Method And Apparatus For Automated Antibiotic Susceptibility Analysis Of Bacteria Samples

Abstract

Method and apparatus for automated antibiotic susceptibility analysis of bacteria samples including a device for feeding in succession a plurality of different samples, each of a significant, unknown bacterium from a respective receptacle to plural respective ones of receiving receptacles in which the sample portions are treated or conditioned, and, in at least certain of the last-mentioned receptacles, the growth of bacteria is challenged by addition of different antibiotics respectively thereto. Samples are incubated and subsequently killed, bacteria from the respective and receiving receptacles are removed by sampling for counting and comparison indicating, with the use of a control, relative antibiotic susceptibility through the proliferation of the bacteria, and recording the results of the analysis. An individual, moveable probe is employed in each of the first mentioned receptacles for transfer therefrom of the respective sample to the plural aforementioned receiving ones of the receptacles and is then returned to the last-mentioned one of the receptacles, effectively reducing the risk of contamination between samples. Such probes may be expendable and disposed of with the remaining corresponding samples, or may be sterilized and reused.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

Method and apparatus for automated antibiotic susceptibility analysis of bacteria samples for information useful in prescribing specific antibiotics to combat infectious diseases without the necessity of identifying the bacteria.

2. Prior Art

Because the identification of bacteria causing infectious diseases takes such a long period of time, usually 72 hours according to present techniques, which identification information may be used in the selection of specific antibiotics for treatment, though such selection based on such information is not considered by microbiologists as having a sound foundation in many circumstances and because this information may be delayed so long as to be no longer relevant when obtained, and because of the risks of secondary or super infection caused by administration of non-specific antibiotics, attempts have been made to develop a useful and standardized antibiotic susceptibility test, which is less time-consuming, for unidentified bacteria. The term unidentified bacteria is used to mean significant bacteria isolated from pathological specimens the identity of which is suspected or established presumptively. Such attempts have been described by World Health Organization Technical Report Series No. 210, Standardization of Methods for Conducting Microbic Sensitivity Tests, Geneva, WHO, 1961; H. Ericsson, Standardization of Methods for Conducting Microbic Sensitivity Tests. Preliminary Report of a Working Group of the International Collaborative Study Sponsored by WHO, Stockholm, Karolinska Sjukhuset, 1964; H. D. Isenberg, a comparison of nationwide susceptibility testing using standardized discs, Health Lab. Sci., 1:185-256, 1964; and A. W. Bauer, W. M. M. Kirby, J. C. Sherris and M. Turk, a standardized single disc method for antibiotic sensitivity testing, Am. J. Clin. Path., 45:493-496, 1966.

Aside from the aforementioned time factor which is very significant in itself, microbiologists have tended to agree that presently acceptable procedures for identification of microorganisms require a high degree of experience with reference to technicians and skilled interpretation of information, and for these reasons these procedures do not lend themselves well to automation at present. While in the past it has been believed that antibiotic susceptibility testing according to acceptable microbiological tenets is better suited to automation for steps subsequent to culture preparation and preparation of an inoculum, to our knowledge no completely automated system has been developed and used heretofore. In fact, though some manual techniques have been mechanized, at least in part, for the performance of biological laboratory jobs of various types automation has not heretofore gained a foothold in the microbiological laboratory.

While antibiotic susceptibility testing is recognized as being of undisputed value to the medical profession, and there is widespread use of the Bauer-Kirby method (see aforementioned referenced article) of agar difusion utilizing for measurement purposes zones of inhibition of bacterial growth around antibiotic discs, users of this method and others have employed their own variations of procedure, which makes for lack of standardization which standardization is so desirable between laboratories and within a single laboratory. In addition, such tests have required 24 hours for completion after preparation of the inoculum, a period much too long.

Another problem which has beset microbiologists in developing a standard antibiotic susceptibility test has been the search for a universal indicator of organism proliferation. It is recognized that only inhibition of active proliferation of bacteria is useful in determining whether or not a bacterium is susceptible to a drug. Such indicators as carbon dioxide production, pH changes and glucose consumption were not found to be universally applicable to the vast majority of fast growing bacteria. While the well known ATP technique for determining the existence of cellular life using the firefly luciferin-luciferase system was found to be universally acceptable as an indicator of bacteria proliferation, the reagents for the performance of the ATP assay are expensive, not always available and not sufficiently uniform from batch to batch to avoid elaborate standardization. Furthermore the ATP methodology was found to be relatively complex leading to serious risks of discrepancies in use.

Still another problem which faced researchers in this field was the discovery that apparently microbiological activity may be determined more readily by changes in a system than in the use of continuous flow techniques employed very successfully in the determination of an amount of a substance in a given volume.

Still another problem of serious consequence to microbiologists in developing any automated apparatus for carrying out antibiotic susceptibility tests was the high risk of contamination between samples, and the further problem of maintaining uniform the additions of any particular inoculum to receiving receptacles for mixture with various antibiotics respectively. Heretofore, these problems were not satisfactorily resolved.

SUMMARY OF THE INVENTION

One object of the invention is to provide a technique for automating antibiotic susceptibility analysis of all steps subsequent to the preparation of a culture and the preparation of a suitably diluted inoculum from the culture and placement of this inoculum in the apparatus. Another object is to provide automated antibiotic susceptibility analysis having much greater speed than any heretofore known, which provides the needed information for the administration on the same day of specific drug or drugs to fight an infectious disease.

It further contemplates a technique of antibiotic susceptibility analysis of high accuracy and reproducibility, and one susceptible of performing tests on more than a hundred samples on a day by day basis, requiring little attention from an attendant and with safety to the attendant and others. The technique has as a significant feature, the reduction of risk of contamination of a sample and between samples. This feature is obtained in part through the use of individual probes for sample receptacles, each of which probes in the analysis is used only once for that particular sample, and which probe may be of the disposable type enabling it to be disposed of in a suitable waste receptacle at the same time a suitable disposition is made to waste of the remaining sample in the sample receptacle.

The individual probes have other advantages. One such advantage is that the probes do not require sterilization as by being heated to incandescence during the analysis of plural samples nor exposed to sterilizing chemicals nor washed during this process, thereby avoiding such problems as the undesirable killing of bacterium in a sample through heat or exposure to chemicals, and further avoiding undesirable sample dilution by probe wash solutions. Another very important feature of the use of such probes is that each will dispense repetitively drops of inoculum of the same volume. This effects analytical results which are reliable with reference to the various antibiotics with which each inoculum is tested, the test with reference to each particular antibiotic being carried out in a respective one of plural discrete chambers.

Still another advantage derived from the use of individual probes for the transfer of each inoculum to the plural testing chambers for the respective antibiotics is that total sterility of the probe is not required. This is a great convenience as well as a time and expense saver. Such total sterility is not required because the probe is used only for a very short period of time, that is, an insufficient time for other bacteria to significantly affect the bacterium of the sample. Other bacteria present, at most in very small numbers, cannot achieve numbers which would interfere with the analysis since the inoculum consists of very large numbers of bacterial particles in an environment favoring this proliferation, according to the invention.

Another object is to provide an antibiotic susceptibility analysis technique which utilizes a very short incubation period for the bacteria, thereby reducing the risk of contamination of the aforesaid type, and also making possible analysis at a rapid rate. In accordance with present practice, analysis utilizing the invention requires but three hours from the time of placement of the inoculum in the apparatus.

This speed of analysis is made possible by the use of a device of a photometric nature of sufficient sensitivity to indicate low or early exponential growth of bacteria by a counting process in a counting device. The first named device is also used to indicate the exponential growth of bacterium in an uninhibited control of each inoculum in a growth medium, such as a broth, employed also with reference to the chambers where the inoculum is challenged by the presence of respective antibiotics.

The use of the last-mentioned photometric device also enables the comparison of the uninhibited growth of the last-mentioned control with a control which is killed after a preincubation stage and subtracted in the counting device from the count of the first-mentioned control to eliminate such factors as unviable organisms and dirt. The counting device, which is actually a particle counter also serves to provide a comparison of the susceptibility of each inoculum to the antibiotics to which it is exposed. The results of the analysis are recorded on a recorder.

In accordance with the invention there is provided a method and apparatus for automated antibiotic susceptibility analysis of bacteria samples including a device for feeding in succession a plurality of different samples, each of a significant, unidentified bacterium from a respective receptacle to plural respective ones of receiving receptacles in which the sample portions are treated or conditioned, and in at least certain of the last-mentioned receptacles the growth of bacteria is challenged by addition of different antibiotics respectively thereto. The last-mentioned feeding device is susceptible of various other uses including the handling in other analytical systems of radioactive substances, or may be employed to transfer a small quantity of any liquid, such as reagent or sample, to a station at which it is utilized. The system of the invention is also suitable for other uses such as providing bacteria counts of uninhibited exponential growth. It is useful for measuring minimum antibiotic inhibitory concentrations, and when used with known bacteria may test various fluids such as body fluids for example for their characteristics of inhibiting bacterial growth. Further objects will be apparent from the following detailed description of the preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a block diagram illustrating sequentially the orientation of stations A-H of apparatus for automated antibiotic susceptibility analysis embodying the invention, wherein A is the nutrient broth dispensing station; B is the inoculum transfer station for transfer of bacterium samples to receptacles containing the broth; C is the station for adding different antibiotics to certain of the last-mentioned receptacles and a bacteria killing agent to another of the last-mentioned receptacles; D is the incubation station; E is the station for adding a bacteria killing agent to those receptacles in which such an agent has not been previously added; F is the station for sampling the contents of each of the receptacles holding the treated samples; G is the photometric station for the examination for counting purposes of the particles in the sample receptacles such as bacteria and foreign matter; and H is the station for receiving signals from station G in order to count the last-mentioned particles and make comparisons of such counts and for recording the results of the analysis;

FIG. 2A is a perspective view illustrating somewhat diagrammatically station A of the apparatus;

FIG. 2B is a similar view illustrating stations B and C of the apparatus;

FIG. 2C is a similar view illustrating stations D-H of the apparatus;

FIGS. 3A--3F are views looking in the direction of the arrows 3A--F of FIG. 2B illustrating different positions of the probe holder and associated parts at the transfer station B;

FIG. 4 is an enlarged sectional view taken on line 4--4 of FIG. 2B, and

FIGS. 5A and 5B, when viewed together, a wiring diagram.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the drawings there is shown a supporting table-like or counter element, indicated generally of 10, providing a supporting surface and of an elongated form. This table-like element is shown associated with stations A-F. Arranged longitudinally on the table 10 in spaced parallel relationship are a pair of lead screws 12 suitably supported from the table for rotation. The screws 12 extend substantially throughout the length of the table and are located a short distance upwardly therefrom in order to clear the table. The table forms part of a conveyor system having a pickup end 14 (FIG. 2A) and a discharge end 16 (FIG. 2C).

In the form of the invention illustrated by way of example, at the discharge end 16 of the table (FIG. 2C) each screw 12 is provided with one of a pair of wheels 18 angularly fixed thereto, interconnected for the drive of one from the other by a belt 20. As shown in FIG. 2C, an electric motor 22 is provided to drive the screws 12 through the belt 20, which motor may be supported from the table. The motor 22 drives a shaft on which is fixed a cam wheel 24 and to which shaft in axially spaced relationship from the wheel 24 is also fixed driving wheel 26. A wheel 28 in angularly fixed relation and axialy spaced from one of the wheels 18 is driven from the wheel 26 through a belt 30. A switch 32 is provided having an actuator cooperating with the cam wheel 24 to de-energize the intermittently operable motor 22 after an appropriate, uniform interval of operation, one revolution of the cam wheel 24.

Samples of different inocula are carried along the table 10 in a stepwise fashion after being transferred thereto at station B for treatment at subsequent stations, and the conveyor system illustrated by way of example includes for the purpose of advancing the samples as aforesaid a plurality of trays 34 which trays have on two opposite sides thereof rack-like gear teeth 36 formed therein for cooperation with the respective screws 12. For ease of handling and to reduce the risk of contamination, disposable cups 38 are provided to receive the sample inocula. These cups 38, which do not require total sterility for the same reasons discussed above with reference to the probes, are suitably supported as in sockets 35 provided in the trays, the cups being laterally spaced apart.

In practice each tray may contain 50 cups of plastic or other suitable material in five rows spaced lengthwise of the table and extending transversely of the table. For the sake of convenience each tray 34 is illustrated as having three transverse rows of cups spaced lengthwise of the table, each row having five cups, the first cup in each row being indicated at 40 and the second cup being indicated at 42. It will be understood from the foregoing that the trays 34 may be advanced in a stepwise manner other than by the use of the lead screws 12. It will be obvious to those versed in the art that sprocket chains, for example, could be employed for this purpose if desired.

As will be more apparent hereinafter, it is essential in order to automate stations A-F that the trays 34 have a predetermined uniform relationship to one another on the table and that the trays be fed into the pickup end 14 of the table in predetermined phased relationship to the rotation of the lead screws 12. In this regard, any suitable conventional mechanism may be employed to properly engage with screws 12 a tray from the entrance ramp 44 (FIG. 2A) at the proper time.

The spacing of the cups 38 in each tray 34 is critical to automation and must be uniform. The spacing of the trays from one another lengthwise of the table has been greatly exaggerated for ease of illustration and better understanding of the apparatus of the drawings. In actual present practice two neighboring trays are spaced apart with reference to the trailing row of one tray and the leading row of the following tray such that the center of a cup in one of the last-mentioned rows is spaced from the center of its corresponding member in the other of the last-mentioned rows a distance which is twice the distance between the centers of neighboring rows of cups in any one tray 34. Trays 34 from the table 10 are discharged by the conveyor system into a suitable collection bin 46 (FIG. 2C) after the sample receiving cups 38 have been emptied as will be described hereinafter.

In the form of the apparatus shown in the drawings only by way of example it is required that each socket 35 of each tray used receive therein one of the disposable cups 38 before the tray is placed on the ramp 44. There is no such requirement in actual practice as those versed in the art will understand from the following description.

The first tray 34 (FIG. 2A) is properly engaged by the screws 12 as aforesaid when received from the ramp 44 after the start button (not shown) is depressed to start main timer 48 (FIG. 10), energizing and re-energizing motor 22 at uniform intervals closely approaching 90 seconds (de-energized once each revolution of the cam wheel 24 coacting with the switch 32) to operate the screws 12 to advance the tray on the right shown in FIG. 2A to the position shown therein at which time the motor 22 is de-energized through actuation of the switch 32 by the cam wheel 24. Movement of the last-mentioned tray to this position is sensed by switch 50 FIG. 10 having an actuator engageable with a protrusion 52 formed on the tray, which protrusion may take the form of an upstanding removeable pin. Each tray is provided with a similar pin 52. Actuation of the switch 50 enables the broth station A.

There is provided at this station a sub-frame 54 including a vertically arranged plate extending transversely across the table 10 in a location thereabove and suitably supported from the table as shown, the sub-frame 54 having at its ends ears 56 which rigidly support therebetween a rod 58 serving as a support and guide as will appear hereinafter.

The frame element 54 supports a reversible electric motor 60 having a driving shaft angularly fixed to a timing wheel 62. In axially spaced relation from the wheel 62, wheel 64 is also angularly fixed to the last-mentioned shaft. A switch 66 has an actuator engageable with the cam wheel 62.

The location of the motor 60 and the above described associated elements is adjacent one end of the frame element 54 as illustrated in the last-mentioned view and, as also shown in this view, there is provided adjacent the other end of the frame element 54 a shaft on which an idler 68 is revolubly provided. A belt 70 is trained over the wheel 64 and the idler 68 and carries fixed thereto a switch actuator 72. The actuator 72 coacts with limit switches 74 and 76 spaced apart as shown and supported from the frame element 54 together with the switch 66 previously described. The belt 70 has fixed thereto, as at 71, a nozzle holder 78, receiving and rectilinearly slidable on the horizontal rod 58, and rigidly supporting a downwardly directed nozzle 80 which in the position of FIG. 2A is directly over the first cup 40 of the first row of cups 38 of the last-mentioned tray.

Connected to the nozzle 80 is one end of a tube 82 having its other end connected for receipt of liquid from a pump designated generally at 84. The inlet of the pump is connected through one end of a tube 86 to a reservoir of liquid receiving the other end of the tube 86, as shown in the last-mentioned view, for aspiration through the tube 86 of liquid from the reservoir. The liquid in the reservoir 88 is sterile bacteria nutrient in the form of a broth. Eugon broth may be used for this purpose.

The pump 84 comprises a suitable plate-like support element 90 horizontally arranged, on which a vertical standard 92 is fixed and on which an upwardly extending plate-like element 94 is also arranged in laterally spaced relation from the element 92. The support element 94 supports an electric motor 96 the driving shaft of which is suitably journaled in the support element 92 and has angularly rigid therewith a cam wheel 98 coacting with the actuator of a switch 100 carried by the plate-like support element 94 which de-energizes the motor 96. The pump motor shaft is indicated at 102.

The pump 84 comprises a syringe and hence is of the expansible chamber type. As shown in FIG. 2A it comprises a barrel element 104 providing the expansible chamber. A plunger 106 is extensible into one end of the barrel 104 and the plunger 106 is operated in the barrel 104 through a linkage 108 connected to one end of the plunger 106 and connected to a crank arm 110 fast on the shaft 102. It is believed made clear from the foregoing that rotation of the motor shaft 102 effects a reciprocating motion of the plunger 106 in the barrel 104. The barrel 104 has an outlet in a conventional valve element 111 secured to the other end of the barrel and supported, as shown in FIG. 2A by a shaft 112 projecting in fixed relation from the support element 92.

The valve element 111, connected to corresponding ends of the tubes 82 and 86, has two ball checks controlling the flow of liquid through the respective tubes 82 and 86. When the plunger 106 is extended from the barrel, liquid from the reservoir 88 is drawn into the barrel through tube 86 past one of the ball checks while the other ball check prevents any flow of fluid through the tube 82. When the plunger 106 is retracted into the barrel 104 to discharge the contents of the syringe the corresponding ball check prevents the flow of fluid through tube 86 while permitting the flow of liquid through the tube 82.

When the last-mentioned tray 34 stops in the position of FIG. 2A and the broth station is enabled, all as aforesaid, the broth pump 84 cycles delivering a precise amount of broth, which may be in the neighborhood of 2 ml., to the first cup 40 through the nozzle 80. When switch 100 is actuated by cam wheel 98 the pump motor 96 is de-energized and operation of motor 60 is commenced to move the nozzle 80 over the cup 42 at which point operation of the motor 60 is stopped through actuation of switch 66 by cam wheel 62.

The pump cycles and the intermittent movement of the nozzle 80 continues in the last-mentioned direction until the last cup 38 in the first row in the tray is filled to the extent indicated above, and the actuator 72 actuates switch 74 de-energizing the motor 60 and placing it in a reversed mode. The filling of the cups 38 of the first row in present practice requires less than 90 seconds. Thereafter, on signal (every 90 seconds) from the main timer 48 the motor 22 is energized to drive the tray-advance screws 12 until the motor 22 is de-energized by operation of the cam wheel acting on the actuator of the switch 32.

After the motor 22 is de-energized as aforesaid, the nozzle 80 is over the corresponding cup of the second row of cups in the last-mentioned tray. The pump 84 cycles on actuation of switch 32 to feed broth to this cup from the nozzle 80 after which the motor 60 is again energized as aforesaid so that the nozzle 80 is brought over the next cup in the secnd row. This operation continues until all the cups in the tray have been filled with broth, to the extent indicated, in the course of which the limit switch 76 also serves to effect stopping and placing in reversed mode the motor 60. After the last cup is filled, on signal from the main timer 48, the motor 22 is energized and advances the tray one increment as aforesaid whereupon the motor 22 is de-energized as aforesaid, and on receipt of a further signal from the main timer 48 the motor is re-energized to advance the tray to the position thereof at the transfer station B shown in FIG. 2B. It is believed made clear that when this tray reaches the last-mentioned position the following tray 34 reaches the starting position previously described with reference to the broth station and shown in FIG. 2A.

As shown in FIG. 2B, there is provided at the inocula transfer station designated B a sub-frame indicated generally at 116 including an elongated vertically arranged plate-like element having end flanges 118 by which it is suitably supported on the table 10 to extend transversely thereof. These flanges 118 rigidly support vertically spaced twin rods 120 extending therebetween and arranged transversely of the table. The lowermost rod 120 is spaced upwardly from the table as indicated and provides clearance for the screws 12 and the trays 34 which move along the table. A carriage, indicated generally at 122, is supported by the rods 120 for horizontal movement therealong. The carriage 122 comprises a vertically arranged plate 124 to which are rigidly secured two vertically spaced pairs of blocks 126 which pairs respectively receive the rods 120 for sliding movement thereon.

The carriage 122 includes a subcarriage indicated generally at 128 arranged to be cammed vertically in one axial position of the carriages 122 and 128 on the rods 120 as will hereinafter appear. The sub-carriage 128 comprises an upper horizontally arranged plate 130 and spaced therebelow a plate 132 having a depending vertically arranged plate part 134 of L-shaped terminating in a cross plate part 136 which is vertically arranged and of elongated form extending longitudinally of the table. The plate parts 130 and 132 are interconnected by a vertically arranged plate 138 (as shown in FIG. 2B) suitably fixed thereto as by welding. The carriage 122 also includes a pair of laterally spaced rods 140 which are vertically arranged and which have their respective ends in assembled form fixed to the respective plates 130 and 132. The rods 140 extend through and are slidable in extensions 142 of the respective blocks 126.

A pair of vertically spaced horizontal rails are arranged transversely of the rods 140 and secured thereto, as by welding, so as to protrude therefrom, the rails being indicated at 144. In the position of the carriages 122 and 128 shown in FIG. 2B the rails receive therebetween a portion of a stationary carrier strip 146. This carrier strip may be formed of relatively heavy strip metal welded in edgewise relation to the frame plate 116 to protrude horizontally therefrom in the manner shown in the last-mentioned view, and as shown here the carrier strip 146 extends transversely of the table. The carrier strip 146, which in the last-named position of the carriage 128 supports the latter, extends from the far one of the flanges 118 shown in FIG. 2B toward the other flange 118 and terminates in a free end in a location approximately over the near one of the tray-advance screws 12.

As shown in FIG. 2B, the sub-frame 116, together with the rods 120 project toward the viewer beyond the main bed of the table to the extent indicated. The construction and arrangement is such that the carriages 122 and 128 may be moved transversely of the table on the support rods 120 to a position in which the vertically moveable sub-carriage 128 may be lowered to the position of FIG. 3B.

To drive the carriages 122 and 128 for horizontal sliding movement on the support rods 120, there is provided a reversible electric motor 148 supported from the frame plate 116 and having its driving shaft angularly fixed to the wheel 150 and angularly fixed to the cam wheel 152 in axially spaced relation from wheel 150. A switch 154 is mounted on the last-mentioned plate which switch is similar to the previously described switch 66 and has its actuator in engagement with the cam wheel 152 serving a similar function to the aforementioned cam wheel 62. The motor 148 and the above described associated parts are disposed adjacent one end of the transverse frame plate 116.

Adjacent the other end thereof, the plate 116 revolubly supports wheel 156 which is an idler similar to the above-described idler 68 and over which a belt 158 is trained which belt is also trained over wheel 150 which drives the belt.

The belt 158 has a switch actuator 160 fixed thereto and the belt 158 is fixed, as at 162, to the least one of the upper pair of the carriage blocks 126. Above the upper run of the belt 158, switches 164, 166 and 168 are spaced from one another transversely of the table and supported by the plate 116, as indicated in FIG. 2 by way of example. The last-named switches have actuators engageable by the switch actuator element 160, previously described, affixed to the belt 158 in the illustrated form.

Also supported on the frame plate 116 is a non-reversible electrical motor 170 (FIG. 5A) for the vertical drive of sub-carriage 128. It is believed made clear that the motor 148 provides horizontal drive for the carriages 122, 128. The driving shaft of the motor 170 has angularly fixed thereto a cam wheel 172, and has a crank arm also angularly fixed thereto in axially spaced relation from the cam wheel 172, the crank arm being indicated at 174. One end of the arm is fixed to the last-mentioned shaft. The other end of the arm 174 carries a cam 176 which is illustrated as a horizontally extending anti-friction roller which roller in the positions of FIGS. 3A--3E is received between the rails 144 of the vertically moveable carriage 128. The frame plate 116 also supports spaced apart switches 178, 180 and 182, each of which has an actuator bearing against cam wheel 172.

There is provided on vertically moveable carriage 128 a vertically arranged probe holder of cylindrical form extending through the plate 132 and fixedly supported by the last-mentioned plate, the proble holder being indicated generally at 184, as shown in FIG. 2B, FIGS. 3A--3F and FIG. 4. The cylindrical body of the holder 184 has an axial bore therethrough into the upper end of which extends a plunger 186. The upper end portion of the plunger 186 receives in fixed relation thereto a transverse pin 188 extending therefrom, as best shown in FIGS. 3A--3F having fixed at its distal end a ball 190 which ball is mounted for universal movement in a socket 192 (FIG. 2B) formed in cam wheel 194 and permitting limited endwise movement of ball pin in the socket. The cam wheel 194 is angularly fixed to the shaft of a reversible electric motor 196 supported by the plate part 138, and switches 198, 200 and 202 spaced from one another are also supported from the plate part 138, each having an actuator engageable with the cam wheel 194.

Communicating with the aforementioned axial bore of the probe holder 184 is a transverse open-ended duct 204 of tubular form best shown in its relationship to the last-mentioned bore in the probe holder 184 in FIG. 2B. Intermediate of its ends the last-mentioned tube is controlled by a solenoid-operated valve 206. The valve normally closes the tube 204 (FIG. 2B) and is open when the last-mentioned solenoid is operated for flow of air through the tube 204 from its right-hand end as shown in FIG. 2B. The amount of air flowing in the tube 204 may be controlled by a needle valve having a body 208 and a manipulating part 210. The air tube 204 and its associated parts previously described may be supported in any suitable manner.

The aforementioned cross plate 136 carried by the depending plate part 134 of the carriage 128 provides a support for a drop counter in the form of a photoelectric device including at one end of the plate 136 a housing 212 for a light source such as an electric bulb and means, not shown, to direct light therefrom along a path toward a light detector housing, indicated generally at 214, supported on the other end of the cross plate 136.

In the position of the drop counter shown in FIG. 2B, the counter is spaced above the tray 34 and the cups thereon and is in operative position to count drops dispensed from the probe, indicated generally at 216, in the holder 184 into the first cup 40 of the first row of cups 38 in the tray 34 shown at the transfer station, and in this position of the tray effected as aforesaid by the operation of the tray-advance screws 12, the pin 52 of the tray is shown engaged with the actuator of switch 218. It is to be noted that the switch 218 is operated by the pin 52 as the tray assumes the position of FIG. 2B and prior to the assumption of the carriage positions shown in FIG. 2B. When the tray 34 assumes the last-mentioned position, the carriage 128 is in the position of FIG. 3A.

The details of the probe holder 184 will now be described with reference to FIG. 4. As previously indicated the holder includes an upright cylindrical body having an axial bore therethrough which bore is indicated at 220 and is shown as receiving through the upper end thereof the aforementioned plunger 186. The last-named bore is counter-bored through the lower end, as at 22. An end cap extends over the outer end of the counterbore 222 and this cap is provided with a tapered orifice 224 through which the upper end portion of the probe is extensible as shown in FIG. 4. The plunger 186 is provided with air tight sealing devices or rings 226. At least two O-rings are provided in the counterbore 22 in axially spaced apart relation to form an air-tight seal around the upper end portion of the probe when it is inserted in the holder and to prevent the probe from cocking in the holder from the vertical position, the two O-rings being indicated at 228.

The O-rings are so constructed and arranged that when they are engaged and sealed against the probe 216 they do not engage so as to be compressed against the wall of the counterbore 222 but rather have clearance with the last-mentioned wall. This tends very effectively to reduce friction when the probe is relatively extended into the holder or removed therefrom. In fact, in the construction shown, at least the lowermost one of the O-rings 228 tends to have only line contact with the probe when it is inserted in the holder and line contact with the aforementioned lower cap of the holder.

A ring-like spacer 230 is interposed between the O-rings 228 and has cam surfaces thereon engageable with the respective O-rings tending to center them. A light compression spring 232 has one end thereof bottoming in the counterbore 222 and has the other end thereof bearing against a sleeve 234 bearing against the upper surface of the uppermost one of the O-rings 228. This spring and the spring follower 234, together with the ring 230, tend to maintain the O-rings 228 in a proper relationship to receive in sealing engagement the upper end of the probe within the holder and provide at least a seal of the lowermost one of the O-rings 228 against the lower end cap in the manner previously indicated. In communication with the bore 220 there is provided an air port, not shown, extending transversely through the cylindrical wall of the holder and in communication with the aforementioned tube 204.

It is believed made clear from the foregoing that when the solenoid operated valve 206 controlling the tube 204 is closed and the plunger descends a distance from its uppermost position, shown in FIGS. 2B and 3B, to the position of FIG. 3C through movement of the cam wheel 194 driving the ball pin 188, air is forced downwardly in the holder 184 and into any probe in communication therewith, while when the plunger 186 is moved in the opposite direction (FIG. 3D) from the position of FIG. 3 in the aforesaid manner by the cam wheel 194 while the solenoid operated valve 206 is closed, an upward aspirating effect is produced in the holder 184 by the plunger 186, which is communicated to any probe in the holder.

Any liquid drawn into the probe 216, which is of open-ended tubular form, by this aspiration is held within the tube 216 while the plunger 186 remains in its upper position, until such time as the solenoid-operated valve 206 opens to admit air into the tube 204. When this occurs and the open upper end of the probe 216 is exposed to atmospheric pressure, drops may be dispensed of the liquid in the probe 216 (FIG. 2B) through the open lower end thereof, and such drops will cease to fall when the solenoid valve 206 is closed.

It is believed also made clear from the foregoing that when the probe 216 is in the holder 184 and contains liquid while the valve 206 is closed, downward movement of the plunger 186, to a position similar to that of FIG. 3C, through movement of the cam 194 connected thereto as aforesaid expels the liquid in the probe 216, and a degree of further downward movement of the plunger 186 from the last-mentioned position effects impingement of the lower end thereof on the upper end of the probe 216 to drive the probe out of the holder 184. As shown in FIG. 4 the lower end portion 238 of the plunger 186 is of reduced diameter to pass through the spring follower 234 and its associated parts to effect this probe ejection.

Returning now to FIG. 2B the probe 216 shown supported in the holder 184 is but one of a plurality of probes employed in the illustrated apparatus for carrying out the above-described type of antibiotic susceptibility tests. There is provided a series of different bacteria samples which may be considered as different cultures developed from different specimens taken from different hospital patients and supplied to the apparatus sequentially. Each sample of a significant bacterium, which is prepared in a manner which will be described hereinafter, is placed in a suitable vessel, such as a test tube, for example, or a cup, which does not require total sterility for the above noted reasons including, primarily, the short length of time which the sample necessarily resides in the vessel so as not to be significantly affected by foreign bacteria in the vessel. These cups, formed of glass or plastic and which may be disposable, may be conveniently supported for the feeding of samples to the last-mentioned tray 34 by a turntable assembly indicated generally at 240.

These cups or tubes, indicated at 242, are supported in a turntable rack 244 (FIG. 2B) by means of peripherally spaced tube-receiving sockets therein. The turntable 244 is shown receiving seven such tubes but in practice the turntable may receive twenty such tubes and when such tubes are placed in the turntable assembly prior to commencement of operation of the apparatus, each is provided with its individual probe 216. As previously made clear total sterility of these probes is not required mainly due to the fact that they are employed in the testing procedure for such a short period of time, as the vessels in which they are supported.

The turntable 244 is suitably supported from a base 246 on a shaft 248 mounting the table 244 for rotation. As indicated diagrammatically the turntable may be driven by a belt 248 trained over the table and also trained over a driving wheel 250. The wheel 250, like the turntable 244, has a vertical axis and the wheel 250 is driven through an electric motor 252 having a vertically arranged driving shaft to which a cam wheel 254 is angularly fixed. The wheel 250 is angularly fixed to the last-mentioned shaft in axially spaced relation from the cam wheel 254. A switch 256 has an actuator engaging the cam wheel 254.

The above-described turntable assembly 240 and associated parts may be supported in any suitable manner with the turntable 244 in the relationship to the table 10 and the transfer station B shown in FIGS. 2B and 4. When the turntable motor 252 is energized, after a dispensing operation utilizing the holder 184, and after the probe 216 shown in the holder 184 in FIG. 2B is returned to the corresponding tube 242, rotation of the wheel 250 drives the belt 248 a distance sufficiently to index the next tube and probe therein with the holder 184 as shown in FIG. 3A.

Each probe 216 may conveniently take the form of a pipette. As the pipettes 216 employed are of identical structure a description of the construction of one pipette 216 will suffice. As shown in FIG. 4 the pipette has an elongated tubular body 258 which is open ended, as previously indicated, and vertically arranged and which narrows somewhat toward a point in the region of its lower end portion. The upper end of the body 258 is slightly tapered to facilitate entrance of the pipette into the holder 184.

Intermediate of its ends the pipette 216 has a radial flange 260 including a depending skirt 262 which terminates downwardly in circumferential interior surface portion which is tapered, as at 264, as shown in FIG. 4, which surface portion is engageable with the mouth of a test tube or the like when a pipette is dropped therein to center the pipette with reference to the test tube. It is important to note that the flange structure 260 supports the pipette in a tube such as one of the tubes 242, with the lower end portion of the pipette being suspended above the bottom of the tube. It will also be apparent that the flange structure provides a cap for the test tube. A wad of cotton or the like may be inserted in the upper end portion of the pipette to prevent foreign material falling down the pipette into the test tube. Such a wad of cotton does not interfere with aspiration of the contents of a test tube employing the pipette. We make no claim to the construction of the pipette per se. The pipette is described and claimed in the co-pending U.S. Pat. Application of Watkin et al. Ser. No. 137,385 filed Apr. 26, 1971, and assigned to the assignee of the instant invention.

The preparation of the inoculum for each test tube is very important. The first step is the isolation of a significant bacterium from a clinical or other specimen by one trained in such microbiological procedure. The next step is inoculum preparation. Portions of five to ten isolated colonies are transferred to two ml. of optically clean broth which may be Eugon broth. The colonies must represent a single bacterial species on the basis of their colonial appearance. The turbidity of this suspension is adjusted visually to the density of the standard in use with the Bauer-Kirby method previously referenced. The aforementioned dilution achieves the desired bacteria to volume proportions in the vicinity of 10.sup.5 per ml. The concentration of bacterium, preferably in the range of 10.sup.6 -10.sup.7 per ml. is checked by a nephelometer, and the concentration is adjusted if necessary. Each inoculum is placed in a tube 242 and the tube is closed by a respective pipette and then placed in the turntable assembly 240 as aforesaid.

The operation of inoculum transfer station B will now be described. When the carriage 128 is in the lateral position shown in FIG. 3A and the carriage 128 is in the raised position thereof, this carriage is supported by the crank arm 174 through the cam 176 which is disposed (FIG. 3A) between the rails 144 of the carriage 128, and the crank arm 174 is in its up position of FIG. 2B. The pipette holder 184 is spaced above and indexed with the first sample receptacle in the turntable assembly 240 and is spaced above the pipette in the last-mentioned sample receptacle. The tray 34 shown at station B has been advanced thereto, and has been stopped by de-energization of the screw motor 22 through switch 32 acting on cam wheel 24, and the tray sensor switch 218 has been energized at station B all as previously indicated. The operation of station B is instituted by actuation of the switch 32.

In the last-mentioned position of the pipette holder 184, the plunger 186 is in its raised position actuating switch 202 which energizes vertical drive motor 170 driving cam wheel 172 in the clockwise direction as viewed in FIGS. 2B and 3A and the crank arm 174, swinging in the same direction, is swung downwardly from its raised position to its lower position or through an angle of 180.degree. at which time the motor 170 is de-energzied by actuation of the switch 180 cooperating with the cam wheel 172. This downward movement of the cam 176 on a crank arm 174 lowers carriage 128 through the rails 144 to its lowermost position (FIG. 3B) in which the pipette holder 184 has engaged and received the corresponding pipette of the first sample receptacle.

Actuation of the switch 180 energizes plunger motor 196 effecting counterclockwise movement of the cam wheel 194 as viewed in FIG. 2B driving the plunger 186 downwardly a distance (FIG. 3C) sufficient to cause air in the pipette holder to be expelled into the pipette and into the sample to thereby agitate it for mixing purposes. During this travel of the plunger 186, solenoid valve 206 controlling the air tube 204 is closed. The last-mentioned movement of the cam wheel 194 controlling movement of the plunger 186 through the ball pin is stopped by actuation of the switch 200 which reverses the motor 196 raising the plunger 186 (FIG. 3D) to its raised or starting position. Movement of the cam wheel 194 in the direction to raise the plunger 186 is stopped by de-energization of the motor 196 through actuation of the switch 202 actuated by the cam wheel 194. During this upward travel of the plunger 126 with the valve 206 closed, the sample in the first cup 40 is aspirated into the pipette.

The last-mentioned actuation of the switch 202 effects energization of the vertical motor 170 swinging the crank arm 174 upwardly from its down position to its raised position, or through an angle of 180.degree., and the motor 170 is de-energized at this point by actuation of the switch 182 controlled by the cam wheel 172. This movement of the crank arm 174 effects through cam 176, acting through the rails 144, movement of the carriage 128 to its raised position. The last-mentioned actuation of the switch 182 energizes horizontal drive motor 148 which through the drive belt 158 traverses the carriages 122 and 128 as a unit, with the support rails 144 leaving the cam 176 and picking up the stationary carrier strip 146 for support by the latter.

A suitable brake, not shown, is provided on the motor 148 to assure that when the carriage 128 leaves the support of the cam 176, the crank arm 174 supporting the cam 176 is left in its raised position. The motor 148 continues to drive the belt 158, overriding the switch 154 coacting with the cam wheel 152 until the switch 166 is actuated by the switch actuator element 160. When the last-mentioned switch is actuated motor 148 is de-energized, and in this position (FIGS. 2B and 3F) the probe holder 184 is over the first cup 40 of the first row of cups 38 in the last-mentioned tray 34.

The actuation of switch 166 causes actuation of the solenoid operated valve 206 to open this valve permitting air to flow in air tube 204 and into the holder 184 at atmospheric pressure, and this enables drops to form and discharge themselves from the lower end (FIG. 2B) of the pipette or probe into the last-mentioned cup 40. In actual practice one drop may be sufficient but the construction and arrangement may be such that ten drops may be dispensed from the probe before the flow is terminated in the manner which will be explained hereinafter.

The actuation of the switch 166 as aforesaid also enables the drop counter 212, 214 to commence counting drops dispensed from the probe 216 until the predetermined number of drops have fallen into the last-mentioned cup at which time the drop counter 212, 214 effects closing of the solenoid-operated valve 206 to terminate the dispensing action in this cup. The drop counter initiates energization of horizontal drive motor 148 to advance the carriages 122 and 128 transversely of the table until the pipette holder 184 is indexed over the second cup, cup 42 in the first row of cups in the last-mentioned tray.

This indexing is accomplished by actuation of switch 154 coacting with cam wheel 152 which de-energizes horizontal drive motor 148. This initiates operation of the solenoid operated valve 206 to open the valve, and the same number of drops is dispensed in cup 42. The probe holder 184 is advanced again in the same manner and drops are dispensed in the same manner in all the subsequent cups of the last-mentioned row.

Here, it should be noted that the probe 216 has a very significant feature in that it effectively tends to dispense drops of equal size due to the pecularities of that particular probe, such as the size of the opening in the lower end thereof, the surface characteristics around this opening affecting surface tension, and also significantly, because of the pecularities of the particular sample such as viscosity. This assures that equal amounts of sample are dispensed in each of the cups in the row which is important to the accuracy of the analysis.

When the horizontal drive motor 148 has been energized to deliver the probe holder 184 over the last cup in the last-mentioned row it is stopped not by the switch 154, but, by switch 164 actuated by switch-actuating element 160. The switch 164 on actuation energizes the solenoid valve 206 to open the same to dispense the same amount of sample into this last cup. Upon completion of the operation of the drop counter 212, 214, horizontal drive motor 148, placed in a reverse mode by switch 164, is energized, overriding switch 154 coacting with cam wheel 152, driving the carriages 122 and 128 in the reverse direction until the switch actuator element 160 on the belt actuates switch 168 at which point the holder 184 has returned to a position (FIG. 3E) over the tube 242 from which the last-mentioned probe 216 was removed.

Switch 168 de-energizes motor 148 and energizes vertical drive motor 170 thereby initiating downward swinging movement of the crank arm 174 which then supports the vertical carriage 128, through an angle of movement of somewhat more than 180.degree. to and beyond the aforesaid lower position of the crank arm 174, that is, on the rise, causing cam wheel 172 to actuate switch 180 during this movement, and allowing sufficient clearance of the probe 216 and the corresponding test tube 242 for the plunger 186 to eject the probe 216 from the holder 184. The probe 216 drops into the tube 242.

It will be understood from the foregoing that in moving to the last-named lateral position of the carriage 128, the support rails 144 run off the free end of the stationary carrier strip 146 and onto the cam roller 176 for support of the carriage. Another roller, not shown, may be employed to aid in the transfer of the carriage 128 from the carrier strip 146 to the cam roller 176.

On the last-mentioned actuation of the switch 180, which serves to de-energize vertical drive motor 148, motor 196 is energized to operate the plunger 186. The motor 196 moves the cam wheel in a direction to drive plunger 186 downwardly while the solenoid valve 206 is closed, thereby expelling liquid in the pipette into the last-mentioned test tube; and further downward movement of the plunger 186 effects, in the above-described manner, ejection of the probe 216 from the holder 184. This movement of the cam wheel 194 operates switch 198 stopping the motor 196 and reversing its direction of rotation, and switch 198 energizes the motor 196 to raise the plunger to the position of FIGS. 2B and 3A. In this position, the switch 202 is actuated stopping the plunger-operating motor 196 and enabling vertical drive motor to continue the last-mentioned swing of the crank arm up to its vertical position at which it is stopped by actuation of switch 182.

The operation of the last-mentioned switch initiates action of the motor 252 of the turntable assembly 240 to index the sample rack 244 in a manner such that the second sample is brought into alignment under the probe holder 184. When, at the end of the last-mentioned movement, switch 256 of the last-mentioned assembly is operated by cam wheel 254, the motor 252 is de-energized. The above-described transfer cycle requires less than 90 seconds.

On the next signal from the 90-second timer 48, the tray-advance motor 22 is energized driving screws 12 to advance the last-mentioned tray 34 one row of cups, at which time the motor 22 is de-energized by the switch 32 coacting with the cam wheel 24. Operation of the switch 32 is effective to initiate the next cycle of the transfer of the inoculum of the second sample to the second row of cups. This is repeated for the transfer of the inoculum of the third sample to the third row of cups.

Advance of the tray 34 from the last-mentioned position causes the tray to leave the station 2B, which movement is effected by the timer 48, which timer is effective to subsequently move the tray one increment and then another increment to bring it into the position shown in FIG. 2C which is the antibiotic dispensing station and wherein the pin 52 of the tray has engaged the actuator of switch 261 to enable the station C, which includes in its features the dispensing of a bacteria killing agent to the first cup 40 of each row of cups in the tray.

The bacteria samples are pre-incubated in the broth in the cups 38 of the tray at 37.degree.C between the time bacterium is deposited in each first cup 40 of the tray at this transfer station B and the time that the bacteria killing agent is added to the respective cups 40 at the dispensing station C. This pre-incubation period is necessary to compensate for any lag in the exponential growth of the bacterium, and the pre-incubation period, which lasts for 30 minutes, is sufficient for the commencement of active exponential growth of all viable fast-growing bacteria.

At the last-mentioned station for adding the bacteria killing agent to each cup 40 and for adding different antibiotics to each of certain sample cups 38 to challenge the growth of bacteria, the antibiotics may be supplied in disc form in solution if desired. The medicated discs require no preparation, are commercially available, easy to handle and easy to dipense. Discs of this type have been certified by the Food and Drug Administration. In actual practice each bacterium sample is challenged by a different one of thirteen antibiotics. For ease and simplicity of illustration and description, only three different antibiotics are shown being dispensed at station C and these are shown in the form of discs 263 illustrated as dropping into the third, fourth and fifth cups of the first row of cups 38 of the last-mentioned tray 34. These discs are commonly used in antibiotic susceptibility studies involving the aforementioned agar diffusion method of analysis also referred to hereinbefore as the Bauer-Kirby method.

There is provided at the last-mentioned station a vertically arranged plate-like part 264 forming a part of a sub-frame supported from the table as shown in FIG. 2B. An elongated plate-part 266 has a body portion which extends across the table and is spaced a distance upwardly from the latter and which may be supported at one end as by being welded to the plate part 264. The plate 266 part has at the other end a downturned flange supported from the table.

As indicated in FIG. 2B, the sub-frame formed by the plate parts 264 and 266 supports an antibiotic-dispensing electric motor 268. A cam wheel 270 is angularly fixed on the shaft of the motor 268 to be rotated thereby and coacts with the actuator of a switch 272. The cam wheel 270 also actuates an antibiotic disc dispensing slide 274 the body of which slides on the plate 266 in flatwise engagement, which plate 266 is suitably provided with longitudinally spaced apertures, not shown, for the discs 263 to fall through respective ones thereof.

The slide 274 has at one end an upstanding cam follower 276 coacting with the cam wheel and is urged thereagainst by a tension spring 278 having one end supported from the plate part 264 and the other supported from the cam follower 276. As shown in the last-mentioned view, the plate part 266 has in depending relation thereto three open-ended tubular guides 280 for the antibiotic discs which terminate a distance above the cups in the tray 34. The other ends of these tubular guides are secured to the underside of the plate 266 in any suitable manner around the respective ones of the last-mentioned apertures in the plate 266 for dispensing the discs 263.

The slide 274 has three apertures 282 formed therein, each receiving one at a time, discs fed, as by gravity, from a respective one of three magazines 284 vertically arranged on cover plate 286 supported from the plate 266 in fixed relation thereto and having an inverted channel in the bottom thereof receiving the slide 274. The magazines 284 extend through the cover plate 286. The magazines 284 may comprise simple open-ended tubes in which the discs 162 are stacked for storage and later dispensing. It is believed made clear from the foregoing that when the slide 274 is in one axial position thereof, it receives in the apertures 282 thereof antibiotic discs from the respective magazines 284, which position of the slide is effected by the spring 278. In another axial position of the slide 274, effected by the cam 270, the apertures 282 register with the aforementioned apertures in the plate 266 and three discs are simultaneously dispensed in the manner shown in FIG. 2B.

The plate 266 has a tubular nozzle 288 extending vertically therethrough and rigidly supported therefrom with its lower discharge end terminating a distance above the cup level in the tray 34 and arranged to index with the cup 40 of each row of cups in the tray. The upper end of the nozzle 288 is connected to one end of a tube 290 having its other end connected to outlet 292 of solenoid-operated valve 294 shown in FIG. 2C.

The valve 294 is of the three-way type, having an outlet at 296, and having an inlet 298. A tube 300 has one end connected to the inlet 298 and has the other end thereof connected, in a manner identical to the above-described tube 82, to a pump, indicated generally at 302, which may be identical to the pump 84 described and which requires no further description here.

An inlet tube 304, similar to the above-described tube 86, has an end connected to the pump in identical fashion and has the other end thereof extending into sealed reservoir 306 which is filled with a liquid bacteria killing agent such as formalin for example. The pump is operated by an electric motor 308, which may be identical to the above-described pump motor 96, which has a cam wheel 310 angularly fixed on the driving shaft thereof coacting with the actuator of a switch 312.

The operation of the dispensing station C, which will be understood from the foregoing is as follows. When the station C is enabled as aforesaid by operation of the tray sensor switch 261 the pump motor 208 is energized and is operative to deliver a precise volume (0.5 ml.) of formalin (25 percent solution) to the solenoid-operated valve 294 which is actuated in a mode to deliver this volume through the tube 290 via the outlet 292, the tube 290 and the nozzle 288 to the first cup 40 of the first row of cups, immediately killing the bacterium therein. At the same time, the dispensing motor 268 is energized dispensing three different antibiotics in the respective last three cups of the row and the motor 268 is de-energized by actuation of the switch 272 coacting with the cam wheel 270. It will be noted that the second cup 42 in this row of cups remains untreated at this station.

Following this operation which requires less than 90 seconds, the 90-second timer 48 actuates the tray-advance motor 22 to advance the last-mentioned tray one row of cups and the cycle is repeated in the second row of cups, and the cycle is repeated again in the third row of cups. Following this the timer 48 again actuates the tray-advance screws 12 to advance the tray one increment after which it is, again, by the timer 48 advanced one increment to the position of the tray shown at incubation station D where the tray actuates tray sensor switch 316 enabling station D shown in FIG. 2C.

At station D, the samples of bacteria are incubated. The presently used incubation time is uniform and is 2 1/2 hours at a temperature of 37.degree.C. Of course, the incubation time at station D may be lengthened somewhat, if desired, and it may even be shortened if circumstances warrant it, and this susceptibility to change applies equally to the incubation temperature. In practice the trays may be conveniently stacked in an elevator for this period of incubation. However, for simplicity and ease of explanation, the tray 34 is merely moved in increments along the table during this incubation period, and it will be noted that in FIG. 2C the apparatus is shown broken at the incubation station D to indicate that the station may be further elongated in this area. As illustrated at station D, a hood 318, which is open-ended longitudinally of the table, extends over the table but this illustration is purely by way of example as no hood is required if the entire apparatus is enclosed is a cabinet which is preferable. It is believed made clear that while the tray 34 is in this incubation station it is advanced periodically by operation of the timer 48 controlling actuation of the tray-advance screws 12 and which advance is periodically terminated by actuation of the switch 32 coacting with the cam wheel 24 on the shaft of the motor 22.

As previously indicated, E is the station for adding a bacteria killing agent to those tray cups 38 in which such an agent has not been added previously, and F is the station for sampling the contents of each of the cups holding the treated samples. Both of these stations E and F are shown in FIG. 2C and share more mechanical elements and control switches than the previously described stations, and hence will be described together. For this purpose, consider that the last-mentioned tray 34 has been treated at station E and has been moved by the tray-advance motor 22 under the influence of the timer 48 to the station F and the tray position shown therein, while the following tray is in the position shown at station E in FIG. 2C.

Stations E and F share a sub-frame 320 which is vertically arranged and extends transversely across the table 10 a distance thereabove as shown in the last-mentioned view. It is supported by end flanges 321, 322 suitably secured to the upper surface of the table 10 in any suitable manner. The end flanges support therebetween in fixed relation horizontally extending vertically spaced rods 324, which are arranged transversely of the table 10 as shown in the last-mentioned view. A carriage is indicated generally at 326 having vertically spaced block-like parts 328 and 330 rigidly interconnected by a bar 332. The blocks 328 and 330 are respectively apertured to receive the respective ones of the rods 324 for support of the carriage 326 on these rods for sliding movement transversely of the table 10.

A carriage-driving motor 334 is supported on the plate part 320 of the sub-frame. The last-mentioned motor has angularly fixed on its driving shaft a cam wheel 336 and, in axially spaced relation to the latter, a wheel 338. A switch 340, supported from the end flange 322 has an actuator engageable with cam wheel 336.

As shown in FIG. 2C the motor 334 and its above-described associated parts are located adjacent one end of the plate 320. Adjacent the other end of plate 320, There is revolubly supported by the latter an idler 342, and a belt 344 is trained over the driving wheel 338 and over the wheel 342. The belt is fixed, as at 346, to the block 330 of the carriage 326 to drive the carriage. The upper run of the belt 344 has switch actuating element 348 fixed thereto which coacts with the respective actuators of limit switches 350 and 352 supported from the plate 320.

On the lower extremity of the carriage 336 there is provided a horizontally extending part arranged longitudinally of the table 10 and extending both forwardly and rearwardly of the block 328 as shown, and fixed thereto, this part part of the carriage being designated at 354 and being shown as formed by a bar. One end of this bar 354 cooperates with station E, while the other end of the bar cooperates with station F as will be more evident hereinafter.

The end of the bar 354 at station E rigidly supports a vertically arranged nozzle 356 extending therethrough and directed downwardly and connected at its upper end to one end of a tube 358. The other end of the tube 358 is connected to outlet 296 of the aforementioned three-way solenoid valve 294 as shown in FIG. 2C.

At the other end of the bar 354 at the station F, there is fixedly supported on the bar 354 a housing provided for a dual probe assembly, indicated generally at 360, which assembly is mounted for vertical rectilinear sliding movement effected by a solenoid in one direction thereof and which may be spring biased in the other direction. Probe 364 of this assembly is longer than companion probe 366. Probe 364 is provided to drain each sample cup after it has first been sampled by the probe 366, which probes are vertically moveable together in a unitary fashion into and out of each sample cup 38. The probes are open ended and on extension of the probes into a sample cup the probe 364 extends close to the bottom of the cup.

Stations E and F are provided with station enabling tray sensor switches 370 and 372, respectively, which are similar to the above described tray sensor switch 316 enabling station D; and the trays 34 at the respective stations E and F simultaneously enable the last-mentioned stations by actuation of switches 370 and 372 by their respective pins 52.

Since the sample probe 266 is in liquid-flow communication with the photometric station G for the examination for counting purposes of the particles in the respective samples such as bacteria and foreign matter, and station H for receiving signals from station G in order to count the last-mentioned particles and make comparisons of such counts and for recording the results of the analysis is electrically linked to the station G to be operated therefrom, a consideration of both of the last-mentioned stations is required at this point in the description. A tube 374 has one end thereof connected to the upper end of sample probe 366 and the other end thereof connected to flow input 376 of a flow cell 378 having a flow output at 380 of the particle counter which may be generally of the type illustrated and described in Isreeli U.S. Pat. No. 3,511,573 issued May 12, 1970. It is of the forward light-scattering dark field type. The counter, which need be described here only generally, may have a support plate 382 supporting a housing 384 for a light source such as a lamp which housing is shown connected to one end of optical tube 386 having the usual condensing and other lenses therein, not shown. The other end of the optical tube 386 communicates with the flow cell 378 in the light path. On the opposite side of the flow cell 378 and in the light path there is housed, as at 388, a light detector. The sensitivity of the particle counter is such that it may detect particles as small as 0.5 microns which is sufficient to enable the device to count cells of bacteria as well as particles of dirt.

The liquid output 380 of the flow cell is connected to one end of a tube 390 the other end of which is received in a sealed waste receptacle 392. Also extending into the waste receptacle 392 is one end of a vacuum tube 394 having the other end thereof connnected to the inlet 396 of an electric motor driven vacuum pump 398 which may be suitably supported on a plate 400. Because of this construction and arrangement operation of the pump 398 causes the sample to flow in tube 374 from the sample probe when the last-mentioned probe is immersed in a sample, and this sample is drawn through the flow cell 378 and exits through the tube 390 to the waste receptacle.

A tube 402 has one end thereof connected to the upper end of the sample drainage probe 364 and the other end extending into the waste receptacle 392. It will be understood from the foregoing that operation of the vacuum pump 398 effects simultaneously flow through tube 402 to the waste receptacle 392 to drain the cup of the last-mentioned sample. It will also be clear from the foregoing that only a small portion of the sample flows through the flow cell 378 of the particle counter.

The aforementioned photodetector in housing 388 is coupled by means of a cable 404 to a computing and recording device at station H which device includes a recorder for recording the results of the analysis which recorder is indicated generally at 406. The recorder 406 is of the type having a moveable stylus which traverses a conventional driven chart strip. The stylus records bacteria counts, including those of a control, as peaks on the chart.

The operation of stations E and F and subsequent stations described above because of the foregoing description should be clearly understood from the following description. Stations E and F are independently operated so that there is no dispensing action at station E if there is a tray at station F but no tray following it. In other words there is no dispensing action at station E unless tray sensor switch 370 is actuated. For present purposes consider that there is a tray at station E actuating tray sensor switch 370 and a tray at station F actuating tray sensor switch 372, these switches being actuated by respective ones of tray pins 52.

The carriage 326 is in a position shown in FIG. 2C in which the dispensing nozzle carried thereby is over the first cup 40 of the first row of cups 38 and, at station F, the probe assembly 362 is over the first cup 40 of the first row of cups in the corresponding tray. It will be recalled that the bacterium sample in the last-mentioned cup of the tray at station E has been previously killed by the addition of formalin solution at station C. No treatment of this cup is effected at station E. At station F the probe assembly 362 is lowered by the energization of the solenoid associated therewith into the corresponding or last-mentioned cup of the tray station F and pump motor 398 is energized to cause a predetermined quantity of sample to flow through sample probe 366 into the flow cell 358 as aforesaid and to the waste receptacle 392, while probe 364 simultaneously aspirates sample from the last-mentioned cup to the waste receptacle 392, emptying the cup.

Actuation of tray sensor switch 372 as aforesaid also serves to energize the particle counter at station G to signal particle counts to the then enabled, also by switch 372, the counting, comparison and recording mechanism of station H. A liquid flow sensor 393 in waste tube 402 from the probe assembly 362 may be used to sense the cessation of liquid flow in the waste line 402 thereupon actuating the solenoid controlling the probe assembly 362 to retract it, and at the same time energizing horizontal drive motor 334 which drives the carriage 326 to place the probe assembly 362 over the second cup 42 of the first row of cups at station F and place the nozzle 356 at station E over the cup 42 of the first row of cups at the last-mentioned station.

When this has taken place the particles in the sample cup 40 of the first row of cups at station F have been counted, and it will be recalled at that in this cup, which is a time zero (T.sub.o) control, the bacterium was killed at station C. All the cells, and all foreign matter such as dirt of a size not less than 0.5 microns, has been counted by the particle counter and information concerning the number of particles has been stored in the recording mechanism H.

It will be further recalled that the second cup 42 in the first row of cups at station F has not received an antibiotic and the bacterium therein has not been challenged but has been incubated along with the rest of the samples in cups three, four and five of the row. The contents of this cup 42 serves as a time 150 minute control which is an indicator the exponential growth of viable bacterium in this cup over an incubation period of 150 minutes.

Movement of the carriage is stopped by de-energization of the horizontal drive motor 334 by the switch 340 coacting with the cam wheel 336. Actuation of switch 340 actuates pump 302 by energizing pump motor 308 at the same time energizing solenoid valve 294 to operate the last-mentioned valve to deliver formalin from the reservoir 306 through the valve outlet 396 to the tube 358 connected to the nozzle 356 delivering the aforementioned volume (0.2 ml of a 25 percent solution) of formalin to the second cup of the first row at station E, which immediately kills the bacterium therein. The pump motor 308 is de-energized by the switch 312 coacting with cam wheel 310.

It will be understood that while formalin is delivered in the last-mentioned manner to the second cup in the first row of cups at station E, the probe assembly 362 has descended into the second cup of the first row at station F, and the contents of this control, T.sub.150, is sampled in the aforementioned manner and the cup drained, with the signal-receiving-and recording mechanism H storing information as to the total number of particles counted in the last-mentioned control cup 42. The last-mentioned mechanism subtracts the first count from the first cup 40 sampled from the total particle count sampled from the second control T.sub.150 giving a figure which represents the total exponential unihibited growth or proliferation of viable bacterium in the sample. This is a highly satisfactory technique for indicating the proliferation of the bacterium of the last-mentioned sample inasmuch as it compensates for those organisms in the sample which were not viable and any particles of foreign matter in sample such as dirt of a size not smaller than 0.5 microns.

The last-mentioned cup 42 is drained by the probe assembly 362 as aforesaid, and upon actuation of the liquid flow-sensing device 393 previously described the probe assembly is retracted from the last-mentioned cup: and the motor 334 is re-energized, and the cycles at stations E and F repeated, until all the bacteria in the first row of cups at station E have been killed and all the cups in the first row at station F have been sampled and the cups drained as aforesaid.

At this time the signal receiving and processing mechanism H has received and stored information as to the proliferation of bacterium in each of the third, fourth, and fifth cups in this row, which it will be recalled have received different antibiotics. It will be understood from the foregoing that in the illustrated form of the apparatus the sample probe 366 will aspirate air in its movement between sample cups. However, it will be understood by those versed in the art that, if desired, the construction and arrangement may be such that the probe assembly 362 may be immersed in a wash liquid between immersions in sample cups.

When formalin has been added to the last cup in the first row at station E and the last cup of the first row at station F has been sampled and drained, switch actuator element 348 on the belt 344 is in engagement with the actuator of switch 352 placing the belt motor 334 in the reverse mode, so that on the next energization of motor 334 as aforesaid the motor 334 drives the carriage 326 in the reverse direction, overriding switch 340, to return it to the position shown in FIG. 2C, wherein switch actuating element 348 engages the actuator of switch 352 de-energizing motor 334 and placing it in reversed mode. On the next signal from the 90-second timer 48, the trays at the stations E and F are simultaneously advanced by the tray-advance screws 12 driven from the motor 22 and the advance is stopped by switch 32 coacting with the cam wheel 24, at which time the probe assembly 362 is indexed with the second row of cups of the corresponding tray at station F and the nozzle 356 is indexed with the second row of cups of the corresponding tray at station E and the aforementioned cycles are repeated until the last cup in each tray at station E and station F and the aforesaid cycle is completed with reference to the same.

Upon completion of the operation at the stations E and F with reference the samples in these trays, the respective trays are advanced on the table 10 as aforesaid with the tray shown at station E in FIG. 2C advancing to station F to the tray position shown therein in the last-mentioned view. The tray, the cups of which have been emptied as aforesaid at station F, travels along the tray as aforesaid to the discharge end 16 of the table and may ultimately be deposited, as by gravity, in a storage bin 46.

Returning to the information receiving storing, computing and recording mechanism at station H, details of which are described in the co-pending U.S. Pat. Application of Caruso et al., titled "Method and Apparatus for Providing Direct Real-Time Determination of a Particular Population," Ser. No. 139,432, filed May 3, 1971, this mechanism is the presently used form, after subtracting the count from cup 40 of each row from the cup 42 of the same row, uses the resultant figure as a divisor for the bacterium count in each sample portion challenged by a different antibiotic. If the computation made by the last-mentioned mechanism results in a figure which is less than 0.1, the particular antibiotic has definitely had an inhibitory effect on the growth of the bacterium. If this figure is between 0.1 and 0.2, the result with reference to inhibition of growth is equivocal. If the figure is more than 0.2, there is a definite indication that the particular antibiotic has not served to effectively inhibit exponential growth of the bacterium and that the latter is resistant to the drug.

The figure which results from the aforementioned computation with reference to the control T.sub.o and each of the other separately tested sample portions is indicated in respective peaks as aforesaid on the chart strip. In connection with this computation, it may be noted that with respect to the unihibited control, five doublings of the viable bacterium present may be anticipated, that is within the aforementioned 150 minute incubation time. The lower limit of acceptability of the findings of the susceptibility to antibiotics is set arbitarily at a one log increase or three doublings in numbers between control time zero and control time 150. To check on the operation of the machine, periodically a sample of a known bacterium is placed in a tube 242, together with a pipette 216, in sampler assembly 240 and run through the analysis apparatus as aforesaid. The susceptibility of this bacterium to the various antibiotics used in the apparatus is known and hence chart readings from the recorder H should accord with this information.

In a manual or semi-automated technique devised to check the operation of the apparatus on randonly selected bacteria, response to the antibiotic cephalothin, present in an arbitrary concentration of 3.75 mcg./ml., Pseudomonas aeruginosa No. 48, as expected, showed a complete resistance to the drug by a growth ratio according to the aforesaid computation of 0.822, while the figure by the same computation for Salmonella No. 72 was 0.018 indicating its susceptibility to the antibiotic. Two representatives of Shigella, Nos. 19 and 20, yielded ratio figures zero and 0.810 respectively indicating the capability of the apparatus to detect resistant and susceptible representatives of a bacterium within the same genus. Klebsiella No. 26 indicated a high susceptibility to the drug by a reading of 0.001 as did Staphylococcus No. 51, to a somewhat lesser extent, with a reading of 0.053.

It will be understood from the foregoing that the above described method and apparatus for automated antibiotic susceptibility analysis achieves the stated objects and may be utilized for other purposes than those described above in detail, such as providing bacteria counts of uninhibited exponential growth or measuring minimum antibiotic inhibitory concentrations. Also as previously stated, it may be used with known bacteria for testing various fluids, such as body fluids for example, for their characteristics of inhibiting bacterial growth. It will be readily apparent to those versed in microbiology that the method and apparatus of the invention may have many other uses in a microbiological laboratory. Still further, at least some sub-combinations of the apparatus, as those various combinations described with reference to particular stations of the apparatus as well as the methods executed thereby, may be utilized outside the field of microbiology.

A specific, purely illustrative example follows. In the analysis of different blood samples fed in succession from the turntable 244, utilizing the test tubes 242 and the pipettes 216, might be transferred utilizing the pipettes 216 to test receptacles as on the table 10, which prior thereto received a suitable diluent and the sample portions in these cups might be treated with different reagents at a later station for analysis of calcium, glucose and albumin for example, and these treated sample portions might be analyzed subsequently in a flow cell of a colorimeter or in a flame photometer, for example. Moreover, it will be obvious that at station B liquid in a probe 216 could be driven so as to be dispensed from the probe by air pressure, without any air bleed, by downward movement of the plunger 186, rather than by dripping from the probe by gravity at atmospheric pressure, it this is desired. Also, stations A and B may be combined.

While several preferred embodiments of the method and apparatus for automated antibiotic susceptibility analysis of bacteria samples have been described and illustrated, it will be apparent to those versed in the art that the invention may take other forms and is susceptible of various changes in detail without departing from the principles of the invention.

Claims

What is claimed is:

1. A method of antibiotic susceptibility analysis of a plurality of liquid samples in sequence, each including a different bacterial inoculum, comprising: supporting each sample in a separate container, transferring each sample sequentially in equal aliquot portions to each one of a series of sample-receiving receptacles provided for each sample and in which a liquid nutrient is supplied, treating with different antibiotics certain ones of said sample aliquot portions in said sample-receiving receptacles simultaneously, while concurrently killing with a bacteria killing agent the bacteria in a first untreated one of said aliquot portions in one of said sample-receiving receptacles, incubating said aliquot portions in said sample-receiving receptacles of said series, killing with a bacteria killing agent a second untreated one of said aliquot portions in one of said sample-receiving receptacles of said series after incubation flowing sequentially each of said samples from the sample-receiving receptacles of said series successively in equal aliquot portions thereof to a counter, counting bacterial cells in each of the aliquot portions of the same sample of said series, comparing the results of the cell counts of the said first and second untreated aliquot portions of said series to quantitatively determine uninhibited bacterial grouwth of the same sample, and comparing the count of bacterial cells in each treated aliquot portion of said series with every other treated aliquot portion of the same sample of said series, utilizing said determination of the uninhibited bacterial growth of the same sample, to quantitatively indicate any inhibition to baterial growth of each sample by each of said antibiotics.

2. The method as defined in claim 1, wherein: said transfer of each sample to the corresponding sample-receiving receptacles of said series is made by a probe, and further including aspirating each sample from the corresponding separate sample container into the probe for sample transfer.

3. The method as defined in claim 1, further including killing with a bacteria killing agent the treated and incubated ones of said sample aliquot portions prior to said flowing of sample from said sample-receiving receptacles.

4. The method as defined in claim 1, wherein: said sample transfer of each sample from the corresponding separate sample container to said sample-receiving receptacles of said series is by an individual probe for each sample.

5. The method as defined in claim 4 wherein: each aliquot portion of each sample during sample transfer to said sample-receiving receptacles of said series is dispensed in drops from the corresponding probe into the corresponding sample-receiving receptacles, and the drops are counted to determine each aliquot portion.

6. Apparatus for antibiotic susceptibility analysis of a plurality of liquid samples in sequence, each including a different bacterial inoculum, comprising: means supporting each sample in a separate container, transfer means transferring sequentially each sample in equal aliquot portions to each one of a series of sample-receiving receptacles provided for each sample and in which a liquid nutrient is supplied including probe means for transfer of each sample from said separate sample containers to the corresponding sample-receiving receptacles of said series, and means aspirating each sample from the corresponding separate sample container into said probe means for sample transfer, means treating with different antibiotics at least certain ones of said sample aliquot portions in said sample-receiving receptacles of said series simultaneously, means introducing a bacteria killing agent into an untreated one of said aliguot portions in one of said sample-receiving receptacles of said series concurrently with said treatment, incubating means for said aliquot portions in said sample-receiving receptacles of said series, means sequentially flowing each of said samples from the corresponding sample-receiving receptacles of said series successively in equal aliquot portions thereof, and means coupled to said flow means and counting the bacterial cells in each of the aliquot portions of the same sample for comparison, utilizing the cell count of the killed aliquot portion as a base, to quantitatively indicate any inhibition to bacterial growth of each sample by each of said antibiotics.

7. Apparatus as defined in claim 6, further including means introducing a bacteria killing agent into a second untreated one of said aliquot portions in one of said sample-receiving receptacles of said series after incubation, the cell counts of said counted ones of said untreated aliquot portions indicating by comparison the uninhibited bacterial growth of the same sample.

8. Apparatus as defined in claim 6, further including means introducing a bacteria killing agent into said ones of said sample-receiving receptacles of said series containing said treated ones of said sample aliquot portions after incubation, prior to said flow of sample from the receptacles of said series.

9. Apparatus as defined in claim 6, wherein said probe means comprises individual probe means for each of said separate sample containers, each individual probe means transferring the corresponding sample in said aliquot portions thereof to the corresponding ones of said sample-receiving receptacles of said series.

10. Apparatus as defined in claim 9, wherein said probe means dispenses the sample in drops from the probe means into the corresponding sample-receiving receptacles of said series to determine each aliquot portion, the probe means including a drop counter.