims in the heading rather than in the text. All changes that fall within the meanings and ranges of equivalents are intended to be embraced therein.

[Brief explanation of the drawing]

1 is a schematic diagram of a preferred embodiment of the system showing the sequence of instrument installation; FIG. 2 is a plan view of a test tray structure according to a preferred embodiment of the present invention; FIG. 3 is a rear view of the test tray of FIG. 1; FIG. 4 is a partial view taken in section along line 4--4 of FIG. 1; FIG. 4a is a view similar to FIG. 4 showing how an improperly oriented tray can become skewed; FIG. 5 is a partially enlarged rear view showing the test tray emplacement recess; FIG. 6 is a dispensing device in which the test trays of FIGS. 2 to 5 are mounted to receive several prepared antibiotic solutions; FIG. Overall perspective view, FIG. 7 shows the slide for the test tray in the dispenser of FIG. 6, where the test tray is precisely positioned with each well placed in accordance with the antibiotic solution or mixture supply device in another part of the instrument. FIG. 8 is a top view of the movable carrier or drawer; FIG. 8 is a general perspective view of the inoculator apparatus in which the test trays of FIGS. 2-5 are bagged after the distribution operations of FIGS. 6 and 7; FIG. FIG. 8 is a plan view showing the test tray of the holder in the inoculator, FIG. 10 is an overall perspective view showing the viewer showing the state of the test tray in FIGS. 2 to 5 after the inoculation operation, and FIG. FIG. 3 is a plan view showing the test tray in the slidable mounting portion of the viewer shown in the figure. 10... Disvensa, 11 """ Inoculator, 1
2... Instrument, 20... Tray, 21.
...Flat nuchal surface, 22 ... Protrusions, 23 ...
...Recess, 24...Well, 25...
Drawer part, 26... Recessed part, 27... Disvenser housing, 28... Handle, 30, 31, 32, 33
... Flange (tap), 45 ... Inoculator device, 46 ... Casing, 47 ... Table, 48 ... Front wall surface, 49 ...・Vertical panel, 51... Mechanism space, 52. ．． ．．
・．．・Tray holder, 53... Furnace cover, 5
4, 55... Concavity, 56, 57... Authentication button, 60... Tray, 61... Tab, 64, 65
, 66, 67... Sekiguchi, 68... Inoculation head, 69...
...Post, 71...Inoculator pin, 72...
...Support structure, T3...Rod, 74...
...Side guide, 75, 78...Motor,
76...Rack bar, 80...Housing, 81,8
2, 83, 84, 85... Wall section, 87...
Reflector, 89...Tray carrier (sliding surface),
91...Guide, 92...Tab. FIG. 1 FIG. 2 FIG. 3 FIG. 4 fTG. 40 FIG. 5 FTG6 FIG. 7 FIG. 9 FIG. 8 FIG. 10 FIG. 11

Claims (1)

[Scope of Claims] 1. A test device consisting of a series of instruments in which a series of test steps is carried out, the series of instruments each administering different concentrations of antibiotics, mixtures or other reagents in a predetermined order. dispensing means for simultaneously dispensing to individual wells of a test tray having a plurality of wells adapted to be transferred to an instrument; dispensing means for distributing inoculum to individual wells of a test tray; observation means in which the test tray is arranged in an optical system suitable for inspection and comparison; Sometimes control means are provided which allow the test tray to be arranged in each instrument in only one direction, said control means cooperating only when the test tray is arranged in said one direction in each instrument. A test device consisting of a tray and means of interaction with the instrument. 2. The test device according to claim 1, wherein the control means comprises a mutually corresponding arrangement and protrusion. 3. The test device of claim 1, wherein the control means comprises an opaque area and a light source-photovoltaic system. 4. A dispensing means having a plurality of wells oriented in a known direction for dispensing a small quantity of an antibiotic from a plurality of supplies having different concentrations of the antibiotic and arranged in known relative positions. Simultaneously and individually dispense into each well of the test tray, then transfer the test tray to the inoculum dispensing means and orient it, inoculate each well with the inoculum, then transfer the test tray to the observation means. A method for determining the minimum inhibitory concentration, comprising the steps of: transferring and placing in a fixed oriented test position, said wells being disposed in the same distinguishable position in all means. 5. The test method according to claim 1, which includes the step of sealing the well after inoculation to prevent evaporation and facilitate handling and storage until observation.