CA1123629A - Antibiotic susceptibility testing - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Method and apparatus are disclosed for detecting antibiotic sus-ceptibilities using serial dilution in liquid media. A plurality of dif-ferent antibiotics are placed in a large number of small wells in a trans-lucent plastic tray, each antibiotic being placed in a column of wells at serially diluted concentrations. A bacteria culture of known uniform con-centration is then inoculated into each well. Following a period sufficient to allow growth, a diffuse uniform light is passed through each of the wells and their contents, and the intensity of the light passing through each well is measured to determine turbidity of the bacterial suspension. A turbidity value for each well is compared with a value corresponding to zero growth, and this information is processed by a computer which calculates which of the antibiotics are effective to inhibit growth of the bacteria, and the minimum concentration of each effective antibiotic necessary to inhibit growth. This information is displayed and printed out and provides inter-pretive information to determine the choice and dosage of an antibiotic to be administered to the patient. The computer is programmed to perform var-ious calibration operations and checking routines to confirm that the in-strument is operating properly and that the test is being conducted properly.

Description

Background of the Invention The invention relates to measurement of the suscep-tibility of bacteria to different antimicrobic drugs, with automatic quantification of the susceptibility to each drug, so that a physician may select a drug that will most effectively treat an infecting bacterium and choose the appropriate dosage for effective treatment.
The physician usually has a choice of about twelve to fifteen types of antimicrobial agents for treating the forty to sixty groups of pathogenic bacteria. Many of these agents are ineffective against a given bacterial strain, but normally some of them will be appropriate for treatment. In order for the physician to choose the best antimicrobic, it is necessary to isolate the pathogenic organism in the laboratory and then test it against a panel of drugs to determine which drugs inhibit growth and which do not. Ideally the doctor should receive susceptibility information the same day the culture is taken, since it is usually necessary to initiate therapy immediately. Unfortunately, it currently takes one day to isolate an organism, and it has required another day to test the susceptibility of the organism to the antimicrobics. Therefore, it has been customary for the physician to institute therapy based on an educated guess at the time the patient is first seen. If the sensitivity studies completed two days later indicate that the guess was incorrect, therapy is changed to the proper drug.

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Clearly an important goal in automating antimicrobic testing would be to diminish the time lag between the initial culture and the obtaining of sensitivity information. An estimated 30 million antimicrobic susceptibility tests are performed annually in the United States by labor in~ensive manual methods. In addition to the potential economic advantages of automation and obvious advantages ~o the patient in receiving only the proper treatment, one could also anticipate better precision, quality control and objectivity.
The most frequently used technique to measure antimicrobial susceptibility has been the standardized disc-diffusion method described by Kirby and Bauer (Bauer, Kirby et al., "Antibiotic Susceptibility Testing by a Standardized Single Disk Method", American Journal of Clinical Pathology, 1966, Vol. 45, No. 4, p. 493). By this method, isolates of bacteria are grown in suspension to a standardized concentra-tion (usually determined by visual turbidity) and streaked onto nutrient agar (culture medium) in a flat glass Petri dish.
Paper discs impregnated with different antim~c^Lobial materials ~a are placed upon the agar streaked with bacteria, and the drug is allowed to diffuse through the agar, forming a gradient halo around the disc. As the bacteria replicate, they form a visible film on the surface of the agar, but in the zones surrounding the antibiotic-impregnated discs, growth is inhibited if the organism is susceptible to that particular antimicrobial agent. Since a concentration gradient has been established, the zone of inhibition around the disc is roughly proporitonal to the degree of susceptibility. Typically, the laboratory classifies an organism as "sensitive'l', "intermediate", or 3Q "resistant" to each drug in the test panel. Thus the results a~
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establish a characteristic profile or "antibiogram" for that organism.
The Kirby-Bauer disc-diffusion method has the advantage of simplicity, but it suffers from several drawbacks.
One problem is that of time efficiency. In order that the initial inoculum become visible on the Petri dish so that zones of growth can be distinguished from zones of inhibition, the bacteria numbers must increase by several orders of magnitude over the original number. However, for determination of whether or not the organism is growing in the antimicrobial milieu, which is the only information required, a period that would allow doubling of all the organisms should be theore-tically sufficient with suitable detection equipment. For most Gram-negative organisms, the doubling period is between twenty and thirty minutes, following a lag phase. Therefore, an automated system should be able to distinguish growth within a thirty-minute period.
Another difficulty with the Kirby-~auer disc method is that of standardization. If an organism is "resistant", does that mean that it cannot be treated with higher than normal doses of the microbial agent? Also, how does this information relate to a site in the body where the antimicrobic is concentrated (such as bile) or decreased in amount (such as cerebrospinal fluid)?
To answer these questions, quantitative data are necessary. To obtain quantitative results, it must be deter-mined that minimum concentration of a drug will inhibit the organism's growth. This quantitation of susceptibility is ~Z;~Ç6Z9 known as minimum inhibitory concentration or MIC. The MIC may be determined by making serial dilutions of the drug in agar or broth, and then inoculating each dilution of each drug with a standardized suspension of bacteria. Since the test procedure may involve as many as 70 to 80 individual tubes, it can become a formidable task if the test is performed in individual test tubes on a macroscale. Systems are available in which the individual dilutions of antimicrobics are made in plastic trays containing small micro-tubes. (Marsh and MacLowry, "Semiautomatic Serial-Dilution Test for Antibiotic Suscepti-bility", Automation and Data Processing in the Clinical Laboratory, Springfield, Illinois, C. C. Thomas 1970). Organisms can be inoculated in a single step using a multi-pronged template.
Thus, setting up the test is simplified, and it takes slightly less time to provide quantitative data than qualitative Kirby-Bauer information. There are now semiautomated devices that dispense antimicrobial solutions into the microtubes.
Trays of microtubes are also commercially available with frozen solutions in the tubes, and the Gram-negative anti-microbial panels have been combined with biochemical tests to identify enteric bacteria as well as to determine their antimicrobic susceptibility.
Although MIC results give quantitative information ~hich allows consideration of multiple doses and multiple sites, the MIC numbers in themselves can be confusing to the clinician. To use MIC data correctly, a physician must refer to tables of achievable antimicrobic levels as a function of dosage and body site. Therapy will be effective if the achievable drug level for a particular dose and site in the .

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body is two to four times the MIC. With the present invention described below, such interpretation of MIC data is accomplished by a computer, which compares the MIC with a table of achievable drug levels at different body sites and different doses.
Optical detection methods have been suggested and have proven to be powerful tools to measure bacterial growth.
A laser light-scattering system can have the sensitivity to detect a single bacterium. Optical methods measure the presence of bacteria either by nephelometry or turbidity measurements.
I Nephelometry measures the ability of the bacteria particles to scatter light, and the detector is aligned at an angle to the axis of ~he light source. Turbidity measures the net effect of absorbance and scatter~ and the transducer is placed on the axis of the radiation source. Nephelometry measurements are significantly more sensitive than turbidity measurements~
but since the nephelometer measures only that fraction of light scattered by bacteria, the signal to the detector is small, and both light source and transducer amplification must be corre-spondingly large.
Automation in microbiology has lagged far behind chemistry and hematology in the clinical laboratory. However, there is presently an intensive effort by industry to develop this field. The best publicized devices for performing automated antimicrobic susceptibility testing use optical detection methods. A continuous flow device fo~ detecting particles 0.5 micron or less has been commercially available since 1970;
however, probably due to its great expense, it has not been widely used in the laboratory. Other devices using laser ., light sources have been suggested but have not proven commercially practicable. Recently, the most attention has been directed to three devices discussed below.
The Pfizer Autobac 1 system (United States Patent No.
RE. 28,801; "AUTOBAC" is a registered trademark of Pfizer, Inc.) measures relative bacterial growth by light scatter at a fixed 35 angle. It includes twelve test chambers and one control chamber in a plastic device that forms multiple contiguous cuvettes. Antibiotics are introduced to the chambers via impregnated paper discs. The antimicrobic sensitivity reader comes with an incubator, shaker, and disc dispenser. Results are expressed as a light scattering index ~LSI), and these numbers are related to the Kirby-Bauer "sensitive, intermediate and resistant". MIC measurements are not available routinely with this instrument. In a comparison with susceptibilities of clinical isolates measured by the Kirby-Bauer method, there was 91% agreement. However, with this system some bacteria strain-drug combinations have been found to produce a resistant Kirby-Bauer zone diameter and at the same time a sensitive LSI.
The Auto Microbic System has been developed by McDonnell-Douglas to perform identification, enumeration and susceptibility studies on nine urinary tract pathogens using a plastic plate containing a 4 x 5 array of wells. The specimen is drawn into the small wells by negative pressure and the instrument monitors the change in optical absorbance and scatter with light-emitting diodes and an array of optical sensors. A mechanical device moves each plate into a sensing slot in a continuous succession so that each plate is scanned once an hour, and an onboard digital computer stores the optical data. The system will process either 120 or 2~0 specimens at a time. One can query the status of each test via a CRT-keyboard console, and hard copy can be made from any display. When the system detects sufficient bacterial growth to permit a valid result~ it automatically triggers a print-out. Fol-lowing identification in four to thirteen hours, a technologist transfers pos-itive cultures to another system which tests for antimicrobic susceptibility.
The results are expressed as "R" (resistant) and "S" (susceptible); no quan-titative MIC data are provided.
The Abbot MS-2 system consists of chambers composed of eleven con-tiguous cuvettes. Similar to the Pfizer Autobac 1, the antimicrobial com-pounds are introduced by way of impregnated paper discs. An inoculum con-sisting of a suspension of organisms from several colonies is introduced into the culture medium, and the cuvette cartridge is filled with this suspension.
The operator inserts the cuvette cartridge into an analysis module which will handle eight cartridges ~additional modules can be added to the system). Fol-lowing agitation of the cartridge, the instrument monitors the growth rate by turbidimetry. I~hen the log growth phase occurs, the system automatically transfers the broth solution to the eleven cuvette chambers; ten of these chambers contain ~ntimicrobial discs, and the eleventh is a growth control.
70 The device performs readings at five-minute intervals, and stores the data in a microprocessor. Following a pre-cut increase of turbidity of the growth control, the processor establishes a growth rate constant for each chamber.
A comparison of the antimicrobic growth rate cons*ant and control growth rate constant forms the basis of susceptibility calculations. The printout pres-ents resuits as either resistant or susceptible; if intermediate, susceptibil-ity information is expressed as an MIC.

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Non-optical methods have also been used or suggested for measuring antimicrobic sensitivity in susceptibility testing.
These have included radiorespirometry, electrical, impedance, bioluminescence and microcalorimetry. Radiorespirometry, based on the principle that bacteria metabolized carbohydrate and the carbohydrate carbon may be detected following its release as CO2, involves the incorporation of the isotope C 4 into carbohydrates. Released C1402 gas is trapped and beta counting techniques are used to detect the isotope. The major difficulty in applying the isotope detection system to susceptibility testing, however, is that an antimicrobic agent may be able to stop growth of a species of bacteria, yet metabolism of carbo-hydrate may continue. Less likely, a given drug may turn off the metabolic machinery that metabolizes certain carbohydrates, but growth may continue. This dissociation between metabolism ; and cell growth emphasizes the fact that measurements for detecting antimicrobic susceptibility should depend upon a determination of cell mass or cell number rather than metabolism.
The electrical impedance system is based on the fact that bacterial cells have a low net charge and higher electrical impedance than the surrounding electrolytic bacterial growth media. A pulse impedance cell-counting device can be used to count the cells; however, available counting devices are not designed to handle batches of samples automatically, and generally do not have the capacity to distinguish between live and dead bacterial cells. Another approach with electrical impedance has been to monitor the change in the conductivity of the media during the growth phase of bacteria. As bacteria utilize the nutrients, they produce metabolites which have a , , greater deg~ee o~ elec~rical condu,ctance th~n the native broth, so that as metabolis,m occursr impedence decreases. ~owever, since this technique measures cell metabolism rather ~han cell mass, its applicability to antimicrobic susceptibility detection suffers ~rom the same drawback as radiorespirometry.
Bioluminescence has also been suggested for the detection of micro organisms. It is based on the principle that a nearly universal property of living organisms is the storage of energ~ in the form of high energy phosphates (adenosine triphosphate, ATP), which can be detected through reaction with firefly luciferase. The reaction results in the emission of light energy which can be detected with great sensitivity by electronic light transducers. Although a clinical laboratory may obtain a bioluminescence system to detect the presence of bacteria in urine~ the technique is expensive due to the limited availability of firefly luciferase, and problems have been encountered in standardizing the system.
Microalorimetry is the measurement o~ minute amounts of heat generated by bacterial metabolism. The principle exhibits certain advantages, but laboratories have not adopted such a system, one serious drawback being that the system measures metabolic activity rather than bacterial mass or number.
S_mmary of the Invention According to the present invention there is provided a method for performing optical density tests, employing a sample tray having a series o~ wells containing liquid samples, said wells having translucent bottoms, one said well being a control well, comprising: holding said tray accurately in a single predetermined stationary reading position without blocking of,~ ht paths throu~h said wells~ sending light down through all said weIls at roughly the same intensity to an array of - g _ ~3~

light_intensity~diet~cti~g ph~toce~ls, there ~eing one photocell ad~acent to each ~e~l, including a control photocell adjacent to said control well~ electronically sequentially comparing the signal from each said photocell o~ said ~rray with the signal from said control photocell and developing a related signal therefrom for each well.
~ ccording to another aspect of the present invention there is provided an automatic scanning apparatus for performing optical density tests, employing a sample tray having a series of wells containing liquid samples, said wells having trans-lucent bottoms, comprising: tray holding means for holding said tray accurately in a single predetermined reading position without blocking off light paths through said wells, a single dif~used light source positioned above the sample tray, for sending light down through all said wells at roughly the same intensity, an array of light-intensity-detecting photcells on the opposite side of the tray holding means from light source means, one photocell adjacent to each well and positioned to .:
receive light from the light source which has been transmitted through the well and its contents, sequential signal receiving means connected to all the photocells for receiving sequentially a signal from each said photocell in a prescribed order, without any physical movement of the tray or photocells, each signal corresponding to the intensity of light received by a said photocell, and electronic sequencing means connected to said signal receiving means for electronically causing it to receive its signals in order.
The present invention provides an optical method and ~ apparatus which can be used for automatically determining bacterial susceptibility to a number of di~erent antimicrobic drugs, utilizing turbidimetry. The preferred system uses broth-dilution ~ ..
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to determine susceptibility. Serial dilutions of the anti-microbic agent are inoculated with the organism and incubated for a period sufficient to allow detectable growth. The disclosed appàratus determines minimu~ inhibitory concentration (MIC) of a particular antimicrobic drwg, which is the lowest concentration of that drug that results in no detectable bacterial growth. Typ-ically, ten antimicrobic drugs are evaluated, with seven different dilutions of each drug being tested. Therefore, to obtain an MIC
determination for ten drugs, seventy tubes or wells must be inoc-ulated and examined. In contrast to previows methods using individ-ual full-sized test tubes, which were cumbersome and expensive, the present system utilizes "micro-tubes", which are presently available as disposable, molded plastic trays, each well of which holds approx-imately 0.5 milliliter.
For measurement of the MIC values in these trays, appropriate dilutions of each antibiotic must be placed in the wells or micro-tubes. Semiautomated devices for making the dilutions and filling the trays in large batches are available commercially. Alternatively, a laboratory may ~0 obtain trays that are already filled with antibiotic dilutions and kept frozen until use. To prepare the bacteria culture for inoculation into the wells, a suspension of the bacterial organisms in water is made in a container. By means of a multiple-pronged device, a technician is able to inoculate a uniform drop of bacterial suspension into each of the large plurality (e.g., seventy) of wells with a single motion.
The bacteria and the various dilutions of the antimicrobic agents are incubated for a time period sufficient to produce detectable bacterial growth, and the MIC may then be determined as the lowest concentration of the effective antimicrobic agents in which there is no evidence of growth.
Previously, the reading of such an MIC tray was done by manual viewing performed by a technician, and was a laborious procedure. An overnight incubation period was generally required in order to produce visually detectable patterns of growth. However, the disclosed apparatus and method provide for the performance of the reading and interpretive task automatically. Moreover, the device has the capability of inter-polating the MIC between twofold dilutions, whereas by visual reading a technician can only detect the difference between growth and no growth and thus can only read MIC to the nearest twofold dilution. With the sensitive photoelectric apparatus described herein, together with the capabilities of a microcomputer the different gradations of growth can be measured even after a rel-atively short incubation period, and a precise MIC can be calculated and displayed on a screen or printed out. Thus, the device makes available continuous numerical data that improves accuracy and allows quantitative quality-control techniques.
Photodetection of bacterial growth is accomplished by passage of uniform intensity light through each of the wells and through the translucent well bottoms following the incubation period. The uniform light may be obtained from plural uniform sources, one at each well, or by a single source of uniform, diffused light over the entire tray. At the opposite side of the tray, preferably below the tray, are an array of sequentially-scanned photoelectric cells, one associated with each well.

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The sensed light intensity level at each well is compared by computer with a light level corresponding to zero bacteria growth to determine a relative value of turbidity. The reference value may be obtained by the reading of a sterile control well.
In addition to the quantitative MIC data, the disclosed apparatus and method provide a graphic interpretive printout to guide the physician's therapy. The computer is programmed to translate the MIC value into dosage ranges that would be necessary to achieve blood levels of the antimicrobic drug effective to in-hibit growth of the organism at a particular site. Por example, a printout of "-" might be used to indicate that the organism is re-sistant and no dosage of a drug can effect the organism. A print-out of "+" may be used to mean that the organism is resistant and may respond to high intramuscular or intravenous doses, with "++"
indicating intermediate sensitivity and that the organism may respond to higher than recommended doses. A printout of "+++" would indicate that the organism may be sensitive to the usual doses of an anti-biotic, and "++++" would indicate a high degree of sensitivity and thus an optimal drug with which to treat the infectious agent.
In one embodiment, a method according to the invention for determining susceptibility of a bacteria culture to various antimicrobic drugs and of determining the minimum inhibitory concentration (MIC) of the bacteria culture to those drugs to which it is susceptible comprises the steps of placing the plurality of different antimicrobic drugs in a plurality of wells in a light-transmissive tray, each drug being included in a series of wells in serially-diluted known concentrations;
establishing a known uniform concentration of the bacteria and placing the uniform concentration in equal volumes of the wells;
following an incubation period, passing ligh~ in substantially equal intensity through each well and determining a turbidity value for the bacterial suspension of each well by sequentially sensing the intensity of light transmitted through the bacterial suspensions of the wells by means of photodetectors adjacent to the wells opposite the light source; and in a computer, comparing turbidity values with a turbidity value corresponding to zero bacterial growth, thereby determining which antimicrobic drugs have inhibited bacterial growth and the minimum concentration of each inhibitory drug required to inhibit growth, and displaying the determined information. The concentration of the bacteria culture may itself be initially determined by turbidimetric measurement utilizing a light source and at least one photo-detector. The antimicrobic drugs may be placed in the tray in a rectangular matrix of wells, with each column of wells containing incrementally varying concentrations of a single drug. Of course, any arrangement of the-wells or of the anti-microbics in the wells is suitable, so long as the computer has the proper information as to what is being tested in each well. Control wells containing only the bacterial suspension, as well as sterile control wells, may be included for self-checking of the system and/or providing a transmitted light value corresponding to zero bacteria growth. The system may, as explained above, provide for translation of the MIC values to dosage ranges necessary to establish the required antimicrobic concentration at the body sites involved.

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Accordingly, it is among the objects of the invention to provide an automated method and apparatus for quantitative antimicrobic susceptibility testing, capable of providing complete MIC data for a plurality of antimicrobics within a very short period of time. An associated objective is to provide in such a system a means for translating MIC results into a complete interpretive therapeutic guide for the patient in question. These and other objects, advantages and features of the invention will be apparent from the following description of the preferred embodiments, taken in conjunction with the accompanying drawings.
Description of the Drawings In the drawings:
Figure 1 is a perspective view showing an automated antibiotic susceptibility apparatus according to the invention.
Figure 2 is an exploded perspective view showing a sample tray and optical detection equipment forming a part of the apparatus of Figure 1.
Figure 3 is a sectional elevational view showing a portion of the apparatus.
Figure 4 is a diagram indicating the operation of the system of the invention.
Figure 5 is a block diagram of an analog to digital converter subsystem of the invention.

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23~29 ~ Figure 6 is a block diagram of a microcomputer portion '~ of the apparatus.
Figures 7A, 7B, and 7C are flow charts of operational steps involved in the method of the invention.
Figure 8 is a schematic elevation view showing an alternative form of optical detection apparatus which may be included in the apparatus of the invention.
Figure 9 shows a form of printout which may be utilized in connection with the apparatus of the invention.

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Description of the Preferred Embodiments In the drawings, Figure 1 shows one example of an external configuration which the susceptibility testing apparatus 10 of the invention may take. The unit 10 com-prises a photo unit or optical detection unit 11 and a processor unit 12. The optical detec~ion unit 11 preferably includes a drawer 13 for receiving and supporting a sample tray 14 whic}l is examined by detection apparatus of the unit ll when the drawer is closed and the testing operation is begun. The detection unit 11 may also include a patient identifica-tion input switch 16, a run switch 17 and a calibrate switch 18.
The processor unit 12 may include a readout display 19, an on/off power switch 21, printer control buttons 22, and a printout exit 23 which dispenses a printed "ticket" 24 bearing the desired sus-ceptibility information.
Figure 2 somewhat schematically represents the con-figuration of the detection apparatus associated with the optical detection unit 11 of the apparatus 10. Within the detection unit 11 above the drawer 13 is a source of uniform, diffuse light which may comprise, for example, a fluorescent light bulb 26, a parabolic reflector 27 positioned thereabove such that the lamp 26 is at the focal point of the reflec~or 27, and a diffuser 28 just below the lamp and reflector. The arrangement of the lamp 26 and the reflector 27 provides a nearly uniform distribution of light over the surface of the diffuser 28, and the diffuser improves uniformity and re-duces intensity to the desired level.

~Z36~9 Within the drawer 13 are a sample tray holder 29 having a matrix of openings 31, and an array of photocells 32 therebelow in a matrix conforming to the position of the openings 31 above. The openings 31 and the photocells 32 also correspond precisely to the position of sample testing wells 33 of a sample tray 14 which is received in registry above the tray holder 29 when a test is to be conducted. The sample tray 14, or at least the bottom of each well 33, is translucent so that light passing through the diffuser 28 penetrates the wells and their contents, passes through the openings 31 in the tray holder 29, and reaches the photocells 32 below, which individually sense thé intensity of the light passing through each well. The photocells may be of the type manufactured by Clairex Electronics of Mt. Vernon, N.Y. as Model CL702L. The tray holder 29 is preferably of a dark, light-absorbing color such as black to reduce light trans-mission between the wells and reflection of diffracted light within any one well. The tray holder arrangement assures that all light passing through the openings 31 is from the wells 33 rather than through other areas of the translucent sample tray 14.
The sample tray 14 is preferably a disposable, molded plastic tray, each well of which holds approximately 0.5 milliliter. Trays of this type are commercially avail-able and have been used previously for simple visual type "reading" techniques as discussed above. The wells 33 are often referred to as "microtubes", since they replace cumber-some full-sized test tubes which were used in the past for this type testing.

Figure 3 shows a portion of the internal apparatus of the optical detection unit 11 in cross section. The drawing is somewhat schematic, without details of the structural supporting arrangement within the unit 11 and the drawer 13, but shows the relationship of the lighting com-ponents 26, 27 and 28 to one another and to the sample tray 14, the tray holder 29 and the matrix of photocells 32. The bottom of a supporting surface of the drawer 13 is shown in this schematic view, with the photocells 32 mounted on that 10surface and the tray holder 29 surrounding and extending above the array of photocells 32. The sample tray 14, several wells 33 of which are indicated in Figure 3 fits snugly over the tray holder 29 with the wells 33 extending down into the openings 31 of the tray holder with little side-to-side tolerance so that registry of the sample wells with the photocells is assured.
The light source illustrated is a convenient and preferred form; however, any light source or a plurality of light sources which will provide light of equal intensity ~0directed into each well 33 of the sample tray 14 is suf-ficient. In this regard, an alternative form of light source and detection system is described below in connection with Figure 8.
As discussed above, the sample tray 14 is pref- I -erably laid out in a rectangular matrix, which may comprise for example eight rows and ten columns. Other arrangements would be adequate, but a rectangular matrix is space-efficient and convenient. The wells 33 contain various dilutions of different antibiotics, and these may be arranged such that each of the ten columns of wells contains a single anti-biotic in a series of different dilutions. There may be seven different concentrations of each antibiotic, with the eighth well of at least several of the columns used for con-trol purposes. For example, one control well might be used for unrestricted growth of bacteria, and another well used to represent no growth, with no bacteria inoculated into the well.
Into the wells containing the various dilutions of different antibiotics is introduced the patient bacteria sample borne within a culture medium. This bacteria culture is uniformly inoculated into each well, and this may be accomplished by commercially available devices having a matrix of prongs (not shown) arranged to register with each well to be inoculated in the commercially available sample tray 14. Of course, the antibiotics and the bacteria cul-ture may be introduced to the wells in the reverse order, but for convenience, efficiency and reliability it is pre-~0 ferred that the antibiotic be introduced first.
Figure 4 indicates diagrammatically the operation of the susceptibility testing apparatus 10. The lamp 26, reflector 27 and diffuser 28 are shown transmitting uniform diffuse light through a sample well 33a of the matrix of wells of the sample tray 14. The well 33a contains one dil-ution of one of the antibiotics being tested, inoculated with a controlled volume and known concentration of the bacteria in a culture sample. The same uniform diffuse .~ - 19 -light is also transmitted through a well 33n containing no bacteria for providing a light intensity reading correspond-ing to zero bacteria growth.
After an incubation period sufficient to allow some detectible growth of the bacteria in the well 33a in the event that growth is not prevented by the particular antibiotic in the particular concentration being tested, a growth culture 36 results therein. The light from the dif-fuser passes through this culture 36 and through the bottom of the well to a photocell 32a of the photocell matrix.
Here the intensity of the light is sensed and converted into an electricàl analog value corresponding to the opacity of the culture 36. This opacity value represents the turbidity of the culture, stemming from the net effect of light ab-sorption and scatter in the well 33a. At the same time, the diffuse light passes through the sterile control well 33n to a photocell 32n of the photocell matrix. Again, the sensed light intensity is converted into an electrical analog reference value.
The photocell 32a is connected to a plus input of a differential amplifier 37 through a noise filter 38 and a multiplexer 39 which functions to select each photocell 33 of the matrix of photocells in a prescribed sequence under direction of a microcomputer 41. Thus, the photocell 32a shown in Figure 4 is connected to the differential amplifier 37 only when the multiplexer 39 momentarily selects that particular photocell. The reference photocell 32n is connected to the minus input of the differential amplifier 37 and ~36~

provides a reference voltage which is subtracted from the plus input to provide an analog differential output. Thus, the light intensity or turbidity value signal emanating from the differential amplifier 37 is in the form of a reference voltage which varies according to turbidity of the sample being sensed, representing the increase in turbidity of that sample since inoculation. Each analog signal is transmitted in its turn to an analog to digital converter 42 which con-verts the analog to a digital signal and sends it to the microcomputer 41.
The microcomputer 41 and its operation are de-scribed below with reference to Figures 5, 6 and 7. It functions to correlate differential digital values ~from the ADC 42) representing bacterial growth for the various wells with the particular drug and its concentration in the subject well.
From this correlation, the microcomputer selects the ~ero growth indication stemming from the weakest concentration of each drug, and this concentration becomes the MIC for that particular drug. If none of the wells containing a particu-lar drug indicates inhibition of growth, the microcomputer prints out the fact that the infectious organism is resistant to that particular drug.
The remaining apparatus indicated in Figure 4 is described below with reference to the other figures.
The analog circuitry associated with this system 10, including the analog to digital converter, is set forth in the detailed block diagram of Figure 5. Figure 5 includes ~3~

the noise filter circuit 38, the multiplexer circuits 39 which are included within the dashed line box, the differ-ential amplifier 37, and the analog to digital converter 42 along with its related supporting circuitry. The ten by eight photocell matrix is also shown in Figure 5 for clarity of understanding of this part of the system 10.
The multiplexer 39 includes a binary coded decimal (BCD) to decimal decoder 101 driving column drivers lQ3, and another BCD to decimal decoder 113 controlling FET
switches lll. A four bit digital line 100 from the micro-computer 41 is connected to the binary coded decimal input of the binary coded decimal to decimal decoder circuit 101 ~which may be preferably implemented as a type 7442 TTL
integrated circuit or equivalent). Ten output lines 102 from the decoder lOl are connected to ten driver circuits 103. The driver circuits are preferably implemented as operational amplifiers type LM 324 or equivalent.
As already explained above, the photocell matrix is arranged as a rectangle with ten columns and eight rows.
Thus, the outputs from the ten driver circuits 103 are ap~
plied to the ten columns respectively via a bus 104 such that when one driver is excited by operation of the decoder 101~ an excitation voltage is provided to one of the column drive lines corresponding to the binary coded decimal column select information input to the decoder 101 via the data line 100 from the microcomputer 41. An eleventh of the drivers 103 applies voltage continuously through a drive line 105 to the reference cell 33n.

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Eight row lines 106 and one line 107 from the reference cell 32n are applied as inputs to nine active filter circuits within the filter 38. Each filter circuit is preferably implemented by an operational amplifier, type LM 324 or equivalent. The filters 38 function to remove power line ripple so that the eight row output lines 108 and a reference output line 109 carry DC voltage levels only.
The eight output lines 108 are applied to eight field effect transistor switches 111, respectively. The switches are preferably implemented as integrated circuits type CD4016 CMOS quad bilateral switch gate chips or equivalent. An output line 110 from the switches 111 is connected directly to the plus input of the differential amplifier 37. The ninth line 109 is applied directly to the minus input of the logarithmic differential amplifier 37.
A three bit digital line 112 from the micro-computer 41 is connected to the input of a second binary coded decimal to decimal decoder 113 which is also preferably implemented as a type 7442 TTL integrated circuit or equiv-alent. The decoder 113 functions to selèct one of eight output control lines 114 which in turn select one of the eight field effect transistor switches 111 to connect one of the filtered row lines to the plus input of the logarithmic differential amplifier 37, in accordance with digital row select information received from the microcomputer 41.
The logarithmic differential amplifier 37 is preferably implemented as an Analog Devices type 756 or equiv-alent, and the purpose of the amplifier 37 is to correct for ~ , ~

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variations in light intensity from the light source 26. The light-variation-corrected analog voltage output from the amplifier 37 is supplied as an input to an operation ampli-fier 116 which is provided with external potentiometers to control gain and DC offset of the incoming signal from the amplifier 37.
An output line 117 from the amplifier 116 is supplied as an analog input to the analog to digital converter 42 which is preferably implemented with a National Semicon-ductor M~15357 integrated circuit or equivalent. A digital control line 118 from the microcomputer 41 is connected as a trigger input to a monostable multivibrator one shot 119, preferably implemented as a type 74121 TTL integrated circuit or the equivàIent. An output pulse from the one shot 119 of appropriate amplitude and duration is supplied via a line 121 to the analog to digital converter 42 to start the con-version process. A timing generator (e.g. type 555) 122 applies timing pulses via a iine 123 to the analog to digital converter 42 to control the sequence of operations thereof.
The analog to digital converter 42 utilizes the timing pulses supplied on the line 123 during a conversion cycle to digitize the analog information on the line 117 and provide an eight bit digital output via an eight bit output bus 124 which is supplied to an input port of the microcomputer 41.
The microcomputer 41 forms the central portion of the system 10. The microcomputer includes a single chip monolithic microprocessing unit (MPU) 140, which is pref-erably implemented as a type 6800 manufactured by Motorola _ 24 -~. ,.

36zg Semiconductor, American Microsystems, and other suppliers.
Although this particular microprocessor was chosen for the described preferred embodiment of the present invention, other types of microprocessors would function equally as well, for example the Intel 8080, the Mostee 6502, the Zilog Z80, the Fairchild F-8, etc. A suitable two-phase clock 141 provides the necessary clock signals to the micro-processing unit 140.
The main system program which is set forth in hexadecimal code in the table following the specification of the present invention is loaded into one and a half kilo-bytes of programmable read only memory 142. The read only memory 142 is preferably implemented with 2708 programmable read only memories produced by Intel and other suppliers.
Other PROMs would be well suited for the program memory 142.
The microcomputer 41 also includes one kilobyte of random access memory ~R~M) 143 which provides volatile storage of data to be processed as well as a stack for the micropro-cessing unit 140. The microprocessing unit 140, the clock ` 141 through the microprocessing unit 140, the program memory 142 and the data storage memory 143 are connected in parallel to the system bus 144 which includes an eight bit data bus, an eight bit control bus, and a sixteen bit address bus.
Input output interface is accomplished with three peripheral interface adapters (PIA) 146, 147 and 148 which are connected to the system bus 144. The interface adapters 146, 147 and 148 are preferably implemented as type 6820 integrated circuits produced by Motorola Semiconductor, ~Z3~2~

American Microsystems, and other suppliers. These inte-grated circuits contain two ports apiece. Each port may be used either to input data to the microprocessing unit 140 or to output data to output devices, as will be explained hereinafter.
The first interface adapter 146 has its first port connected to receive the eight bit digitized information via the bus 124 from the analog-to-digital converter 42, as shown in Figure 5. The first port of the interface adapter 146 also provides the control signal line 118 which is connected to the one shot 119 which functions to start the analog to digital conversion process of the converter 42. The line 118 will be further explained hereinafter. The second port of the interface adapter 146 is connected to the multiplexer 39 with four bits provided for the column select control signal via the bus 100, and the three remaining bits provide for the row select control signal via the bus 112.
The second peripheral interface adapter 147 includes a first port which controls the printer 20. Two ~0 bits of data are input from status indicators in the printer 20 via a line 149. One of these bit positions is from a microswitch which indicates that the paper form has been properly inserted and that a printout can be made. The other bit is a signal from the printer electronics which indicates that the printer is either in a "print" or a "wait" opera-tional mode. Four bits of the first port of the interface adapter 147 are also used to control the printer and shift data to be printed into the printer 20. The data is entered 3~iX9 serially via a line 151 from the first port of the adapter 147 to the printer 20. Other control functions carried out by the four bits on the line 151 include line feed (advance the paper one line), print (cause the print solenoid to make an impression on the paper), and shift (move the next data bit into position for printing). The second port of the interface adapter 147 is not used in the present embodiment.
The third peripheral interface adapter 148 in-cludes a first port which reads the thumbwheel switch 16 for patient identification information via a four-bit line 153. The upper four bit positions of this first port of the adapter 148 are used to select and enable one of the four thumbwheel positions via a four bit line 152. One bit position of the line 152 is low to enable one of the four switching positions. The lower four bits of the first port of the adapter 148 are used to read data via a bus 153 from the switch position selected by the upper four bits. The data from the switch represent a binary number between zero and nine. The second port of the interface adapter 148 is used to supply data to the alpha-numeric display readout 19.
The display 19 is the Burroughs model SSD0132-0070 self-scan display unit with built-in electronics. As explained, it is controlled via a line 154 from the second port of the third peripheral interface adapter 148. Data to be displayed on the display 19 are entered into the unit via a line 156 in a six bit code for all alpha-numeric'characters as well as some special symbols.,,The data are read in from left to right and appear on the display until new data are entered.
Thus, the upper two bits are provided via the line 154 to _ 27 -~Z3~i2~

control the display, wi~h one of the bits being a clear line and the other being an enable line. The lower six bits are provided via the line 156 for the purpose of sending parallel data to the display presented to the user in accordance with the operation of the system 10.
In addition to the characteristics of the inter-face adapters 146, 147 and 148 described hereinabove, each adapter also has an interrupt function. The interrupt is an additional line which is available for monitoring the status of external devices. In the presently described system 10, the interrupts are used to monitor operator actions of several types. Interrupt capabili~y which re-sults in an output rather than an input is termed a strobe.
Strobes are utilized in the system 10 as well as interrupts.
Thus, the first peripheral interface adapter 146 controls the conversion of data from analog to digital format via the analog to digital converter 42 by utilizing a strobe line 118 which is connected to the one shot 119 (Figure 5) to start the analog to digital conversion operation.
The second peripheral interface adapter utilizes an interrupt from the printer 20 via a line 155 and utilizes one interrupt each from the run switch 17 via a line 158 and calibrate switch 18 via a line 159. The second port of the second adàpter 147 utilizes the output strobes via a line 157 to cause the printer 20 to execute a print cycle.
A third peripheral interface adapter 148 has two interrupt inputs: one from a microswitch indicating that ~3~Z~

the photo uni~ drawer is open via a line 161 and one in-dicating that the drawer is closed via a line 162.
The printer 20 is preferably implemented as an MFE model TKllE with built-in electronics package. Data is fed from the microcomputer 41 via the line 151 which generates the proper control signals to enable the printer electronics to cause the printer 20 to print, line feed or shift data into internal registers. The data is fed to the printer 20 in serial format, stored in buffers in the printer electronics, and is then printed in parallel. The command to print is generated as a strobe output of the second port of the second peripheral inter~ace adapter 147 via the line 157. The printer is a commercially available unit presently being sold for the original equipment manufacturer (OEM) market.
The operation of the system 10 is explicated by the flowchart set forth in Figure 7. Therein, at a power on step 166, the operator turns the power on to the system 10.
At that point, the display 19 informs the operator to insert the calibration tray. At insertion step 168, the operator inserts the tray, and at step 169, the operator closes the drawer. At a logical step 170, thè system checks the identifica-tion of the tray in the drawer. For this purpose a binary code is implemented using the uppermost right two wells of the tray, either of these wells belng either opaque or transparent, thus providing identification of four possible types of trays. This code is made to correspond to the combination antibiotics which the tray contains.

~36~3 In the event that the type of tray is not iden-tified at step 171, the system asks whether the tray is inserted backwards at step 172. If so, the readout 19 dis-plays a tray backwards indication at step 173, and the oper-ator opens tile drawer at a step 174 and removes the tray, orients it correctly, and reinserts it, then repeats steps 168, 169, 170 and 171.
Once the tray is identified at step 171, the readout 19 displays the tray type at step 175, and directs the operator to press the calibration switch 18 at a step 176. At step 177, the operator presses the calibration switch 18 whereupon the system tells the operator to wait at step 178. The wait signal remains until the system informs the operator to remove the tray at step 179. The operator opens the drawer at step 180. In the event that the tray is not in backwards, and yet the tray remains unidentified at step 181, the operator is then instructed to open the drawer to manually inspect the tray to find out why the system 10 is unable to identify it.
At step 182, the readout 19 tells the operator to close the drawer, and at step 183 the operator removes the tray and closes the drawer. The readout 19 then tells the operator that if a next test is desired, he should press the run or calibrate button at step 18~. At a step 185, the operator actually presses the run or the calibrate switch.
If the system has been previously calibrated at step 186, then the readout 19 directs the operator to insert the test tray at step 187. However, if the system 10 has not been , ~Z3~

calibrated at step 186, the program returns to step 167 and the calibration procedure is carried out as set forth in steps 167 through 185.
At step 188, the operator opens the drawer and inserts the test tray. The display 19 than tells the operator to close the drawer at step 189. The operator closes the drawer at step 190 and the tray identification is determined at step 191. In the event that the tray is not identified, the system then determines whether the tray is in backwards at step 192. If so, the system informs the operator that the tray is in backwards by a readout display at step 193. In the event that the tray remains unidentified and it is not in backwards, then at step 194, the operator is informed that the tray is unidentified and the program loops back to step 180 whereupon the operator opens the drawer and repeats steps 180 through 191.
Once the identification of the tray has been determined at logical step 191, the system 10 displays the type of tray at the readout with step 195. Then the oper- :
ator is informed to set the patient identification informa-tion into the identification switch 16 and insert the form to be printed into the printer`20 at step 196. The operator performs these operations at step 197 and when they are completed, the display 19 tells the operator to press the run switch 17 at step 198. The operator presses the run switch 17 at step 199 and the patient identification infor-mation is displayed at step 200. Then, the patient identi-fication is printed on the form at a step 201 and then the '' ' l~Z3Çi~9 MIC values and interpretive information are printed on the form in step 202 to produce the form 203.
Once the form is printed with the patient identi-fication MIC values and interpretive information the dis-play tells the operator to remove the tray at step 204.
The operator opens the drawer and actually removes the tray at step 205 whereupon the display 19 tells the operator to close the drawer at step 206. The operator closes the drawer at step 207 and the apparatus 10 then instructs the operator to perform the next operation of either "run" or "calibrate" at step 208 whereupon the program loops back to step 185 where the run or calibration switches are operated and the program is repeated as heretofore described until all of the samples have been evaluated by the system lO.
Figure 8 shows schematically an alternative arrange-ment for passing light through the wells 33 of the sample tray 14 and detecting the resultant light intensity passing through each well. The apparatus of Figure 8, which utilizes fiber optics to transmit light, would replace the form of light source and diffuser 26, 27 and 28 shown in Figures 2, 3 and 4. Tt would also eliminate the need for a large plurality of photocells 32 in a matrix as shown in Figure 2, and would replace the multiplexing unit 39 (Figure 4) with a substitute arrangement which selects one cell at a time for receipt of a penetrating quantum of light.
The apparatus of Figure 8 includes a light source 221 and a reflector 222, directing light through a lens 223 llZ36Z~

toward a rotatable selector plate 224 driven by a stepper motor 225. The selector plate 224 has a single opening 226 (dashed lines) which sequentially directs light to different fiber optic fibers 228 of a fiber optic bundle 229. The stepper motor 225 is under the control of the microcomputer 41 via the lines 100 and 112 ~Figures 4 and 6), in lieu of and to perform the same function as the multiplexer 39 indicated in Figure 4 and 6. The fiber optic fibers 228 of the bundle 229 each go to individual testing wells 33 of the tray 14.
The fibers are indicated only schematically, as is the bundle 229.
Below the wells 33 are a second plurality of fiber optic fibers 230 of a second bundle 231. Transmitted light from each well is collected by a fiber 230 of the bundle 231 and fed via a lens 232 to a single photocell detector 233. A value corresponding to the intensity of incident light is then fed to the filter 38, then to the plus input of the differential amplifier 37, as in the apparatus of the other embodiment described above.
In order to provide a control or reference value which may be fed into the minus input of the differential amplifier 37 to represent a base iight intensity corre-sponding to zero bacterial growth, there must be one optical fiber which always carries light through a reference sterile control well, i.e. the well 33n of Figure 4, also indicated in the schematic representation of Figure 8. Accordingly, a single optical fiber 228n is positioned at the lens 223 in such a way that it receives and carries light continuously .~

3~2~

whenever the lamp 221 is energized, i.e. whenever any of the wells 33 is being tested. The fiber 228n extends to a position adjacent to the sterile control well 33n as shown, and a receiving fiber 230n carries the transmitted light to second lens 232n. The resultant analog light intensity value for the control well is fed through the filter 38 to the minus input of the differential amplifier 37~ so that the differential amplifier yields a differential analog signal corresponding to increased turbitity in the tested well from bacterial growth.
The remainder of the system remains the same as described above. The principal advantage of the form illus-trated in Figure 8 is the use of a single light source focused on the fiber optic bundle and a single detector for all test wells of the sample tray, providing a more uniform measure-ment over the matrix of test wells in the tray. Light is transmitted through only two wells of the tray at any given time: the well currently being tested for turbidity, and the sterile reference well 33n. The subsystem of~Figure 8 allows for close standardization and easy calibration and checking.
Figure 9 shows a form of printout ticket 24 which may be used in conjunction with the present invention, with exemplary MIC susceptibility information and therapy information. As discussed above, the apparatus of the invention provides a graphic interpretive printout to guide the physician's therapy, an example of this type information being located in the right column of the ticket 24. The computer algorithm translates the MIC values (left column) .

~ 34 -3'~

to dosage ranges that would be necessary to achieve blood levels of each antimicrobic drug to effectively inhibit growth of the organism. Figure 9 indicates one form that the "therapy guide" information may take. With this format, "-" indicates that the organism tested is resistant to that particular antimicrobic, and that no dosage of the ~nti-microbic can affect the organism. "+" indicates resistance but that the organism may respond to high intra-muscular intra-venous doses. "++" indicates that the organism is intermediate in sensitivity to the particular antimicrobic, and may respond to higher than recommended doses. A printout of "+++~' indicates sensitivity to the usual recommended doses of the antibiotic, and "++++" means that the organism ex-hibits a high degree of sensitivity and thus is an optimum drug with which to treat the infectious organism. A print-out of "****" tells the physician that a dosage of that particular antibiotic necessary for therapy may be toxic to the patient.

~Z36~

TABLE OF P~ROGRAM FOR ANTIBIOTIC SUSCEPTIBILITY TESTING
The follo~ing listing cons~itutes the program for antibiotic susceptibility testing in hexadecimal code for direct loading into the programmable read only memory ; 5 142.
Address Program Instructions 0100 CE 80 00 6F 05 6F 04 86 2C A7 05 6F 07 ~6 7F A7 0130 FF A7 OE C6 2C E7 OF 7F 87 E~ 7F 87 E9 01 01 0`1 0140 7E Fl 3B;01 01 01 01 01 01 01 01 01 01 01 01 01 0150 01 01 01 01 01 01 7F 87 Dl 7F ~7 EO 7F 87 El 4F

0170 2E 43 84 FO 27 07 CE F5 61 B7 ~7 Dl 39 86 47 8D
0180 lE 43 84 FO 27 08 CE F6 10 B7 ~7 EO 20 07 CE F6 0190 lF 43 B7 87 El BD F3 36 BD F2 F5 BD F2 CF 39 B7 OlBO 39 01 01 01 BD FO 56 B6 ~7 Dl 26 6C CE F5 D4 BD
OlCO F2 4A 20 03 BD F2 50 B6 ~7 D5 27 03 7E F2 98 B6 OlDO 87 D6 27 FO BD F3 36 BD F2 F5 CE F6 2E BD F2 CF
OlEO 86 OA BD F2 BD CE 84 00 B6 87 BO 26 03 CE 84 AO

0200 B6 87 E0 27 03 7F 87 E8 B6 87 El 27 03 7F 87 E9 0210 7E F2 98 01 01 B6 87 EO 27 03 B7 ~7 E~'B6 87 El 0230 CE F5 9B BD F2 47 B6 %7 D4 27 F8 CE F5 29 BD F2 0240 47 B6 87 D6 27 03 7E FO Bl B6 37 D7 26 03 7E Fl 0250 3B 01 01 01 BD-FO 43 B6 87 Dl 27 03 7E Fl 28 B6 0260`~7 EO 27 OB B6 87 E8 26 10 CE F5 4B 7E Fl 28 B6 0270 87 El 27 F5 B6 87 E9 27 FOi~6 03 BD F2 BD CE F5 02A0 87 D7 27 FO 86 FF B7~7 AO B7 87 A3 B7;~7 A4 BD

~3~

0300 CE ~7 A4 B6 ~0 08 85 20 26 F9 B6 80 08 85 10 27 0310 FO C6 08 64 oa 25 04 ~6 OE 20 02 36 OF B7 ~ a8 0320 B6 80 OA B7 80 OA 4F 4C 26 FD 5A ~6 E6 09 8C 37 0350 FF 87 D2 CE 87 D4 6F 00 0~ 3C 37 D9 26 F6 FE 87 0360 D2 OE 3E 39 CE ~0 00 A6 CD 35 30 27 03 B7 87 D4 0370 85 40 27 03 B7 87 D5 E6 OC A6 09 ~5 ~0 27 03 B7 0380 87 D6 85 40 27 03 B7 ~7 D7 E6 08 A6 OB 85 80 27 03AO OE C,6 ~0 FD F2 B9 BD F3 36 BD F2 CF C6 ~0 BD F2 03BO B9 32 4A 26 E5 7E Fl 25 5F B6 01 20 01 5F 37 5F

03DO ~7 D2 E6 OO 27 OA 86 AA B7 80 OE gD 1~ SA 26 F6 03EO E6 Ol 08 A6 Cl 84 3F 8B ~O B7 80 OE ~D 07 5A 26 03FO Fl FE 87 D2 39 4F 4A 26 FD 39 BD F3 36 g6 F7 CE
0400 87 Al OD 49 25 OC BD F3 47 BD F2 00 BD F3 47 01 0470 F5 B6 80 04 43 A7 00 08 7C 80 06 B6 80 06 &4 o 0490 BD F3 47 09 FF 87 D2 A6 50 AO 00 2B lE BD F3 C

04CO D3 26 ~2 F7 87 AO 01 01 20 D3 FF 87 EE 5F CE F6 04FO A6 00 01 01 CE OF FF 85 07 27 lF CE 4F FF 85 06 0500 27 18 CE 44 FF 85 05 27 11 CE 44 4F 85 04 2~ OA
0510 CE 44 44 85 03 27 03 CE 38 ~ FF 87 A3 84 F8 44 0530 87 EF FE 87 EE EE 00 FF 87 Al B6 87 AC 26 03 7A

36~9 0610 41 59 05 15 49 4E ~3 45 52 54 20 43 41 4C 49 42 0620 52 41 54 45 20 34 52 41 59~ 0~) 20 4E 45 5~` 54 20 06g0 43 4B 57 41 52 44 53 OD 05 45 52 52 4F 52 OA OB

06A0 53 45 20 44 52 41 57 45 52 01 lE 53 45 54 20 50 06BO 41 54 4q 45 4E 54 20 49 44 20 41 4E 44 20 49 4E
06CO 5~ 45 52 54 20 46 4F 52 4D OB 09 50 52 45 53 53 06EO ~2 52 41 54 45 06 13 49 4E 53 55 46 46 49 43 49 06FO 45 4E 54 20 47 52 4F 57 54 4~ 06 14 53 54 45 52 0700 49 4C 45 20 43 4F 4E 54 41 4C~ 49 4E 41 54 45 44 0740 SA 00 4A 00 2A 00 lA 00 OA 50 OA 25 OA 12 OA 06 0780 11 19 22 2A lB 05 20 29 32 3B 43 4C 54 05 lD 20 0790 29 32 3B 43 4C 05 2~ 30 39 42 4B 53 5C 05 00 08 07AO 10 lB 23l 2B 33 05 70 78 ~1 8A 93 9C A4 05 28 30 07FO 10 lB 23 2B 33 05 70 78 81 3A 93 96 A4 05 CE 84 0800 5C B6 87 EO 26 03 CE 84 FO PF ~7 D2 BD F3 60 B6 0830 7E Fl 28 A6 OF AO 5F 2A 01 40 CE F5 E5 81 OA 2D
0840 EF BD F3 8C 7E Fl 25 01 01 01 01 01 01 01 01 01 0890 01 01 01 01 01 01 01 ûl 01 01 01 01 01 01 01 01 08DO, 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 08FO 01 01 01 01 01 01 01 01 01 01 01 01 01 0]. 01 01 ~ ~ ~ 36 Z~

1 The above described preferred embodiments provide

2 apparatus and a method for automatically determining the

3 minimum inhibitory concentration of a plurality of different

4 antibiotics necessary to stop growth of an infective orga-nism being tested. Minimum inhibitory concentration infor-6 mation is also transferred to dosage information by the 7 apparatus and method of the invention. The required time 8 to perform such a test is greatly reduced in comparison to 9 other methods, a great deal more information is provided, 10 and accuracy is improved. Various other embodiments and 11 variations to the preferred embodiments will be apparent to ~2 those skilled in the art and may be made without departing 13 from the spirit and scope of the following claims.

SUPPLEMENTARY DISCLOSURE
16 Collimation means, preferably beneath the tray 17 holding means, collimates the light from each well after it 18 has passed through the wells. For some tests there is 19 light filter means below the tray holding means, for 20 filtering the color values of the light passing through the 21 wells. The photocells may also be set at a,n inclination to 22 the vertical lines through the wells an~ operate as nephelo- ' 23 meters.
24 The single diffuse source need not put out a 25 uniform light. The light need only be roughly even. Also, 26 the photodetectors may be inexpensive ones, providing 27 signals of different strengths for the same light intensity, 28 so long as the invention is practiced with an initial cali-29 bration step. In this step, the light source directs 30 light over all the photode~ectors either without a tray , ~ ~ 3 ~

1 positioned above them or with an empty tray, to take any 2 variations in the plastic material of the tray into account 3 in the calibration. As another alternative, the tray wells 4 may be filled and then run through before any culture, at S zero time relative to growth. In the calibration, a scan 6 is made and all values, i.e. photodetector output signal 7 values, are stored. When each actual test is run, a dif-8 ference or ratio signal is created for each photocell, so 9 that only the difference in sensed light intensity is used, disregarding effects of localized differences and intensi~y 11 and difference in the photocells themselves.
12 With the filters the invention makes it possible to 13 use an optical-electrical method for automatically reading 14 the color changes of a plurality of biochemical reactions in small microtubes. As noted in the main disclosure the micro-~6 tubes are, preferably, all part of a unitary sample tray, made 17 of suitable translucent material. Each microtube is a well of 18 this tray. In each well and in a standardized manner, is 19 placed a suitable chemical reagen~ or reagents; then each well 20 is inoculated with the sample. Photodetection of color 21 changes is accomplished by the passage of uniform intensity 22 light through each of the wells and through the translucent 23 well bottoms following an incubation period. At the opposite 2~ side of the tray, preferably below the tray, is an optical 25 filter designed to pass only certain wavelengths of light. ~e-2~ neath the filter is an array of sequentially-scanned trans-27 ducers such as photoelectric cells, one associated with each 28 well. The optical filter is designed so that a shift in color 29 in the wells will result in a predictably greater or lesser 30 amount of light passing through to the photoelectric cells.

~ 6 ~

1 The e~act filter used depends on the test con-2 cerned. The filter may be made to be easily removable and 3 replaceable. For example, a large number o~ tests may be 4 run using only three filters one at a time; these three being (for example) filters numbers 809, 863, and 878, 6 of Edmund Scientific Co., 785 Edscorp Building, Barrington, 7 New Jersey 08007.
~ The sequential signal-receiving means is con-9 nected to all the photocells for receiving sequentially 10 a signal from each photocell in a prescribed order, each 11 signal corresponding to the intensity of light received 12 at the photocell. Electronic sequencing means is con-13 nected to the signal-receiving means and electronically 14 causes it to receive its signals in order, all without 15 any mechanical movement of anything.
16 The sequencing, being automatic, is very fast, 17 going through 80 or 96 wells of a tray ill about five 1~ seconds or less. The automatic electronic scan has no 19 moving parts--an important feature.
Electronic sequencing is much more reliable than ~1 mechanical movement of a tray or o~her mechanical sequenc-22 ing. Multiplexing has the advantages of speed, accuracy, 23 reliability and maintainability, i.e. easy maintenance.
24 ~or at least these reasons, the inven~ion is a significant 25 improvement over mechanical scanning.
26 As noted in the main disclosure, first comparator 27 means is connected to the signal-receiving means and 28 sequentially compares the signal from each photocell of 29 the array with the signal from the reference-detecting 30 photocell and then develops a different signal therefrom.

, ~ - 41 -z~

1 The data storage means holds data values corresponding to 2 zero reaction or other base comparison values and holds 3 data relating to various organisms or tests. Second comparator means is connected to the first comparator means and to the data storage means, and sequentially makes a 6 comparison of each different signal value with a value 7 corresponding to that of the same well when empty or at 8 zero time or zero growth or reacts or develops at a result-9 ant value from that comparison.
The third comparator means may be connected to 11 the second comparator means and to the data storage means 12 sequentially compares said resultant values with a large 3 number of stored values and for determining such conclusory 14 values as the probability values for the presence of 15 selected organisms in khe sample or the minimum inhibitory 16 concentratiOn desired.
17 Finally, the output means connected to the third 18 comparator means gives the results obtained, displaying 19 them or printing them out.
It will be apparent that ~he tray itself might 21 be a source of error. That is, its own light transmissivity 22 and opaqueness and f~aws can substantially affect the light 23 transmissivities received by the photocells, in addition to 24 the light transmissivity of the liquid in the wells. The 25 trays can vary from tray to tray, and they can also vary 26 in a tray from well to well. This could, of course, lead 27 to substantial errors that would give false impressions 28 and false results if not compensated or corrected.
29 The present invention accomplishes the needed 30 correction by the two different types of comparison stages - 42 _ ~ ~ ~ 3 ~ ~

1 already explained in the main disclosure.
2 First, for each reading in any sequence of wells 3 in the tray, each well is immediately compared with the 4 value obtained by direct light transmission to the ref-S erence photocell. From this comparison, the device 6 provides an after culture value for each well, which is a 7 function of the after culture signal values (or amplifi-8 cation thereof) for the tested well and for the reference g photocell. This, of course, represents a comparison of 10 the light received at each photocell in the main array and 11 the intensity of the light received at the reference 12 photocell. The signal may be amplified and is used as 13 the operative signal, as shown in Fig. 4. The after 14 culture value for each well may be called a "difference"
15 signal value, regardless of the type of function which is 16 used in comparing the two values (tes~ well vs. reference 17 photocell) to produce this value. In the embodiment of 18 Fig. 4 the subtractive difference preferably is taken 19 between the two values, and the differential amplifier 20 amplifies the difference signal. However, the signal 21 value may instead be a ratio, and the signal from each 22 well compared with the reference photocell signal by 23 means of a log ratio module. In other words, there is 24 again a "difference" signal, but it is a difference in ~5 logarithms, so that the subtraction is really a division, 26 and a quotient or ratio is obtained instead of a difference 27 expressed as a logarithm.
28 Thus a first comparator may incorporate a log 29 ratio module and send out its related signal as an 30 amplitude ratio between each signal Sw obtained through a .~ - 43 -h ~ "

1 well and its photocell and a signal SR obtained from the 2 reference photocell. This related signal Sx = kl SW

4 where kl is a constant. This first comparator may also incorporate a log ratio module and sends out its resultant 6 value Sv as a ratio k~ SX , where DV is the data reference 8 value and k2 a constant.
9 The first and second comparators may use the same 10 log ratio module. The second comparator may utilize as its 11 data reference value Dv, stored ratios read earlier from an 12 empty tray, so that DV = kl SWE for each well, where SwE

14 is the signal coming from an empty well.
The second comparator may utilize as its data 16 reference value Dv, stored ratios read earlier from a tray 17 containing the same liquid from which the signals Sw are 18 generated, but read at a time when there has been zero 19 growth, so that DV = kl SW for each well, where SwO is n 1~
21 the signal coming from a well containing the liquid at zero 22 growth time.
23 Also the preliminary comparing means may include 24 a log ratio module for sending as its derived signal a 25 signal based on the ratio of the two signals it compares.
26 Thus, in the invention, each reading of each well, 27 at each stage where readings are taken, is compared by a 28 first comparator means with the reading at the reference 29 photocell, and a difference or ratio signal developed from 30 it. By this procedure, variations in light intensity from ~_ - 44 -~ ~ ~ 3 ~ ~

1 the source over time, as would be induced by supply voltage 2 fluctuations, have no effect on the readings. Such varia-3 tions will vary the reference and well photocells propor-4 tionately, so that a ratio will cancel the errors out.
This is the purpose of the reference photocell.
6 Second, to further reduce the possibility of 7 error particularly due to flaws in the tray, and in view 8 of the fact that each tray is positively identified in g the apparatus, as has already been described, a prior 10 reading may be taken through the tray before the reading 11 after bacterial culture; this prior reading is stored and 12 is later compared with the sample reading.
j 13 One way of taking the prior reading is to take 14 a reading of the tray in its empty state, before it is 15 filled with fluid, to compare the reading through each 16 empty well with the reading of the reference photocell, 17 as above, and to store ~he resulting difference signal or 18 ratio signal in the data storage portion of the microcom-19 puter. Then the ratio signal (or difference signal) 20 derived from the liquid at the time of the after culture 21 reading is compared with the ratio signal (or difference 22 signal) of the empty wells. Thereby, each well is compared 23 with itself when full and when emp-ty, and errors due to the 24 wells are substantially eliminated.
Another way of taking this prior reading is to 26 take the prior reading, not of the empty tray but of the 27 tray just after its wells have been filled with the solu-28 tion and prior to the culture; in other words, at sub-29 stantially zero time so far as growth or culture is con-30 cerned. This means that the reading is taken ~ .
~ - 45 -~ Z 3 ~ 2~

1 through the actual solution~ and the ratio of that reading 2 to the reference electrode is stored in the data storage 3 bank for the later use.
4 With the zero based signal (however obtained) in the data bank, and with the ratio or difference signal 6 provided for each well for the liquid after culture, then, 7 before proceeding further~ the next step is to compare by 8 a second comparator means the two ratio (or difference~
9 values, that is, to compare the ratio of the signal derived 10 from the light transmissivity of the specimen after culture 11 to the direct light reception by the reference cell, with 12 the ratio of the empty tray or tray with the same liquid 13 at zero time to the signal from the reference cell. This 14 second comparison may also be made by calculating a ratio 15 of the two ratios, which is preferably accomplished by ;~
16 taking the difference in logarithms of the two ratios, 17 resulting in another logarithm which is the log of the 18 comparison ratio, or of what may be called the comparison l9 signal In the next step, a third comparison depends 21 upon what test is being run. Basically, it is a compari-22 son of the ratio signal obtained from the second comparator 23 means, which preferably is the logarithm of the comparison 24 signal, with values that are stored in the data storage 25 means to determine the final asked-for result.
26 For good results in this last step, especially 27 when applied to MIC procedure, a distinction is made 28 between a growth state and a no-growth state. The instru-29 ment determines at the output from the second comparator 30 means, a voltage level or logarithm value that represents : .

-~ 2 ~ ~2 ~

1 the extent of bacterial growth, when that voltage level is 2 compared to voltages that are obtained from known sterile 3 and growth controls, these voltage values being stored in 4 the data bank of the microcomputer. A first step here is to determine whether there is an adequate voltage (logarithm 6 value~ difference between the readings obtained from the 7 sterile and the growth control wells. This is done pre-8 ferably by comparing the ratios for the two wells, i.e.
9 the products of the first comparator means for the two 10 wells, which are logarithms of ratios of well readings 11 vs. reference readings. The comparison of the two con~
12 trol values is done by taking a difference between the 13 two logarithms. The resulting difference is compared to 14 a predetermined, stored value representing adequate 15 growth-sterile difference for the test. If there is an 16 inadequate difference, this means either one of two things, 17 either that there had not been sufficient growth to pro-18 vide an adequate difference, or that the sterile well had 19 been contaminated and that there had been growth there.
20 In either case, the instrument will display a reading such 21 as "insufficient growth-sterile difference", and the 22 computer returns to the beginning of the program. The 23 operator then checks to see which of these two possi-24 bilities is the one that is present. If there is in-25 sufficient growth, it may be due to a lack of time or 26 because there was nothing to grow. If there were con-27 tamination, that would show and be readily detectable, and 28 the test must be re done.
29 Once the computer has established that there is 30 an adequate difference between the sterile condition and ... . .
'-: ; .; ' l the expected growth condition from one well to another, 2 the calculated logarithm values and their difference are 3 used for computation of a break point, or a limit compari-4 son signal value. Preferably, the break point is biased toward the sterile value to achieve more sensitivity to 6 growth detection, via a preselected fraction of the sterile-7 growth logarithm difference. The break point may, for ~ example, be placed at 25% of the determined sterile-g growth difference (preferably a logarithm value as above), 10 added to the log value for sterility. For all wells where ll there has been less growth than that represented by 25%
12 of the determined growth-sterile difference for the test 13 being conducted, then the concentration of those wells is 14 considered as inhibitory. For each drug being tested, the 15 concentration closest to the break point, but on the in-16 hibitory side, is selected as the minimum inhibitory con-17 centration value. Thus, supposing that there are a series 18 of wells of different dilutions and that the operation is 19 moving from wells of greater growth towards those of 20 lesser growth and toward the sterile condition, then the ~1 minimum inhibitory concentration is not found until the 22 irst well is reached which shows less than 25V/o of the 23 determined difference between the sterile and growth con-24 trol wells. In this way, a "floating threshold" is Z5 utilized, i.e. one which is calculated from controls in 26 the very test being conducted and with the same organism 27 being tested, rather than a fixed threshold which has been ~8 calculated based on prior information and stored.
29 Another important comparison which should be 30 performed preferably at least once a day, before series 'i :~z~

1 of tests are performed, is an initial calibration step.
2 This initial calibration is in lieu of the empty tray 3 (or just filled tray~ reading procedure described above.
4 Like that procedure, this calibration procedure is impor-

5 tant in that it enables the use of a light source which

6 is not tatally uniform for each photodetector, but only

7 generally uniform, and also the use of ine~pensive photo-

8 detectors which may not be uniform or totally constant,

9 over a long period of time, in their sensitivity. By

10 this procedure the light is first passed directly (no

11 tray) to all photodetectors, including the reference

12 photocell, and ratio readings (preferably their logarithms)

13 are taken as above and recorded, These values are stored

14 and give a relative base line or initial calibration

15 value for each photocell. All subse~uent after culture

16 values (which are preferably logarithms of ratios as above)

17 are compared to these base line readings, and expressed

18 as "difference" (or log ratio) readings. Thus, any

19 differences in sensitivities of the various pho~ocells,

20 or differences in light intensity due to position, are

21 "zeroed out" by comparison of after culture ratios with

22 initial calibration ratios, the comparisons being sepa-

23 rate for each well.

24 As indicated above, the apparatus of this

25 invention is capable of performing various types of com-

26 parisons. Any specific comparison depends upon what

27 method is being used and which types of comparison are

28 appropriate.

29 In some instances, there may be only one com-

30 parison, the specific type of comparison depending on .~
~ .

, 1 the particular apparatus or particular method belng used.
2 For example, it is advisable to relate for every sample 3 the signal received from each well with a signal of a 4 reference transducer. ~uch comparison negates the effect of the variation of light intensity or power fluctuation 6 with time.
7 A second type of comparison is often made, in 8 addition to the first one. This may be considered as a 9 type of calibration procedure aimed at negating the vari-ation of response of the different photosensors. The com-11 parison may be achieved by storing the signals from each 12 of the photosensors before the tray is introdu~ed, and then 3 subtracting from this the corresponding signals generated 14 by each of the wells after the filled tray with its cultured samples has been read. This technique of elimi-16 nating transducer-to-transducer variation is important.
17 Further refinements, which are not necessarily 18 crucial, may be added to eliminate further well-to-well 19 variation. For example, variations in the plastic trays 20 or their contents may affect ~he accuracy of a reading.
2~ One way-to eliminate this problem is to calibrate with an 22 empty trày instead of calibrating without any tray in the 23 holder. A single empty tray may be used, assuming that all 24 trays to be used are substantially identical. Another 25 approach is to compare the wells of each individual tray 26 when empty with the results obtained a~ter filling them 27 with li~uid and culturing the liquid. This is more time 28 consuming and not usually necessary, but it is more accu-29 rate. With suitable multiplexing wired into the device, 30 however, this becomes quite practical. Thus, it is possible ~ 5 ~ ~::

; ' , 3~

1 to eliminate the variations in the signal fluctuating 2 with time, to eliminate the variation of one sensor versus 3 another, and also to compensate for tray-to-tray and well-to-well variations.
A third type of comparison may be used ~or 6 certain tests, such as the MIC test, where the signal level 7 indicating bacterial growth is differentiated from the 8 signal level indicating no growth. This may be accom-9 plished by comparison between various wells on the tray;
that is, some wells may be control wells or sterile no-11 growth wells, in which there is no growth or which are 12 inoculated with suitable inhibitors. There is a possible 13 interpolation between the values of growth and no-growth, 14 as dîscussed above. Alternatively, by experimentation, one 15 can determine a signal value that differentiates between 16 growth and no-growth, and this-decision point may be used 17 instead of one derived by controls on board each tray.
18 We claim:

~0 , ; ~ .
., .

Claims (43)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for performing optical density tests, employing a sample tray having a series of wells containing liquid samples, said wells having translucent bottoms, one said well being a control well, comprising:
holding said tray accurately in a single predetermined sta-tionary reading position without blocking off light paths through said wells, sending light down through all said wells at roughly the same intensity to an array of light-intensity-detecting photocells, there being one photocell adjacent to each well, including a control photocell adjacent to said control well, electronically sequentially comparing the signal from each said photocell of said array with the signal from said control photocell and developing a related signal therefrom for each well.

2. The method of claim 1 including the steps of sending said light to a reference detecting photocell without passing the light through a said well, and electronically sequentially transmitting the signals from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell.

3. The method of claim 2, including the steps of sequentially making a comparison of each said related signal value with at least one data reference value and developing and reading out a resultant value from that comparison.

4. Automatic scanning apparatus for per-forming optical density tests, employing a sample tray having a series of wells containing liquid sam-ples, said wells having translucent bottoms, com-prising:
tray holding means for holding said tray accurately in a single predetermined reading posi-tion without blocking off light paths through said wells, a single diffused light source positioned above the sample tray, for sending light down through all said wells at roughly the same intensity, an array of light-intensity-detecting photo-cells on the opposite side of the tray holding means from light source means, one photocell adjacent to each well and positioned to receive light from the light source which has been transmitted through the well and its contents, sequential signal receiving means connected to all the photocells for receiving sequentially a sig-nal from each said photocell in a prescribed order, without any physical movement of the tray or photocells, each signal corresponding to the intensity of light received by a said photocell, and electronic sequencing means connected to said signal receiving means for electronically causing it to receive its signals in order.

5. The apparatus of claim 4, including a reference detecting photocell for receiving light directly from said light source means without passing through a said sample, and first comparator means connected to said signal receiving means, for sequentially comparing the signal from each said photocell of said array with the signal from said reference detecting photocell and de-veloping a related signal therefrom.

6. The apparatus of claim 5 wherein said first comparator means includes a log ratio module for producing as the related signal the amplified ratio of the two signals.

7. The apparatus of claim 5 wherein said first comparator means includes a difference amplifier for producing as the related signal the amplified difference of the two signals.

8. A method for performing optical density tests, employing a sample tray having a series of wells containing liquid samples, said wells having translucent bottoms, comprising:
holding said tray accurately in a single pre-determined reading position without blocking off light paths through said wells, sending light from a single light source down through all said wells at roughly the same in-tensity to an array of light-intensity-detecting photocells, there being one photocell adjacent to each well, sending light directly from said light source means to a reference detecting photocell without passing the light through a said sample, electronically sequentially transmitting the signals from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell, sequentially comparing the signal from each said photocell of said array with the signal from said reference detecting photocell and developing a related signal therefrom for each well, sequentially making a comparison of each said related signal value with a data reference value and developing a resultant value from that comparison, sequentially comparing said resultant values with other stored values and for determining a desired result therefrom, and reading out the desired results thereby ob-tained.

9. The method of claim 8 wherein the step of developing a related signal comprises generating a signal at an amplified value of the difference between each signal derived via a well and the signal derived from the reference photocell.

10. The method of claim 9 wherein the step of developing a resultant value comprises generating a signal at an amplified value of the difference be-tween said related signal and a similarly derived signal taken with the tray wells empty.

11. The method of claim 9 wherein the steps of developing a resultant value comprise generating a signal at an amplified value of the difference between said related signal and a similarly derived difference signal taken with the tray wells filled with the liquid but before any growth or culture thereof.

12. Automatic scanning apparatus for perform-ing optical density tests, employing a sample tray having a series of wells containing liquid samples, said wells having translucent bottoms, comprising:
tray holding means for holding said tray accurately in a single predetermined reading position without blocking off light paths through said wells, light source means positioned above the sample tray, for sending light down through all said wells at roughly the same intensity, an array of light-intensity-detecting photo-cells on the. opposite side of the tray holding means from light source means, one photocell adjacent to each well and positioned to receive light from the light source which has been transmitted through the well and its contents, a reference light-intensity-detecting photo-cell for receiving light from said light source means without passing through a said sample, sequential signal receiving means connected to all the photocells for receiving sequentially a signal from each said photocell in a prescribed order, each signal corresponding to the intensity of light received by a said photocell, electronic sequencing means connected to said signal receiving means for electronically causing it to receive its signals in order, first comparator means connected to said sig-nal receiving means, for sequentially comparing the signal from each said photocell of said array with the signal from said reference detecting photocell and developing a related signal from those two signals, data storage means for holding data reference values, and second comparator means connected to said first comparator means and to said data storage means for sequentially making a comparison of each said related signal value with at least one data reference value and developing and indicating a resultant value from that comparison.

13. The apparatus of claim 12 wherein said light source means is a single unitary source of dif-fused light.

14. The apparatus of claim 12 having collimator means between said tray bottom and said photocells.

15. Automatic scanning apparatus for per-forming optical density tests, employing a sample tray having a series of wells containing liquid samples, said wells having translucent bottoms, comprising:
tray holding means for holding said tray accurately in a single predetermined reading position without blocking off light paths through said wells, light source means positioned above the sample tray, for sending light down through all said wells at roughly the same intensity, an array of light-intensity-detecting photo-cells on the opposite side of the tray holding means from light source means, one photocell adjacent to each well and positioned to receive light from the light source which has been transmitted through the well and its contents, a reference light-intensity-detecting photo cell for receiving light from said light source means without passing through a said sample, sequential signal receiving means connected to all the photocells for receiving sequentially a signal from each said photocell in a prescribed order, each signal corresponding to the intensity of light received by a said photocell, electronic sequencing means connected to said signal receiving means for electronically causing it to receive its signals in order, first comparator means connected to said signal receiving means, for sequentially comparing the signal from each said photocell of said array with the signal from said reference detecting photocell and developing a related signal from those two signals, data storage means for holding data refer-ence values, second comparator means connected to said first comparator means and to said data storage means for sequentially making a comparison of each said re-lated signal value with a data reference value and developing a resultant value from that comparison, and third comparator means connected to said second comparator means and to said data storage means for sequentially comparing said resultant values with other stored values and for determining a desired result therefrom, and output means connected to said third compar-ator means for giving the results obtained by said third comparator means.

16. The apparatus of claim 15 wherein said light source means is a single unitary source of diffused light.

17. The apparatus of claim 15 wherein said light source means comprises a plurality of fiber optic filaments, one for each said well and one for said reference photocell.

18. The apparatus of claim 15 in which the photocells are located vertically to read light passing vertically through said wells.

19. The apparatus of claim 15 in which said first comparator means incorporates a difference ampli-fier and sends out said related signal corresponding to the difference in amplitude between each signal SW
obtained through a well and the signal SR through the reference photocell, said related signal being k1(SW - SR) where k1 is the amplification.

20. The apparatus of claim 19 in which said second comparator means also incorporates the same difference amplifier and said resultant value SV = k2[k1 (SW - SR)- DV] where k2 is the amplification and DV is the data reference value.

21. The apparatus of claim 20 in which said second comparator means utilizes as said data reference value DV stored difference signals read earlier from an empty tray, so that DV = k1(SWE - SR) for each well, where SWE is the signal coming from an empty well.

22. The apparatus of claim 20 in which said second comparator means utilizes as said data reference value DV stored difference signals read earlier from a tray filled with the liquid from which the final readings are made but at zero growth time, so that DV =
k1(SWO - SR) for each signal where SWO is the signal coming from a well at zero growth time.

23. The apparatus of claim 15 wherein said electronic sequencing means comprises multiplexing means.

24. Apparatus for determining susceptibility of a bacteria culture to various antimicrobic drugs and for determining the minimum inhibitory concentration of the bacteria culture to those drugs to which it is susceptible, said apparatus having a sample tray with a plurality of light-transmissive wells for containing uniform samples of the bacteria culture and series of varied concentrations of a plurality of antimicrobic drugs, comprising:
tray holder means for supporting the sample tray in an accurate predetermined position assuring proper transmission of light through said wells, single light source means positioned above the sample tray for sending light of generally uniform intensity generally vertically through all wells, an array of light intensity detecting photo-cells below the sample tray, one adjacent to each well and positioned to receive light from the light source which is transmitted through the well and its contents, signal receiving means connected to each photocell for receiving from each photocell a signal corresponding in amplitude to the intensity of light transmitted through its adjacent well and thus to the turbidity of the contents of the well, electronic sequencing means for delivering the photocell signals to said signal receiving means in an automatic sequence, rapidly, one at a time, comparator means connected to said signal receiving means for comparing the amplitude of each signal with a value corresponding to inhibited bacterial growth, and determination means connected to said com-parator means for determining from said comparisons which antimicrobic drugs inhibit growth of the bacteria and for determining and indicating for each inhibitory drug the minimum concentration of that drug which will inhibit such growth.

25. The apparatus of claim 24, having a reference photocell receiving light direct-ly from said single light source and connected to said signal receiving means, preliminary comparing means connecting a said signal receiving means to said comparator for comparing each signal of said array with the signal from said reference photocell and sending to said comparator a signal derived from a mathematical rela-tion between those signals.

26. The apparatus of claim 25 wherein said preliminary comparing means includes a difference amplifier for sending as its derived signal a signal based on the difference between the two signals it compares.

27. A method for performing optical density tests, employing a sample tray having a series of wells, said wells having translucent bottoms, comprising:
filling said wells with culturable liquid prior to any culture thereof, holding said tray accurately in a single predetermined reading position without blocking off light paths through said wells, sending light from a single light source down through all said wells at roughly the same inten-sity to an array of light-intensity-detecting photo-cells, there being one photocell adjacent to each well, sending light directly from said light source means to a reference detecting photocell without pass-ing the light through a said sample, electronically sequentially transmitting the signals from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell, sequentially comparing the signal from each said photocell of said array with the signal from said reference detecting photocell and developing a first related signal therefrom for each well, culturing the liquid in the tray wells, again holding said tray after culture accurately in said single predetermined reading posi-tion without blocking off light paths through said wells, sending light after culture from a single light source down through all said wells at roughly the same intensity to an array of light-intensity-detecting photocells, there being one photocell adjacent to each well, again sending light directly from said light source means to a reference detecting photocell without without passing the light through a said sample, electronically sequentially transmitting the signals after culture from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell, sequentially comparing the signal after culture from each said photocell of said array with the signal then obtained from said reference detecting photocell and developing a second related signal therefrom, sequentially making a comprison of each said second related signal value with the correspond-ing said first related signal value, well by well, and developing a resultant value from that comparison, sequentially comparing said resultant values with other stored values and for determining a desired result therefrom, and reading out the desired results thereby obtained.

28. The method of claim 27 wherein the step of developing a related signal comprises generating a signal at an amplified value of the difference be-tween each signal derived via a well and the signal derived from the reference photocell.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

29. The method of claim 8 wherein the step of developing a related signal comprises generating a signal as a ratio of each signal derived via a well to the signal derived from the reference photocell.

30. The method of claim 29 wherein the step of developing a resultant value comprises generating another ratio signal as the ratio of said related signal to a similarly derived ratio signal obtained by initially reading the photocells unobstructed by the tray.

31. The method of claim 29 wherein the step of developing a resultant value comprises generating another ratio signal as the ratio of said related signal to a similarly derived ratio signal obtained by reading the tray wells filled with the liquid but before any growth or culture thereof.

32. The method of claim 8 having the step of color-filtering the light between the light source and the photocells.

33. The method of claim 27 wherein the step of developing a related signal comprises generating a signal as a ratio of each signal derived via a well to the signal derived from the reference photocell.

34. The apparatus of claim 14 having color filter means between said light source and all said photocells.

35. The apparatus of claim 15 having collimator means between said tray bottom and said photocells.

36. The apparatus of claim 35 having color filter means between said light source and all said photocells.

37. The apparatus of claim 15 in which the photocells are set at an inclination to the vertical lines through the wells and operate as nephelometers.

38. The apparatus of claim 15 in which said first comparator means incorporates a log ratio module and sends out is related signal as an amplitude ratio between each signal SW obtained through a well and its photocell and the signal SR obtained from the reference photocell, said related signal SX = , where k1 is a constant.

39. The apparatus of claim 38 in which said comparator means also incorporates a log ratio module and sends out its resultant value SV as a ratio , where DV is the data reference value and k2 a constant.

40. The apparatus of claim 39 in which said first and second comparator means use the same log ratio module.

41. The apparatus of claim 39 in which said second comparator means utilizes as said data reference value DV, stored ratios read earlier from an empty tray, so that DV = for each well, where SWE is the signal coming from an empty well.

42. The apparatus of claim 39 in which said second comparator means utilizes as said data reference value DV, stored ratios read earlier from a tray con-taining the same liquid from which the signals SW are generated, but read at a time when there has been zero growth, so that DV = for each well, where SWO is the signal coming from a well containing the liquid at zero growth time.

43. The apparatus of claim 25 wherein said preliminary comparing means includes a log ratio module for sending as its derived signal a signal based on the ratio of the two signals it compares.