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S P E C I F I C A T I 0 M page 1
1 Background of the Inven_ion
2 Thls invention relates to automatic scanning
3 apparatus, which in rapid sequence performs a series of
4 related densitometric or optical density tests on samples
contained in a large number of wells in a tray. It also
6 relates to measurement o~ the susceptibility of bacteria
7 to different antimicrobic drugs, with automatic quan-ti-
8 fication of the susceptibility to each drug, so that a
9 physician may select a drug that will most effectively
treat an infecting bacterlum and choose the appropriate
11 dosage for effective treatment. It further relates to
12 the identification of microorganisms that have been
13 isolated from patients.
14 In the clinical laboratory, the -bacteriology
department has two major functions: 1) the identifica-
16 tion of organims that are isolated from patients and
17 2) the determination of the susceptibility of these
18 organisms to antimicrobic medication. Both of these
19 are involved here.
~l Identification of rliicroorganisms
22 Organism identification has generally been
23 accomplished by noting both the microscopic appearance
24 of the bacteria and their gross appearance (colonial
25 morphology) as they grow on a solid medium. In addi-
26 tion to morphologic examination, a technologist sometimes
27 tested the organism with immunological techniques and
28 special stains to gain further information on the micro-
29 organism's identity.
However, the most important technique for
31 bacterial identification relates to that organism's bio-
32 chemical properties.
33 Each organism possesses a set of enzymes that
34 act as chemical catalysts or fermentors. By performing a
35 series of chemical reactions in a medium where an organism
36 is growing, a technologlst is able to identify a combi-
37 nation of positive and negative reactions that effectively
38
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9L:13~71~
-- 2
1 provide a chemical fingerprint for that organism. Typi~
2 cally, these reactions include fermentation of a wide
3 range of carbohydrates, citrate utilization, malonate
4 utilization, phenylalanine deaminase production, beta
5 galactosidase production, indole production, hydrogen
6 sulfide production, lysine decarbonxylase production,
7 ornithine decarboxylase production, urease production,
8 sucrose utilization, and arginine dehydroxylase pro-
9 duction. A reaction result is determined by a visual
1~ color change in the medium. The color reagent in most
11 cases is pH indicator which measures the alkalinity or
12 acidity resulting from the chemical reactions.
13 variety of indicators such as bromphenol bllle and phenol
14 red may be used to measure pH changes over a wide range
15 of the pH scale. Another mechanism for chemical color
16 development is the enzymatic splitting of a chromogen
17 (color producing chemical) off the original substrate,
18 thus signalling a positive chemical reaction.
19 A combination of color reactions as just
20 described forms a profile that may be used to identify
21 the organism. For this purpose, identification can pro-
22 ceed in either a parallel mode in which a large number
23 of tests are performed at one time, or in a serial mode,
24 also known as a sequential or "branching" mode, in which
subsequent tests are chosen on the basis of previous
26 results. The serial mode saves reagents since only those
27 tests are performed which will directly affect the final
28 results; however, it is quite time consuming, since each
29 subsequent test cannot be performed until the results
from the preceding test have been obtained. In a medical
31 setting, each test may take 24 hours to obtain a result,
32 and where time is of the essence, the system may be far
33 too slow for practical application. Additionally, should
34 the technologist misread one result early in the decision
tree, then all subsequent results could possibly be
36 misleading~
37 For these reasons, the parallel mode employing
38
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1 the performance of a large number of biochemical tests on
~ bacteria is presently preferred by most workers. In the
3 parallel mode, large numbers of known organisms are tested
4 with a battery of biochemical substrates, and the proba-
5 bilities of each of these bacterial taxa having a positive
6 reaction are tabulated. With this information, the
7 probability of each organism occuring for each combi-
8 nation of chemical reactions can be computed by standard
9 statistical methods.
Systems presently available comTnercially pro-
11 vide a convenient battery of biochemical substrates and
12 indicators to test a given organism. A "book" (typically
13 a computer printout) is provided to enable a technologist
14 to translate combinations of chemical reactions into the
correspondingly most probable organism. These commercial
16 systems provide a plurality of microtubes (e.g., 15 to
17 30) which contain substrate indicators. The microtubes
18 are inoculated with the bacteria and, after an appropriate
19 time for the organism to grow and elaborate its enzymes,
20 the reactions can be read as a color change. Combinations
21 of these reactions can then be transposed into a unique
22 numerical code.
23 A standard way of transposing these reactions
24 into numbers involves reading the reactions in groups
25 of threes and e~pressing the reactions as an octal code.
26 This octal code ranges from 0-7 with 0 representing no
27 positive reaction and 7 indicating that all 3 reactions
28 were positive. This octal number or "biotype" can be
29 found in a book where the most probable organisms are
30 listed for each biotype.
31 With the present art it is necessary for a
32 technologist to read each reaction visually, record
33 each of the results, compute a biotype number, and then
34 find this biotype number in a computer printout, in order
35 to make the organism identification. Thus, although they
36 are much more convenient than the original serial branch-
37 ing technique the present manual multi-test battery
38
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1 methods are still laborious and time-consuming.
3 Minimum inhibitory concentration
4 The physician usually has a choice of about
5 twelve to fifteen types of an~imicrobial agents for
6 treating the forty to sixty groups of pathogenic bacteria.
7 Many of these agents are ineffective against a given
8 bacterial strain, but normally some of them will be
9 appropriate for treatment. In order for the physician
10 to choose the best antimicrobic, it is necessary to iso-
11 late the pathogenic organism in the laboratory and then
12 test it against a panel of drugs to determine which drugs
13 inhibit growth and which do not. Ideally, the doctor
14 should receive susceptibility information the same day the
15 culture is taken, since it is usually necessary to
16 initiate therapy immediately. Unfortunately, it currently
17 takes one day to isolate an organism, and it has required
18 another day to test the susceptibility of the organism to
19 the antimicrobics. Therefore, it has been customary for
20 the physician to institute therapy based on an educated
21 guess at the time the patient is first seen. If the
22 sensitivity studies completed two days later indicate that
23 the guess was incorrect, therapy is changed to the proper
24 drug.
Clearly an important goal in automating anti-
26 microbic testing would be to diminish the time lag between
27 the initial culture and the obtaining of sensitivity infor- ;
28 mation. An estimated 30 million antimicrobic susceptibil-
29 ity tests are performed annually in the United States by
30 labor intensive manual methods. In addition to the
31 potential economic advantages of automation and obvious
32 advantages to the patient in receiving only the proper
33 treatment, one could also anticipate better precision,
34 quality control and objectivity.
The most frequently used technique to measure
36 antimicrobial susceptibility has been the standardized
37 disc-diffusion method described by Kirby and Bauer
38 (Bauer, Kirby et al, "~ntibiotic Susceptibility Testing
. , ,
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1 by a Standardized Single Disk Method", America _Journal of
2 Clinical Pathology, 1966, Vol. 45, No. 4, p. 493). By
3 this method, isolates of bacteria are grown in suspension
4 to a standardized concentration (usually determined by
5 visual turbidity) and streaked onto nutrient agar (culture
6 medium) in a f~at glass Petri dish. Paper discs impreg-
7 nated with different anti-microbial materials are placed
8 upon the agar streaked with bacteria, and the drug is
9 allowed to diffuse through the agar, forming a gradient
10 halo around the disc. ~s the bacteria replicate, they
11 form a visible film on the surface of the agar 7 but in the
12 zones surrounding the antibiotic-impregnated discs,
13 growth is inhibited if the organism is susceptible to that
14 particular antimicrobial agent. Since a concentration
gradient has been established, the zone of inhibition
16 around the disc is roughly proportional to the degree o~
17 susceptibility. Typically, the laboratory classifies an
18 organism as "sensitive", "intermediate", or "resistant"
19 to each drug in the test panel. Thus the results estab-
20 lish a characteristic profile or "antibiogram" for that
21 organism.
22 The Kirby-Bauer disc-diffusion method has the
23 advantage of simplicity, but is suffers from several
24 drawbacks. One problem is that of time efficiency. In
25 order that the initial inoculum become visible on the
26 Petri dish so that zones of growth can be distinguished
27 from zones of inhibition, the bacteria numbers must
28 increase by several orders of magnitude over the original
29 number. ~owever, for determination of whether or not the
30 organism is growing in theantimicrobial milieu, which is
31 the only information required, a period that would allow
32 doubling of all the organisms should be theoretically
33 sufficient with suitable detection equipment. For most
34 Gram-negati~e organisms, the doubling period is between
35 twenty and thirty minutes, following a lag phase. There-
36 fore, an automated system should be able to distinguish
37 growth within a thirty-minute period.
38
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1 Another difficulty with the Kirby-Bauer disc
2 method is that of standardization. If an organism is
3 "resistantl', does that mean that it cannot be treated
4 with higher than normal doses of the microbial agent?
5 Also, how does this information relate to a site in the
6 body where the antimicrobic is concentrated (such as
7 bile) or decreased in amount (such as cerebrospinal
8 fluid)?
9 To answer these questions, quantitative data
10 are necessary. To obtain quantitative results, it m~lst
11 be determined what minimum concentration o a drug will
12 inhibit the organism's growth. This quantitation of
13 susceptibility is known as minimum inhibitory concentra-
14 tion or MIC. The MIC may be determined by making serial
15 dilutions o~ the drug in agar or broth, and then inocu-
16 lating each dilution of each drug with a standardized
17 suspension of bacteria. Since the test procedure may
18 involve as many as 70 to 80 individual tubes, it can
19 become a formidable task if the test is performed in
20 individual test tubes on a macro scale. Systems are
21 available in which the individual dilutions of anti-
22 microbics are made in plastic trays containing small
23 micro-tubes. (March and MacLowry, "Semiautomatic
24 Serial-Dilution Test for Antibiotic Susceptibility",
25 Automation and Data Processing the the Clinical Labora-
26 ~2~ Springfield, Illinois, C. C. Thomas 1970). Orga-
27 nisms can be inoculated in a single step using a multi-
28 pronged template. Thus, setting up the test is simpli-
29 fied, and it takes slightly less time to provide
30 quantitative data than qualitative Kirby-Bauer infor-
31 mation. There are now semiautomated devices that dispense
32 antimicrobial solutions into the microtubes. Trays of
33 microtubes are also commercially available with frozen
34 solutions in the tubes, and the Gram-negative anti-
35 microbial panels have been combined with biochemical tests
36 to identify enteric bacteria as well as to determine
37 their antimicrobic susceptibility.
38
7E~;
1 Although MIC results give quanti~ative infor-
2 mation which allows consideration of multiple doses and
3 multiple sites, the MIC numbers in themselves can be
4 confusing to the clinician. To use MIC data correctly,
5 a physician must refer to tables of achievable anti-
6 microbic levels as a function of dosage and body site.
7 Therapy will be effective if the achievable drug level
8 for a particular dose and site in the body is two to
9 four times the MIC. With the present invention
10 described ~elow, such interpretation of MIC data is
11 accomplished by a computer, which compares the MIC
12 with a table of achievable drug levels at different
13 body sites and different doses.
14
15 Optical testing methods and ~pparatus
16 Optical detection methods have been suggested
17 and have proven to be powerful tools to measure bacterial
18 growth. A laser light-scattering system can have the
19 sensitivity to detect a single bacterium. Optical
20 methods measure the presence of bacteria either by
21 nephelometry or turbidity measurements. Nephelometry
22 measures the ability of the bacteria particles to scatter
23 light, and the detector is aligned at an angle to the
24 axis of the light source. Turbidity measures the net
25 effect of absorbance and scatter, and the transducer
26 is placed on the axis of the radiation source. Nephelo-
27 metry measurements are significantly more sensitive than
28 turbidity measurements, but since the nephelometer
29 measures only that fraction of light scattered by
30 bacteria, the signal to the detector is small, and both
31 light source and transducer amplification must be corres-
32 pondingly large.
33 Some apparatus heretofore relied direct inspec-
34 tion by the human eye, as did Astle in U.S. Patent No.
3,713,985. Aware of inaccuracies involved, Astle sug-
36 gested, but did not disclose details of automatic equip-
37 ment for reading and recording the results of densitometric
3~
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1 tests and suggested tl~at his tur'bidity data could be fed
2 to a device that would translate the data to machine
3 language for ~ecording on computer punch cards. Astle
4 does not teach how to do that. His own device is a strip
5 having a series of wells, all in a single line which
6 involve mechanical position shifts.
7 Automation in microbio'Logy has lagged far behind
8 chemistry and hematology in the clinical laboratory.
9 However, there is presently an intensive e~ort by indus-
10 try to develop this field. The best publicized devices
11 for performing automated antimicrobic susceptibility
12 testing use optical detecti.on methods. A continuous
13 flow device for detectin~ particles 0.5 micron or less
14 has been commercially available since 1970; however,
15 probably due to its great expense, it has not been widely
16 used in the laboratory. Other devices using laser li~ht
17 sources have been suggested but have not proven com-
18 mercially practicable. Recently, the most attention has
19 been directed to three devices discussed below.
The Pfizer ~utobac 1 system (U~S. Patent No.
21 RE. 28,801) measures relative bacterial growth by light
22 scatter at a fixed 35 angle. It includes twelve test
23 chambers and one control chamber in a plastic device
24 that forms multiple contiguous cuvettes. Antibiotics are
25 introduced to the chambers via impregnated paper discs.
26 The antimicrobîc sensitivity reader comes with an
27 incubator, shaker, and disc dispenser. Results are
28 expressed as a light scattering index (LSI~, and these
29 numbers are related to the Kirby-~auer "sensitive,
30 intermediate and resistant''. MIC measurements are not
31 available routinely with this instrument. In a compari-
32 son with susceptibilities of clinical isolates measured
33 by the Kirby-Bauer method, there was 91~/~ agreement.
34 However, with this system some bacteria strain-drug ~-
combinations have been found to produce a resistant
36 Kirby-Bauer zone dlameter and at the same time a sensi- '-'
37 tive'LSI.
38
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g
1 The Auto Microbic System has been developed by
2 McDonnell-Douglas to perform identification, enumeration
3 and susceptibility studies on nine urinary tract patho-
4 gens using a plastic plate containing a 4 x 5 array of
5 wells. See &ibson et al~ U.S. Patent No. 3,957,583;
6 Charles et al, U.S. Patent No. 4,118,280, and Charles
7 et al, U.S. Patent No. 4,116,775. The specimen is
8 drawn into the small wells by negative pressure and the
9 instrument monitors the change in optical absorbance and
10 scatter with light-emitting diodes and an array of
11 optical sensors. A mechanical device moves each plate
12 into a sensing slot in a continuous succession so that
13 each plate is scanned once an hour, and an onboard digi-
14 tal computer stores the optical data. The system will
15 process either 120 or 240 specimens at a time. One can
16 query the status of each test via a CRT-keyboard console,
17 and hard copy can be made from any display. When the
18 system detects sufficient bacterial growth to permit a
19 valid result, it automatically triggers a print-out.
20 Following identification in four to thirteen hours, a
21 technologist transfers positive cultures to another
22 system which tests for antimicrobic susceptibility.
23 The results are expressed as "R" (resistant) and "S"
24 (susceptible); no quantitative MIC data are provided.
It should be noted that Gibson et al, U.S.
~6 Patent No. 3,957,583 do not include automation, but
27 use naked-eye inspection or a manually-operated color-
28 imeter. Scanning is therefore a hand or a mechanical
29 operation. Charles et al, Patents Nos. 4,116,775 and
30 4,118,280 also require mechanical movement of their
31 cassette for reading different rows.
32 The Abbott MS-2 system consists of chambers
33 composed of eleven contiguous cuvettes. Similar to the
34 Pfzier Autobac 1, the antimicrobial compounds are intro-
35 duced by way of impregnated paper discs. An inoculum
36 consisting of a suspension of organisms from several
37 colonies is introduced into the culture medium, and the
38
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1 cuvette cartridge is filled with this suspension. The
2 operator inserts the cuvette cartridge into an analysis
3 module which will handle eight cartridges (additional
4 modules can be added to the system). Following agitation
5 of the cartridge, the instrument monitors the growth rate
6 by turbidimentry. When the log growth phase occurs, the
7 system automatically transfers the broth solution to the
8 eleven cuvette chambers; ten of these chambers contain
9 antimicrobial discs, and the eleventh is a growth control.
10 The device performs readings at five-minute intervals,
11 and stores the data in a microprocessor. Following a
12 pre-set increase of turbidity of the growth control,
13 the processor establishes a growth rate constant for each
14 chamber. A comparison of the antimicrobic growth rate
15 constant and control growth rate constant forms the basis
16 of susceptibility calculations. The print,out presents
17 results as either resistant or susceptible; if inter-
18 mediate, susceptibility information is expressed as an
19 MIC.
Non-optical methods have also been used or
21 suggested for measuring antimicrobic sensiti~ity in
22 susceptibility testing. These have included radio-
23 respirometry, electrical impedance, bioluminescence and
24 microcalorimetry. Radiorespirometry, based on the
25 principLe that bacteria metabolized carbohydrate and
26 the carbohydrate carbon may be detected following its
27 release as C02, involves the incorporati~n of the isotope
28 C14 into carbohydrates. Released C1402 gas is trapped
29 and beta counting techniques are used to detect the
30 isotope. The major difficulty in applying the isotope
31 detection system to susceptibility testing, however, is
32 that an antimicrobic agent may be able to stop growth of
33 a species of bacteria, yet metabolism of carbohydrate
34 may continue. Less likely, a given drug may turn off
35 the metabolic machinery that metabolizes certain carbo-
36 hydrates, but growth may continue. This dissociation
37 between metabolism and cell growth emphasizes the fact
38
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1 that measurements for detecting antimicrobic susceptibility
2 should depend upon a determination of cell mass or cell
3 number rather than metabolism,
4 The electrical impedance system is based on
the fact that bacterial cells have a low net charge
6 and higher electrical impedance than the surrounding
7 electrolytic bacterial growth media. A pulse impedance
8 cell-counting device can be used to count the cells;
9 however, available counting devices are not designed
10 to handle batches of samples automatically, and generally
11 do not have the capacity ~o distinguish between live
12 and dead bacterial cells. Another approach with electri~
13 cal impedance has been to monitor the change in the
14 conductivity of the media during the growth phase of
15 bacteria. As bacteria utilize the nutrients, they pro-
16 duce metabolites which have a greater degree of electri-
17 cal conductance than the native broth, so that as
18 metabolism occurs, impedance decreases. However, since
19 this technique measures cell metabolism rather than
20 cell mass, its applicability to antimicrobic suscepti-
21 bility detection suffers from the same drawback as
22 radiorespirometry.
23 Bioluminescence has also been sugges~ed for the
24 detection of microorganisms. It is based on the princi-
25 ple that a nearly universal property of living organisms
26 is the storage of energy in the form of high energy
27 phosphates (adenosine triphosphate, ATP), which can be
28 detected through reaction with firefly luciferase. The
29 reaction results in the emission of light energy which
30 can be detected with great sensitivity by electronic
31 light ~ransducers. Although a clinical laboratory may
32 obtain a bioluminescence system to detect the presence
33 of bacteria in urine, the technique is expensive due to
34 the limited availability of firefly luciferase, and
35 problems have been enco~mtered in standardizing the
36 system.
37 Microcalorimetry is the measurement of minute
38
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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.
Summary of the Invention
The present invention employs optical methods and
apparatus for automatically identifying microorganisms and auto-
matically determining bacterial susceptibility to a number of
different antimicrobic drugs, utilizing turbidimetry.
The invention provides apparatus for identifying a micro-
organism, employing a sample tray having a series of wells for con-
taining uniform samples of microorganism culture and a reagent,
said wells having translucent bottoms, comprising:
tray holding means for holding said tray accurately in
a predetermined position without blocking off light paths -through
said wells,
a single diffused light source means positioned above the
sample tray, for sending light down through all said wells at
approximately uniform intensity,
collimation means beneath said tray holding means for
collimating the light from each well after it has passed through
the wells,
light filter means below said tray holding means for
filtering the color values of the light passing through the wells,
an array of light-intensity-detecting photocells on the
opposite side of the filter means from the tray holding means, one
adjacent to each well and positioned to receive light from the
light source which has been transmitted through the well and its
.~ - 12 -
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contents,
a reference detecting photocell for receiving light
directly from said single diffused light source means without pass-
ing through a said tray,
sequential signal receivi.ng 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 transmitted through the ad~acent well and thus
to the opacity of the contents of t.he well,
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 receiviny
means, for sequentially comparing the signal from each said photo-
cell of said array with the signal from said reference detecting
photocell and developing a difference signal therefrom,
data storage means for holding data values corresponding
to inhibited growth and for holding data relating to various
organl sms,
second comparator means connected to said first com-
parator means and to said data storage means for sequentially
making a comparison of each said difference signal value with a
value corresponding to inhibited growth and developing a
resultant value from that comparison,
third comparator means connected to said second com-
parator means and to said data storage means for sequentially
comparing said resultant values with a large number of stored
values and for determining the probability values for the presence
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of selected orcJanisms in the sample, and
output means connected to said third comparator means
for giving the results obtained by said third comparator means.
The invention, from another aspect, provides a method
of identifying microorganisms, comprising:
placiny a series of different reagents in series of wells
in a light-transmissive sample tray,
establishing a known uniform concentration o~ a culture
of the microorganism and placing the uniform concentration in equal
volumes in the wells,
following a predetermined period for bacterial growth,
passing light from a single light source in substantially equal
intensity through all said wells and through a color filter and
collimator, according to the opacity value for each well,
automatically sensing the intensity of the collimated
light transmitted through each well by photodetector means adjacent
to the wells and filter and opposite the light source,
automatically and sequentially comparing the opacity
values for each well with an opacity for light from the same source
not passing through any well but passing through the filter, and
for generating a signal from such comparisonl
automatically and sequentially comparing that signal with
a value corresponding to inhibited reaction for each well, and
automatically and sequential~y comparing the opacity
values from different tests to obtain probability values for
various suspected organisms.
The invention also provides a method for performing
optical density tests, employing a sample tray having a series of
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wells, said wells having translucent bottoms, comprising:
holding said tray, with the wells empty, accurately in
a single predetermined reading poisition without blocking off light
paths through said wells,
sending light 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,
sending light directly from said light source means to a
reference detecting photocell without passing the light through
the tray,
electronically sequentially transmitting the signal from
all said photocells in a prescribed order, each signal correspond-
ing to the intensity of light received by a said photocell,
sequentially comparing the signal from each said photo-
cell of said array with the signal from said reference detecting
photocell and developing a first related signal therefrom for each
well,
storing said first related signals,
filling the wells with liquid samples,
culturing said liquid for a predetermined length of time,
holding said filled tray, after culture, accuratsly in
said single predetermined reading position without blocking off
light paths through said wells,
sending light from said single light source down through
all said filled wells at roughly the same intensity to said array
of light-intensity-detecting photocells, there being one photocell
adjacent to each well,
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7~i6
sending light directly from said light source means to
said reference detecting photocell without passing the light
through a said sample,
electronically sequentially transmitting the signals for
filled wells from all said photocells in a prescribed order, each
signal corresponding to the intensi.ty of light received by a said
photocell,
sequentially comparing the signal for the filled wells
from each said photocell of said array with the signal from said
reference detecting photocell and developing a second related
signal therefrom for each well,
sequentially making a comparison of each said second
related signal value with the corresponding Eirst related signal
value for the same well, and developing a resultant value from
that comparison,
se~uentially comparing said resultant values with other
stored values and for determining a desired result therefrom, and
reading out the desired results thereby obtained.
The invention also provides a method for determining
susceptibility of a bacteria culture to various antimicrobic drugs
and of determining the minimum inhibitory concentration of the
bacteria culture to those drugs to which it is susceptible, com-
: prising:
providing a sample tray having a series of light-trans-
missive wells, and a series of photodetectors, including a
reference photodetector not associated with a tray well, each
photodetector being adapted to provide a signal corresponding to
the sensed light intensity,
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7~
initially calibrating the photodetectors by passing
light from a source of generally uniform intensity over the photo-
detectors and electronically sequencing the photodetectors to read
a signal from each photodetector, comparing the values of the
signals obtained for each well-associated photodetector sequential-
ly with the value of the reference signal obtained for the refer-
ence photodetector, and providing an initial calibration value for
each well-associated photodetector which is a function of the well
photodetector signal and the reference signal, and storing and
retaining the calibration value for each well-associated photo-
detector,
placing in the wells a plurality of different antimicrobic
drugs, each drug being included in a series of wells in serially
diluted known concentrations, and samples of equal volumes of a
known uniform concentration of the bacteria, with the wells adjacent
to the well-associated photodetectors,
following a period for bacterial growth, passing light of
generally uniform intensity simultaneouialy through each well and to
the reference photodetector and reading the intensity of the trans-
; 20 mitted light with the photodetectors by electronically sequencing
the photodetectors by electronically sequencing the photodetectors
to read an after culture signal from each,
comparing the values of the after culture signals
obtained from the well-associated photodetectors with the value of
the after culture signal from the reference photodetector and pro-
viding an after culture value for each well-associated photo-
detector which is a function of the after culture well photo-
detector signal and the after culture reference signal,
13d -
comparing, for each well, the after culture value with
the initial calibration value and providing a comparison signal for
each well which allows for variations in the intensity of the light
directed from the source onto the clifferent wells and for varia-
tions in the sensitivities of the photocells,
automatically and sequentially comparing each comparison
signal value with a limit comparison signal value which represents
a cutoff between inhibition and growth, correlating the comparisons
with stored data identi~ying the antimicrobic drug and concentra-
tion in each well, and obtaining therefrom an indication of which
antimicrobic drugs inhibit growth of the bacteria, and automatical-
ly selecting the minimum inhibitory concentration of each
inhibitory drug by selecting the minimum concentration of each
drug which produced a comparison signal value on the inhibition
side of the limit comparison signal value, and
automatically displaying the minimum inhibitory concen-
tration for each inhibitory drug, and for each drug that does not
inhibit growth, displaying that the bacteria is resistant to that
drug.
The in~ention further provi~es a method of identifying
microorganisms, comprising:
placing a series of different reagents, including one
for determining whether an organism is a dextrose fermenter in a
series of we.~ls in a light-transmissive sample tray,
establishing a known uniform concentration of a culture
of the microorganism and placing the uniform concentration in equal
volumes in the wells,
following a period for bacterial growth, passing light
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in substantially equal intensity through each well and through
appropriate color filter means to determine an opacity value for
each well by automatically sensing the intensity of the light
transmitted through each well with photodetector means adjacent to
the wells and to the filter opposite the light source,
comparing the obtained opacity values with a value corre-
sponding to zero reaction,
determining from the opacity ~alue from the dextrose
reagent well whether the microorganism is a dextrose fermenter,
depending on whether the microorganism is or is not so
found to be a dextrose fermenter, comparing the opacity values from
appropriate wells to tables of values indicative of microorganism
to obtain probability values for various suspected microorganisms,
cumulatively multiplying each such probability value by
other probabilities for a given taxon to give the non-normalized
frequency for each taxon,
adding the non-normalized frequencies for all such taxa,
determining the three most probable taxa fxom said non-
normalized frequencies,
determining from the values for the three most probable
taxa whether any of them have a frequency greater than 1 x 10 6
and, if they do,
normalizing the frequencies o the three most pro~able
organisms, and
indicating the normalized frequencies of the most
probable microorganism and the identity of that microorganism and
the probability percentage, if the probability is greater than 75%.
The pre.sent invention makes it possible to use an optical-
~à~ - 13f -
electrical method for automatically .readinc3 the color changes of a
plurality of bicchemical reactions in small microtubes and for
calculating and printing out the most probable organism by means
of an inboard computer and probability data stored in the computer
memory.
The microtubes are, preferably, all part of a unitary
sample tray, made of suitable translucent material. Each micro-
tube is a well of this tray. In each well and in a standardized
manner, is placed a suitable chemical reagent
~' - 13g -
~:3~t71~
1 or reagents; then each well is inoculated with the sample.
2 Photodetection o~ color changes is accomplished by the
3 passage of uni~orm intensity light through each of the
4 wells and through the translucent well bottoms following
5 an incubation period. At the opposite side of the tray,
6 preferably below the tray, is an optical filter designed
7 to pass only certain wavelengths of light. Beneath the
8 filter is an array of sequentially-scanned transd~tcers
9 such as photoelectric cells, one associated with each
10 well. The optical filter is designed so that a shift in
11 color in the wells will result in a predictably greater
12 or lesser amo~nt of light passing through to the photo-
13 electric cells.
1~ Previously, the reading of an identiEication
system required a technologist manua].ly to record visual
16 impressions oE color changes indicating either positive or
17 negative biochemical reactions generated by the enzymes
18 contained in the bacteria to be tested. The apparatus
19 in the present invention provides this reading auto-
20 matically and objectively. With present manual methods
21 and apparatus, after the reactions had been determined,
22 it was necessary for the technologist to calculate a
23 numerical summation of these reactions and to express
24 them as an octal number or "biotype". This biotype
25 number was then searched out in a large book containing
26 various biotypes and corresponding organism probabilities.
27 Once a biotype was found, the most probable organism
28 was noted and reported. With the present invention, the
29 computer which is an integral part of the instrument,
30 computes the probability for each organism and prints
31 out the identification on a laboratory form.
32 Signals from the transducers (photoelectric
33 cells) are transmitted to a computer which contains an
34 algorithm that transforms the reaction results to
35 organism identification. The following description
36 presents in detail the algorithm used by the computer
37 to convert the reaction colors to organism identification.
~9 3~
:....... , - . , , .::~ .~:
- i . . ...
~37'~
- 15 -
1 This algorithm is also summarized in the accompanying
2 flow diagram.
3 For each biochemical reaction, a voltage value,
4 which discriminates between a positive and negative
5 result, is or has already been determined by experi-
6 mentation. Each of these "cutoff" points is stored in
7 the computer's memory together with a module that indi-
8 cates if a given value above that point is negative or
9 positive.
The computer is programmed to compile a table
11 of the probability of occurrence for each biochemical
12 reaction with each of the organisms (taxa) in the data
13 base. This probability assumes a positive reaction.
14 If, in act, a negative reaction occurs, then the proba-
15 bility of the observa~ion would be l.000-P. For example,
16 i a given biochemical with a given organism has a proba-
17 bility of 0.005 of occurring, and the reaction was found
18 to be negative, then the probability would be .995 (1.000-
19 .005) that this reaction would not occur. So the program
20 at this point calls for converting all the negative
21 probabilities to l.000-P values for the table. The
22 positive reactions are left unchanged, and are manipulated
23 exactly as they occur in the table.
24 In addition to printing out the most probable ;
25 organism, the instrument provides the operator wlth
26 several indices of reliability. First, the overall non- ;
27 normalized probabllity of the reaction is computed. If
28 this probability is very low, this may mean that there
29 was an error in reading or that the suspension of test
30 bacteria contained more than one taxon. Second, the
31 relative normalized probabilities of three most likely
32 organisms are computed and displayed to the technologist.
33 Clearly, if several organisms have equal probability of
3~ occurring with a given set of biochemical reactions,
35 further testing is necessary to discriminate between
3~ them. Third, the instrument measures the susceptibility
37 of the test organism to several antimicrobics. If the
38
~'~31~78
- 16 -
1 known susceptibility is in conflict with the identification
2 by biochemicals, a warning is given to the operator.
3 Thus, after the individually observed proba-
4 bilities have been determined, each of the biochemical
5 probabilities is cumulatively multiplies by the other
6 probabilities for a given taxon. For example, the
7 observed probability (P) for the organism, E. Coli,
8 with dextrose is multiplied by the P for sucrose, and
9 this product is multiplied by the P of sorbitol, and
10 so on. This continues until a p:roduct of, for example,
11 twenty-one multiplica~ions is obtained for each organism.
12 Each of these products is the non-normalized frequency Eor
13 each taxon. As these non-normalized frequencies are
14 being computed, they are added to each other, so that a
sum of all of the non-normalized frequencies for each
16 organism is obtained.
17 Rare combinations of biochemical reactions can
18 occur with organisms, but more commonly, a very low
19 frequency will indicate a technical error. The most
20 common technical errors are due either to a mixed culture
~1 or to areading error. The instrument software is
22 designed so that an organism frequency (non-normalized)
23 of less than 1 x 10- 6 Will be read out as unacceptable.
24 If the organism with the greatest frequency is computed
25 to have a frequency of less than this value, the display
26 indicates: "VERY RARE BIOTYPE", and the program goes
27 back to the beginning. If the first organism frequency
28 is greater than 1 x 10- 6 but less than or equal to 1 x 10- 5,
29 the display says: "RARE BIOTYPE~PRINT? (1 or 0)". If the
30 operator wishes to go ahead and print, then he presses
31 "1" on the keyboard; if he wishes to go back to the main
32 program, then he presses "0". The instrument waits for
33 either of these keys to be pressed.
34 Normalization is accomplished by dividing each
35 of the three highest frequencies by the sum of all of the
36 frequencies. If the most probable organism has a normal-
37 ized frequency between .950 and .999, then the display
38
... . . . . . . .
.. ~ .
- 17 -
1 shows "MOST PROBABLE--XX.~%". In this case, the proba-
2 bility is converted to a percent figure. The program
3 then returns to check the dextrose fermenter flag. If
4 the organism is a dextrose fermenter, then the program
5 goes on to print the name o~ the most probable organism
6 and the biotype in appropriate spaces on the form. If
7 it is a non-fermenter it is compared with Colisti.n and
8 nitrofurantoin (Furadantin) results, as outlined below.
9 If the most probable organism has a normalized frequency
10 between .850 and .950, then the display indicates
11 "VERY PRORABLE--XX.X%". The program again checks for
12 fermenter or non-fermenter status as above. If the
13 relative (normalized) frequency is between .750 and
14 .850, the display indicates: "PROBABLE--X~.X%" and
15 loops through the fermenter/non-fermenter check as
16 above. If the relative probability is less than .750,
17 the display outputs three messages in sequence at one-
18 second intervals: "LOW SEJ.ECTIVITY-RECHECK"; followed
19 by "000000000000000000000--XX.X%" where 000 is the
20 organism name, and XX.X is the percentage as above.
2~ The third display is "STILL WAMT TO PRINT? (1 or 0)".
22 As stated above, if the organism is a non-
23 fermenter, the instrument also measures the suscepti-
24 bility of the test organism to several antimicrobics.
25 Thus, identification may be evaluated for its sensitivity
26 to the two antibiotics Colistin and Nitrofurantoin. If
27 there is growth in these wells (hex voltage less than
28 threshold), then this means the organisms are resistant
29 ("R"). If there is no growth (hex voltage greater than
30 threshold~, then the organism is sensitive or "S".
31 Once the sensititvity or resistance for Colistin
32 has been determined, the program looks up a table to see
33 if the result is correct; if not, t~en it displays on
3~ the visual display: "REC~ECK I.D. & COLISTIN DISAGREE";
35 the most probable organism is then printed out, and the
36 routine returns to the main program. If the table and
37 results agree with Colistin, then a similar procedure is
38 -
' .' , . ' , ' ':
3-~t~
- 18 -
1 performed with Nitrofurantoin. If there is disagreement,
2 the display says: "RRCHECK-ID & FURAMTOIN DISAGREE", If
3 there is agreement, then the result is printed out as
4 above.
In the method for determining bacterial suscep-
6 tibility to various antimicrobic drugs, the system o the
7 invention uses broth-dilution to determine susceptibility.
8 Serial dilutions of the antimicrobic agent are inoculated
9 with the organism and incubated for a period sufficient
lO to allow detectable growth. The apparatus of the inven-
11 tion determines minimum inhibitory concentration (MIC)
12 of a particular antimicrobic drug, which is the lowest
13 concentration of that drug that results in no detectable
14 bacterial growth. Typically, ten antimicrobic drugs
are evaluated, with seven different dilutions of each
16 drug being tested. Therefore, to obtain an MIC deter-
17 mination for ten drugs, seventy tubes or wells must be
18 inoculated and examined. In contrast to previous methods
19 using individual full-sized test tubes, which were
20 cumbersome and expensive, the present system utilizes
21 "micro-tubes", which are presently available as dis-
22 posable, molded plastic trays, each well of which holds23 approximately 0.5 milliliter.
24 For measurement of the MIC values in these
25 trays, appropriate dilutions of each antibiotic must be
26 placed in the wells or micro-tubes. Semiautomated
27 devices for making the dilutions and filling the trays
28 in large batches are available commercially. Alterna-
29 tively, a laboratory may obtain trays that are already
30 filled with antibiotic dilutions and kept frozen until
31 use. To prepare the bacteria cultures for inoculation
32 into the wells, a suspension of the bacterial organisms
33 in water is made in a container. By means of a multiple-
34 pronged device, a technician is able to inoculate a
35 uniform drop of bacterial suspension into each of the
36 large plurality (e.g., seventy) of wells with a single
37 motion. The bacteria and the various dilutions of the
" ~
~ 3~
~3~7~i
- 19 -
l antimicrobic agents are incubated for Q time period suf-
2 ficient to produce detectable bacterial growth, and the
3 MIC may then be determined as the lowest concentration
4 of the effective antimicrobic agents in which -there is
5 no evidence of growth.
6 Previously the reading of such an ~IIC tray was
7 done by manual viewing performed by a technician, and was
8 a laborious procedure. An overnight incubation period was
9 generally required in order to produce visually detectable
10 patterns of growth. However, the apparatus and method of
11 the present invention provided for the performance of the
12 reading and interpretive task automatically. Moreover,
13 the device has the capability of interpolating the MIC
14 between twofold dilutions, whereas by visual reading a
technician can only detect the difference between growth
16 and no growth and thus can only read MIC to the nearest
17 twofold dilution. With the sensltive photoelectric appa-
18 ratus described herein, together with the capabilities of
19 a microcomputer the different gradations of growth can be
20 measured even after a relatively short incubation period,
21 and a precise MIC can be calculated and displayed on a
22 screen or printed out. Thus, the device makes available
23 contînuous numerical data that improves accuracy and allows
24 quantitative quality-control techniques.
Photodetection of bacterial growth is accom-
26 plished by passage of uniform intensity light throu~h each
27 of the wells and through the translucent well bottoms
28 following the incubation period. The uniform light may
29 be obtained from plural uniform sources, one at each well,
30 or by a single source of uniform, diffused light over the
31 entire tray. At the opposite side of the tray, preferably
32 below the tray, are an array of sequentially-scanned photo-
33 electric cells, one associated with each well. The sensed
34 light intensity level at each well is compared by computer
35 with a light level corresponding to zero bacteria growth
36 to determine a relative value of turbidity. The reference
37 value may be obtained by the reading of a sterile control
38 well.
- . :,
~.~ 3~7~6
- 20 -
1 In addition to the quantitative MIC data,
2 the apparatus and method of the invention provide a
3 graphic interpretive printout to guide the physician's
4 therapy. The computer is programmed to translate the
5 MIC value into dosage ranges that would be necessary to
6 achieve blood levels of the antimicrobic drug effective
7 to inhibit growth of the organism at a particular site.
8 For example, a printout of "-" might be used to indicate
9 that the organism is resistant and no dosage of a drug
10 can effect the organism. A printout of "+" may be used
11 to mean that the organism is resistant and may respond
12 to high intramuscular or intravenous doses, with "-~t"
13 indicating intermediate sensitivity and that the
14 organism may respond to higher than recommended doses.
15 ~ printout of "+++" would indicate that the organism
16 may be sensitive to the usual doses o~ an antibiotic,
17 and "++++" would indicate a high degree of sensitivity
18 and thus an optimal drug with which to treat the infec-
19 tious agent.
In one embodiment, a method according to the
21 invention for determining susceptibility of a bacteria
22 culture to various antimicrobic drugs and of determining
23 the minimum inhibitory concentration (MIC) of the bacteria
24 culture to those drugs to which it is susceptible com-
25 prises the steps of placing the plurality of different
26 antimicrobic drugs in a plurality of wells in a light-
27 transmissive tray, each drug being included in a series
28 of wells in serially-diluted known concentrations;
29 establishing a known uniform concentration of the bacteria
30 and placing the uniform concentration in equal volumes of
31 the wells: following an incubation period, passing light
32 in substantially equal intensity through each well and
33 determining a turbidity value for the bacterial suspension
34 of each well by sequentially sensing the intensity of
35 light transmitted through the bacterial suspensions of the
36 wells by means of photodetectors adjacent to the wells
37 opposite the light source; and in a computer, comparing
38
~L~3'~71~
- 21 -
1 turbidity values with a turbidity value corresponding
2 to zero bacterial growth, thereby determining which anti-
3 microbic drugs have inhibited bacterial growth and the
4 minimum concentration of each inhibitory drug required
5 to inhibit growth, and displaying the determined infor-
6 mation. The concentration of the bacteria culture may
7 itself be initially determined by turbidimetric measure-
8 ment utilizing a light source and at least one photo-
9 detector. The antimicrobic drugs may be placed in the
10 tray in a rectangular matrix of wells, with each column
11 of wells containing incrementally varying concentrations
12 of a single drug. Of course, any arrangement of the wells
13 or of the antimicrobics in the wells is suitable, so long
lh as the computer has the proper in~ormation as to what ls
15 being tested in each well. Control wells containing only
16 the bacterial suspension, as well as sterile control
17 wells, may be included for self-checking of the system
18 and/or providing a transmitted light value corresponding
19 to zero bacteria growth. The system may, as explained
20 above, provide for translation of the MIC values to
21 dosage ranges necessary to establish the required anti-
22 microbic concentration at the body sites involved.
23 There are other applications for the instru-
24 ment. For example, heretofore, very sensitive techniques
25 have used bacteria as biological indicators for detecting
26 trace amounts of chemicals. Strains of bacteria are
~7 obtained by mutation that manifest growth that is ~ -
28 directly proportional to the quantity of a given sub-
29 stance so that calibration curves are easily made. Such
30 a substance and the amount thereof can therefore be
31 detected in a cultured sample by using the apparatus of
32 the lnvention, preferably using optical filters. Suitable
33 programs can, of course, be preferred.
34 Another example is the instrument's applica-
35 bility to the technique of enzyme-linked immuno absorbent
36 assay (E~ISA) to detect the presence of a specific
37 species of protein molecules (e.g. bacteria, virus, or
38
..... . . . . .
;. ~ .~ ., ,. . - ' `,' :
.. . . ... .. .
,................... . . ... . . . . .
.:
. ~ - .. . ..
L3~ 6
l hormone) which is detected by its combina~ion with an
2 antibody. An antigen-antibody reaction is detected
3 (in this technique) by a color change caused by an
4 enzyme or enzymes and detected by the instrument.
S
6 Brief Description of the Drawings
; 7 In the drawings:
9 Fig. 1 is a perspective view showing an auto-
10 mated apparatus embodying the principles of the invention.
11
12 Fig. 2 is an exploded perspective view showing
13 a sample tray and optical detection equipment forming a
14 part o:E the apparatus of Fig. 1.
16 Fig. 3 is a schematic sectional elevational
17 view showing a portion of the apparatus of Fig. 1.
18
19 Fig. 4 is a block diagram of the apparatus of
20 Fig. 1.
21
22 Fig. 5 is a block diagram of an analog-to-
23 digital converter subsystem usable in the apparatus
24 of Fîgs. 1-4.
26 Fig. 6 is a block diagram of a microcomputer
27 portion of the apparatus.
28
29 Fig. 7A, 7B, and 7C are flow charts of
30 operational steps involved in the method of determining
31 minimum inhibitory concentration.
32
33 Fig. 8 is a schematic elevation view showing an
34 alternati~e~form of optical detection apparatus which may
35 be included in the apparatus of the i.nvention.
36
37 Fig. 9 shows a form of printout which may be
~`' 38
,. ., . . .. . ,. ~
' ~ ' , " . ~ I . '. . . ' " ., ' `, ' , ., ' `' . ' " ., ` ' ' ' ' ' ", '` ' ~ .
3~
- 23 -
1 utilized in connection with the apparatus of the invention
2 when determining minimum inhibitory concentration.
4 Figs. lOA, lOB, and lOC (which are on the same
S sheet as Fig. 1) are a set of three spectral absorbent
6 responses for three respective optical filters that may
7 be used in the invention.
9 Figs. llA, llB, and llC are flow charts of
10 operational steps involved in the method for identifying
11 microorganisms according to the principles of the
12 invention.
13
14 Description of the Preferred Embodiments
15 The apparatus of Figs. 1-6:
16 Fig. 1 shows one example of an external con-
17 figuration which the susceptibility testing apparatus 10
18 of the invention may take. The unit 10 comprises a photo
19 unit or optical detection unit 11 and a processor unit 12.
20 The optical detection unit 11 preferably includes a
21 drawer 13 for receiving, supporting, and correctly posi-
22 tioning a sample tray 14 which is examined ~y detection
23 apparatus of the unit 11 when the drawer is closed and
24 the testing operation is begun. The detection unit 11 may
25 also include a patient identification input switch 16,
26 a run switch 17 and a calibrate switch 18. The processor
27 unit 12 may include a readout display 19 J an on/off
28 power switch 21, printer control buttons 22, and a
29 printout exit 23 which dispenses a printed "ticke-t" 24
30 bearing the desired susceptibility information.
31 Fig. 2 somewhat schematically represents the
32 configuration of the detection apparatus associated
33 with the optical detection unit 11 of the apparatus 10.
34 Within the detection unit 11 above the drawer 13 is a
35 source of uniform, diffuse light which may comprise,
36 for example, a fluorescent light bulb 26, a parabolic
37 reflector 27 positioned thereabove such that the lamp 26
3~
~ .. .. .. . .
... : ~ , . . . . .
- 24 -
1 is at the focal point of the reflector 27, and a diffuser
2 28 just below the lamp and reflector. The arrangement
3 of the lamp 26 and the reflector 27 provides a nearly
4 uniform distribution of light over the surface of the
5 diffuser 28, and the diffuser împroves uniformity and
6 reduces intensity to the desired level.
7 Within the drawer 13 are a sample tray holder
8 29 having a matrix of openings 3:L, and an array of photo-
9 cells 32 therebelow in a matrix conforming to the posi-
10 tion of the openings 31 above. The openings 31 and the
11 photocells 32 also correspond precisely to the position
12 of sample testing wells 33 of a sample tray 14 which is
13 received in registry above the tray holder 29 when a test
14 is to be conducted. The sample tray 14, or at least the
lS bottom of each well 33, is translucent so that light
16 passing through the diffuser 28 penetrates the wells and
17 their contents, passes through the openings 31 in the
18 tray holder 29 (and usually through a collimator~ and
19 reaches the photocells 32 below, which individually
20 sense the intensity of the light passing through each
21 well. The photocells may be of the type manufactured
22 by Clairex Electronics of Mt. Vernon, N.Y. as Model
23 CL702L. The tray holder 29 is preferably of a dark,
24 light-absorbing color such as black to reduce light
25 transmission between the wells and reflection of dif-
26 fracted light within any one well. The tray holder
27 arrangement assures that all light passing through the
28 openings 31 is from the wells 33 rather than through other
29 areas of the translucent sample tray 14. The embodiment
30 as described is particularly suitable for determining
31 MIC.
32 In another embodiment adapted for bacterial
33 identification, a collimator 34 and an optical filter 35
34 are placed between the sample tray 14 and the photocells
35 32. The exact filter 35 used depends on the test con-
36 cerned. The filter 35 is made to be easily removable
37 and replaceable. For example, a large number of tests
38
, , , .,. . ~ . . .. .. .. . . .
71~
- 25 -
1 may be run using only three filters one at a time; these
2 three being (for example) ~ilters numbers 809, 863, and
3 878, of Edmund Scientific Co., 785 Edscorp Building,
4 Barrington, New Jersey 08007. Fig. 14 shows the spec-
5 tral absorber responses of these three filters, 809 at
6 A, 863 at B, and 878 at C.
7 The sample tray 14 is preferably a disposable,
8 molded plastic tray, each well of which holds approxi-
9 mately 0.5 milliliter. Trays of this type are commer-
10 cially available and have been used previously for
11 simple visual type "reading" techniques as discussed
12 above. The wells 33 are often referred to as "micro
13 tubes", since they replace cumbersome full-sized test
14 tubes which were used in the past for this type
testing.
16 Fig. 3 shows a preferred arrangement for ~he
17 light source, the sample tray, and the means of holding
18 the sa~ple tray and collimating the light through the wells
19 to the photocells. A partially broken-away, schematic
20 sectional view in Fig. 3 shows the lamp or bulb 26
21 with the reflector 27 above and the diffuser 28 below.
22 The drawer arranement slides in and out of the appratus
23 10 above and independen-tly of the array of photocells 32.
24 As indicated, the photocells 32 are mounted fixedly below
25 the drawer 13 and of course positioned to receive light
26 passing through each well 33 when the drawer is fully
27 inserted, in the testing position.
28 The tray 14 rests on a tray block 40 secured
29 within the drawer 13 and having a matrix of openings
30 31 for receiving the depending sample wells 33. Below
31 the tray block 40 is a drawer plate 44, also bored at
32 each location of a well as indicated, the drawer plate
33 bores 45 being directly in registry with the openings
34 31 above. The drawer plate 44 with its bores 45 serves
35 as the collimator 34.
36 Alternatively, the matrix of photocells 32 may be
37 slidab~y mounted with a tray holder 29 having a matrix of
38
., ~ . . .. . .
., . , , . -. ~
.
13~'7~6
- 26 -
1 openings 31 into which the wells 33 extend, with little
2 side-to-side tolerance so that the registry of the ~ample
3 wells with the photocells is assured. In this embodiment,
4 which may be used particularly for determining MIC, the
5 tray holder and photocell assembly slides in and out of
6 the apparatus 10 with the drawer 13. The collimator 34
7 may be omitted.
8 As indicated in Fig. 3, there is a space left
9 between the tray block 40 and the drawer plate 44 for an
10 optical filter 35. The filter 35 preferably is slidably
11 received between the two drawer-attached components 40
12 and 44 above and below, and different filters can be
13 used.
14 Another feature illustrated in Fig. 3 is the use
15 of a microswitch 46 which is tripped by the back end of
16 the drawer 13 as it is fully inserted into the testing
17 position. This starts the test automatically, and the
18 testing cycle proceeds to completion.
19 The light source illustrated is a convenient and
20 preferred form; however, any light source or a plurality
21 of light sources which will provide light of equal inten-
22 sity directed into each well 33 of the sample tray 14 is
23 sufficient. In this regard, an alternative form of light
24 source and detection system is described below in con- -~
25 nection with Fig. 8.
26 The single diffuse source 26, 27, 28 need not put
27 out a uniform ligh~. The light need only be roughly even.
28 Also, the photodetectors 32 may be inexpensive ones,
29 providing signals of different strengths for the same
30 light intensity, 90 long as the invention is practiced
31 with an initial calibration step. In this step, the
32 light source 26, 27, 28 directs light over all the photo-
33 detectors 32 either without a tray ~4 positioned above `
34 them or with an empty tray 14, to take any variations in
35 the plastic material of the tray into account in the
36 calibration. As another alternative, the -tray wells 33
37 may be filled and then run through before any culture,
38
'71~i
1 at zero time relat:ive to growth. In the calibration,
2 a scan is made and all values, i.e. photodetector out-
3 put signal values, are stored. When each actual test
4 is run, a difference or ratio signal is created for each
5 photocell, so that only the difference in sensed light
6 intensity is used, disregarding effects of localized
7 differences and intensit~ and differences in the photo-
8 cells themselves.
9 The reference photocell 32n is preferably
10 outside the area of the tray (as shown in Fig. 4),
11 although it may be beneath a sterile or empty well 33n
12 of the tray.
13 As discussed above, the sample tray 14 is
14 preferably laid out in a rectangular matrix, which may
comprise for example eight rows and ten columns. Other
16 arrangements would be adequate, but a rectangular matrix
17 is space-efficient and convenient. The wells 33 may, as
18 for obtaining MIC values, contain various dilutions of
19 different antlbiotics, and these may be arranged such
20 that each of the ten columns of wells contains a single
21 antibiotic in a series of different dilutions. There
22 may be seven different concentrations of each anti-
23 biotic, with theeighth well of a~ least several of the
24 columns used for control purposes. For example, one
25 control well might be used for unrestricted growth of
26 bacteriaj, and another well used to represent no growth,
27 with no bacteria inoculated into the well.
28 Into the wells containing the various dilutions
29 of diferent antibiotics ~for determining ~IIC values) is
30 introduced the patient bacteria sample borne within a
31 culture medium. This bacteria culture is uniformly
32 inoculated into each well, and this may be accomplished
33 by commercially available devices having a matrix of
34 prongs (not shown) arranged to register with each well
35 to be inoculated in the commercially available sample
36 tray 14. Of course, the antibiotics and the bacteria
37 culture may be introduced to the wells in the reverse
38
:
- 28 -
1 order, but for convenience, efficiency and reliability
2 it is preferred that the antibiotic be introduced first.
3 Fig. 4 indicates diagrammatically the operation
4 of the susceptibility testing apparatus 10. The lamp 26,
5 reflector 27 and diffuser 28 are shown transmitting
6 uniform diffuse light through a sample well 33a of the
7 matrix of wells of the sample tray 14. The well 33a
8 contains one dilution of one of the antibiotics being
9 tested, inoculated with a controlled volume and known
10 concentration of the bacteria in a culture sample. The
11 same uniform difEuse light may be transmitted through a
12 well 33n containing no bacteria or providing a light
13 intensity reading corresponding to zero bacteria growth.
14 Alternatively, the sterile control well 33n may be
eliminated, as shown, the diffuse light passing through
16 a collimator 34 directly to the reference photocell 32n.
17 An optional color filter 35 may also be added for bacterial
18 identification purposes.
19 After an incubation period sufficient to allow
20 some detectible growth of the bacteria in the well 33a
21 in the event that growth is not prevented by the particular
22 antibiotic in the particular concentration being tested,
23 a growth culture 36 results therein. The light from the
24 diffuser passes through this culture 36 and through the
25 bottom of the well to a photocell 32a of the photocell
26 matrix. Here the intensity of the light is sensed and
27 converted into an electrical analog value corresponding
28 to the opacity of the culture 36. This opacity value
29 represents the turbidity of the culture, stemming from
30 the net effect of light absorption and scatter in the
31 well 33a. At the same time, ~he diffuse light passes
32 through the steri~e control well 33n to a photocell 32n
33 of the photocell matrix. Again, the sensed light
34 intensity is converted into an electrical analog
35 reference value.
36 The photocell 32_ is connected to a plus input
37 of a differential amplifier 37 through a noise filter
38
- 29 -
l 38 and a multiplexer 39 which functions to select each
2 photocell 33 of the matrix of photocells in a prescribed
3 sequence under direction of a microcomputer 41. Alterna-
4 tively, the differential amplifier 37 may be replaced by
5 a log ratio module 237, Thus, the "difference" is made
6 into a quotient, giving more sensitivity. The sequencing,
7 being automatic, is very fast, going through 80 or 96
8 wells of a tray 14 in about five seconds or less. The
9 automatic electronic scan has no moving parts--an
lO important feature.
11 Electronic sequencing :is much more reliable than
12 mechanical movement of a tray or other mechanical seque~c-
13 ing. Multiplexing has the advantages of speed, accuracy,
14 reliability and maintainability, i.e. easy maintenance.
15 For at least these reasons, the invention is a signifi-
16 cant improvement over mechanical scanning. Thus, the
17 photocell 32a shown in Fig. 4 is connected to the dif-
18 ferential amplifier 37 only when the multiplexer 39
19 momentarily selects that particular photocell. The
20 reference photocell 32n is connected to the minus input
21 of the differential amplifier 37 and provides a reference
22 voltage which is subtracted from the plus input to provide
23 an analog differential output. Thus, the light intensity
24 or turbîdity value signal emanating from the differential
25 amplifier 37 is in the form of a reference voltage which
26 varies according to turbidity of the sample being sensed,
27 representing the increase in turbidity of that sample
28 since inoculation. Each analog signal is transmitted in
29 its turn to an analog-to-digital converter 42 which
30 converts the analog to a digital signal and sends it to
31 the microcomputer 41.
32 The microcomputer 41 (See Figs. 5 and 6) func-
33 tions to correlate differential digital values (from the
34 ADC 42) representing, for example, bacterial growth for
35 the various wells witll the particular drug and its con-
36 centration in the subject well. From such correlation,
37 the microcomputer selects, for example, the zero growth
38
,: . :.1: . . .~ :
30 -
1 indication ste~ming from the weakest concentration of
2 each drug, and this concentration becomes the MIC for
3 that particular drug. If none of the wells containing
4 a particular drug indicates inhibition of growth, the
5 microcomputer prints out the fact that the infectious
6 organism is resistant to that particular drug.
7 The remaining apparatus indicated in Fig. 4 is
8 described below with reference to the other figures.
9 The analog circuitry associated with this
10 system 10, including the analog-to-digital converter,
11 is set forth in the detailed block diagram of Fig. 5.
12 Fig. 5 includes the nolse filter circuit 38, the multi-
13 plexer circuits 39 which are included within the dashed
14 line box, the di~ferential amplifier 37, and the analog-
to-digital converter 42 along with its related supporting
16 circuitry. A ten by eight photocell matrix is also shown
17 in Fig. 5 for clarity of understanding of this part of
18 the system 10. A twelve-b~-eight system (or other such
19 system) may be used instead.
The multiplexer 39 includes a binary coded
21 decimal (BCD) to decimal decoder 101 driving column
22 drivers 103, and another BCD to decimal decoder 113 con-
23 trolling FET switches 111. A four bit digital line 100
24 from the microcomputer 41 is connected to the binary
25 coded decimal input of the binary coded decimal to deci-
26 mal decoder circuit 101 (which may be pre~ereably imple-
27 mented as a type 7442 TTI. integrated circuit or equiva-
28 lent). Ten output lines 102 from the decoder 101 are
29 connected to ten driver circuits 103. The driver circuits
30 are preferably implemented as operational amplifiers type
31 LM 324 or equivalent.
32 As already explained above, the photocell
33 matrix is arranged as a rectangle with ten columns and
34 eight rows. Thus, the outputs from the ten driver
35 circuits 103 are applied to the ten columns respectively
36 via a bus 104 such that when one driver îs excited by
37 operation of the decoder 101, an excitation voltage is
38
~3~7~
- 31 -
1 provided to one of the column drive lines corresponding
2 to the binary coded decimal column select information
3 input to the decoder 101 via the data line 100 from the
4 microcomputer 41. An eleventh of the drivers 103 applies
5 voltage continuously through a drive line 105 to the
6 reference cell 33n.
7 Eight row lines 106 and one line 107 from the
8 reference cell 32n are applied as inputs to nine active
9 filter circuits within the filter 38. Each filter cir-
10 cuit is preferably implemented by an operational ampli-
11 fier, type LM 324 or equivalent. The filters 38 function
12 to remove power line ripple so that the eight row output
13 lines 108 and a reerence output line 109 carry DC voltage
14 levels only. The eight output lines 108 are applied to
15 eight field effect transistor switches 111, respectively.
16 The switches are preferably implemented as integrated
17 circuits type CD4016 CMOS quad bilateral switch gate
18 chips or equivalent. An output line 110 from the switches
19 111 is connected directly to the plus input of the dif-
20 ferential amplifier 37. The ninth line 109 is applied
21 directly to the minus input of the logarithmic differen-
22 tial amplifier 37.
23 A three bit digital line 112 from the micro-
24 computer 41 is connected to the input of a second binary
25 coded decimal to decimal decoder 113 which is also
26 preferably implemented as a type 7442 TTL integrated
27 circuit or equivalent. The decoder 113 functions to
28 select one of eight output control lines 114 which in
29 turn select one of the eight field effect transistor
30 switches 111 to connect one of the filtered row lines to -
31 the plus input of the logarithmic differential ampli-
32 fier 37, in accordance with digital row select information
33 received from the microcomputer 41. .
34 The logarithmic differential amplifier 37 is
35 preferably implemented as an Analog Devices type 757 or
36 equivalent, and the purpose of the amplifier 37 is to
37 correct for variations in light intensity from the light
3~
~ . , . , .. " , - , ~ , ~, ; ., . ,-, .
1..13~7'~
- 32 -
1 source 26. The light-variation-corrected analog voltage
2 output from the amplifier 37 is supplied as an input to
3 an operational amplifier 116 which is provided with
4 external potentiometers to control gain and DC offset
5 of the incoming signal from the amplifier 37.
6 An output line 117 from the amplifier 116 is
7 supplied as an analog input to the analog-to-digital
8 converter 42 which is preferably implemented with a
9 National Semiconductor MM5357 integrated circuit or
10 equivalent. A digital control line 118 from the micro-
11 computer 41 is connected as a trigger input to a mono-
12 stable multivibrator one shot 119, preferably implemented
13 as a type 74121 TTL integrated circuit or the equivalent.
~ An output pulse from the one shot 119 of appropriate
15 amplitude and duration is supplied via a line 121 to the
16 analog-to-digital converter 42 to start the conversion
17 process. A timing genera-tor (e.g. type 555) 122 applies
18 timing pulses via a line 123 to the analog-to-digital
19 converter 42 to control the sequence of operations thereof.
20 The analog to-digital converter 42 utilizes the timing
21 pulses supplied on the line 123 during a conversion cycle
22 to digitize the analog information on the line 117 and
23 provide an eight bit digital output via an eight bit
24 output bus 124 which is supplied to an input port of the
25 microcomputer 41.
26 The microcomputer 41 forms the central portion
27 of the system 10. The microcomputer includes a single
28 chip monolithic microprocessing unit (MPU) 140, which is
29 preferably implemented as a type 6800 manufactured by
30 Motorola Semiconductor, American Microsystems, and other
31 suppliers. Although this particular microprocessor
32 was chosen for the described preferred embodiment of
33 the present invention, other types of microprocessors
34 would function equally as well, for example the Intel
35 8080, the Mostec 6502, the Zilog Z80, the Fairchild F-8,
36 etc. A suitablle two-phase clock 141 provides the neces-
37 sary clock signals to the microprocessing unit 140.
38
, ~ . :,- ,., , ~ . .,
3~7~7~6
- 33 ~
1 A main system program like that which is set
2 forth in hexadecimal code in the table following the
3 specification of the present invention may be loaded
4 into one and a half kilobytes of programmable read only
S memory 142. The read only memory 142 is preferably
6 implemented with 2708 programmable read only memories
7 produced by Intel and other suppliers. Other PROMs
8 would be well suited for the program memory 142. The
9 microcomputer 41 also includes one kilobyte of random
10 access memory (RAM) 143 which provides volatile storage
11 of data to be processed as well as a stack for the
12 microprocessing unit 140. The microprocessing unit 140,
13 the clock 141 through the microprocessing unit 140, the
14 program memory 142 and the data storage memory 143 are
15 connected in parallel to the system bus 144 which
16 includes an eight bit data bus, an eight bit control
17 bus, and a sixteen bit address bus.
18 Input output interface is accomplished with
19 three peripheral interface adapters ~PIA) 146, 147 and
20 148 which are connected to the sys~em bus 144. The
21 interface adapters 146, 147 and 148 are preferably
22 implemented as type 6820 integrated circuits produced
23 by Motorola Semiconductor, ~merican Microsystems, and
24 other suppliers. These integrated circuits contain two
25 ports apiece. Each port may be used either to input
26 data to the microprocessing unit 140 or to output data
27 to output devices, as will be explained hereinafter.
28 The first interface adapter 146 has its first
29 port connected to receive the eight bit digiti~ed
30 information via the bus 124 from the analog-to-digital
31 converter 42, as shown in Fig. 5. The first port of the
32 interface adapter 146 also provides the control signal
33 line 118 which is connected to the one shot 119 which
34 functions to start the analog-to-digital conversion
35 process of the converter 42. The line 118 will be
36 further explained hereinafter. The second port of the .
37 interface adapter 146 is connected to the multiplexer 39
38
, ~ . , . . ; ! ' ' I ' ' . ' ' ;
'7~
- 34 -
1 with four bits provided for the column select control
2 signal via the bus 100, and the three remaining bits
3 provide for the row select control signal via the bus
4 112.
The second peripheral :interface adapter 147
6 includes a first port which controls the printer 20.
7 Two bits of data are input from status indicators in
8 the printer 20 via a line 149. One of these bit posi-
9 tions is from a microswitch which indicates that the
10 paper form has been properly inserted and tha~ a print-
11 out can be made. The other bit is a signal from the
12 printer electronics which indicates that the printer is
13 either in a "print" or a "wait" operational mode. Four
14 bits of the first port of the interface adapker 147 are
15 also used to control the printer and shift data to be
16 printed into the printer 20. The data is entered seri-
17 ally via a line 151 from the first port of the adapter
18 147 to the printer 20. Other control functions carried
19 out by the four bits on the line 151 include line feed
20 (advance the paper one line), print (cause the print
21 solenoid to make an impression on the paper), and shift
22 (move the next data bit into position for printing~. The
23 second port of the interface adapter 147 is not used in
24 the present embodiment.
The third peripheral interface adapter 1~8 in-
26 cludes a first port which reads the thumbwheel switch 16
27 for patient identification information via a four bit
28 line 153. The upper our bit positions of this first
29 port of the adapter 148 are used to select and enable
30 one of the four thumbwheel positions via a four bit line
31 152. One bit position of the line 152 is low to enable
32 one of the four switching positions. The lower four bits
33 of the first port of ~he adap~er 148 are used to read
34 data via a bus 153 from the switch position selected by
35 the upper four bits. The data from the switch repre-
36 sent a binary number between zero and nine. The second
37 port of the in~er:Eace adapter 148 is used to supply data
38 to the alpha-numeric display readout 19. The display 1 is
, , :.
- , ::
..
37"~3~;
- 35 -
l the Burroughs model SSD0132-0070 self-scan display unit
2 with built-in electronics. As explained, it is controlled
3 via a line 154 from the second port of the third peripheral
4 interface adapter 148. Data to be displayed on the
5 display 19 are entered into the lmit via a line 156 in a
6 six bit code for all alpha-numeric characters as well
7 as some special symbols. The data are read in from left
8 to right and appear on the display until new data are
9 entered. Thus, the upper two bits are provided via the
10 line 154 to control the display, with one of the bits
11 being a clear line and the other being an enable line.
12 The lower six bits are provided via the line 156 for the
13 purpose of sending parallel data to the display presented
14 to the user in accordance with the operation of the
system 10.
16 In addition to the characteristics of the
17 interface adapters 146, 147 and 148 described hereinabove,
18 each adapter also has an interrupt function. The
19 interrupt is an additional line which is available for
20 monitoring the status of external devices. In the pres-
21 ently described system 10, the interrupts a~e used to
22 monitor operator actions of several types. Interrupt
23 capability which results in an output rather than an
24 input is termed a strobe. Strobes are utilized in the
25 system 10 as well as interrupts. Thus, the first peri-
26 pheral interface adapter 146 controls the conversion of
27 data ~rom analog--to-digital format via the analog-~o
28 digital converter 42 by utilizing a strobe line 118 which
29 is connected to the one shot 119 (Fig. 5) to start the
30 analog-to-digital conversion operation.
31 The second peripheral intsrface adapter utilizes
32 an interrupt from the printer 20 via a line 155 and
33 utilizes one interrupt each from the run switch 17 via a
34 line 158 and calibrate switch 1& via a line 159. The ;-
35 second port of the second adapter 147 utilizes the output
36 strobes via a line 157 to cause the printer 20 to execute
37 a print cycle.
38
:: , ,, ~ -
3~'7~i
- 36 -
1 A third peripheral interface adapter 148 has
2 two interrupt lnputs: one from a microswitch indicating
3 that the photo unit drawer is open via a line 161 and one
4 indicating that the drawer is closed via a line 162.
The printer 20 may be implemented as an MFE
6 model TKllE or Practical Automation DMPT-9, both with
7 electronics package. Data is fed from the microcomputer
8 41 via the line 151 which generates the proper control
9 signals to enable the printer electronics to cause the
10 printer 20 to print, line feed or shift data into internal
11 registers. The data is fed to the printer 20 in serial
12 format, stored in buffers in the printer electronics, and
13 is then printed in parallel. The command to print is
14 generated as a strobe output of the second port of the
second peripheral interface adapted 147 via the line 157.
16 The printer is a commercially available unit presently
17 being sold for the original equipment manufacturer (OEM~
18 market.
19
20 Determining minimum inhibitory concentration (MI~) (Fi~. 7):
21 One method using the system 10 is explicated by
22 the flow chart set forth in Fig. 7. Therein, at a power
23 on step 166, the operator turns the power on to the system
24 10. At that point, the display 19 informs the operator
25 to insert the calibration tray. At insertion step 168,
26 the operator inserts the tray, and at step 169, the
27 operator closes the drawer. At a logical step 170, the
28 system checks the identification of the tray in the
29 drawer. For this purpose a binary code may be implemented
30 using the uppermost right two wells of the tray, either
31 of these wells being Qither opaque or transparent, thus
32 providing identification of four possible types of trays.
33 This code is made to correspond to the combination
34 antibiotics which the tray contains.
In the event that the type of tray is not
36 identified at step 171, the system asks whether the tray
37 is inserted backwards at step 172. If so, the readout 19
38
'' ' ' ~ ' '` ' 'i` ' ' .` '', ;;~ ' ' " ` . ' ' .;: '.' ~ `"` " '' ' .. . .
3~ 6
- 37 -
1 displays a ~ray backwards indication at step 173, and
2 the operator opens the drawer at a step 174 and removes
3 the tray, orients it correctly, and reinserts it, then
4 repeats steps 168, 169, 170 and 171.
Once the tray is identified at step 171, the
6 readout 19 displays the tray type at step 175, and
7 directs the operator to press the calibration switch 18
8 at a step 176. At step 177, the operator presses the
9 calibration switch 18 whereupon the system tells the
10 operator to wait at step 178. The wait signal remains
11 until the system informs the operator to remove the
12 tray at step 179. The operator opens the drawer at step
13 180. In the event that the tray is not in backwards, and
14 yet the tray re~lains unidentified at step 181, the
15 operator is then instructed to open the drawer to manually
16 inspect the tray to find out why the system 10 is unable
17 to identify it.
18 At step 182, the readout 19 tells the operator
19 to close the drawer, and at step 183 the operator removes
20 the tray and closes the drawer. The readout 19 then tells
21 the operator that if a next test is desired, he should
22 press the run or calibrate button at step 184. At a step
23 185, the operator actually presses the run or the cali-
24 brate switch. If the system has been previously cali-
25 brated at step 186, then the readout l9 directs the
26 operator to insert the test tray at step 187. However,
27 if the system 10 has not been calibrated at step 186, the
28 program returns to step 167 and the calibration procedure
29 is carried out as set forth in steps 167 through 185.
30 At step 188, the operator opens the drawer and ~-
31 inserts the test tray. The display 19 then tells the
32 operator to close the drawer at step 189. The operator
33 closes the drawer at step 190 and the tray identification
34 is determined at step 191. In the event that the tray is
35 not identified, the system then determines whether the
36 tray is in backwards at step 192. If so, the system
37 informs the operator that the tray is in backwards by a
38
3~
- 38 -
1 readout display at step 193. In the event that the tray
2 remains unidentified and it is not in backwards, then at
3 step 194, the operator is informed that the tray is
4 unidentified and the program loops back to step 180
5 whereupon the operator opens the drawer and repeats
6 steps 180 through 191.
7 Once the identification of the tray has been
8 determined at logical step 191, the system 10 displays
9 the type of tray at the readout with step 195. Then the
10 operator is informed to set the patient identification
11 information i.nto the identification switch 16 and insert
12 the form to be printed into the printer 20 at step 196.
13 The operator performs these operations at step 197 and
14 when they are completed, the display 19 tells the operator
to press the run switch 17 at step 198. The operator
16 presses the run switch 17 at step 199 and the patient
17 identification information is displayed at step 200.
18 Then, the patient identification is printed on the form
19 at a step 201 and then the MIC values and lnterpretive
20 information are printed on the form in step 202 to produce
21 the form 203.
22 Once the form is printed with the patient
23 identification MIC values and interpretive information
24 the display tells the operator to remove the tray at
25 step 204. The operator opens the drawer and actually
26 removes the tray at step 205 whereupon the display 19
27 tells the operator to close the drawer at step 206. The
28 operator closes the drawer at step 207 and the apparatus
29 10 then instructs the operator to perform the next ~-
30 operation of either "run" or "calibrate" at step 208
31 whereupon the program loops back to step 185 where the
32 run or calibration switches are operated and the program
33 is repeated as heretofore described until all of the
34 samples have been evaluated by the system 10.
36 Comparisons to reduce errors due to the tray and to light
37 intensity and_photodetector differences:
38 It will be apparent that the tray 14 itself might
31~l3~ 9
- 39 -
1 be a source of error. That is, its own light trans-
2 missivity and opaqueness and flaws can substantially
3 affect the light transmissivities received by the photo-
4 cells 32, in addition to the light transmissivity of the
5 liquid in the wells. The trays 14 can vary from tray to
6 ~ray, and they can also vary in a tray from well to well.
7 This could, of course, lead to substantial errors that
8 would give false impressions and false results if not
9 compensated or corrected.
The present invention accomplishes the needed
11 correction by two different types of comparison stages.
12 First, for each reading in any sequence of wells
13 33 in the tray 14, each well 33 is immediately compared
14 with the value obtained by direct light transmission to
15 the reference photocell 32_. While this may be done
16 through a sterile control well, as shown in Fig. 4, it
17 is preferably done directly, completely outside the tray
18 14, as shown in Figs. 10 and 13, with the light to the
l9 reference photocell not passing through any portion of the
20 tray. F'rom this comparison, the device provides an after
21 culture value for each well, which is a function of the
22 after culture signal values (or amplification thereof) for
23 the tested well and for the reference photocell. This,
24 of course, represents a comparison of the light received
25 at each photocell in the main array and the intensity of
26 the light received at the reference photocell. The signal
27 may be amplified and is used as the operative signal, as
28 shown in Fig. 4. The after culture value for each well
29 may be called a "difference" signal value, regardless
30 of the type of function which is used in comparing the
31 two values (test well vs. reference photocell) to produce
32 this value. In the embodiment of Fig. 4 the subtractive
33 difference preferably is taken between the two values,
34 and the differential amplifier 37 amplifies the difference
35 signal. However, the signal value produced in the other
36 embodiment of Fig. 4 is a ratio, and the signal from each
37 well is compared with the reference photocell signal by
38
: . . : . ~ ~: ~ .
. .. :. ; ~ ; ,
r - ~
- 40 -
1 means of a log ratio module 237. In other words, there
2 is again a "difference" signal, but it is a difference
3 in logarithms, so that the suhtraction is really a
4 division, and a quotient or ratio is obtained instead
5 of a difference expressed as a logarithm.
6 Thus a first comparator may incorporate a log
7 ratio module and send out its related signal as an ampli-
8 tude ratio between each signal Sw obtained through a well
9 and its photocell and a signal SR obtained from the
10 reference photocell. This related signal Sx =
11 R
12 where kl is a constant. This first comparator may also
13 incorporate a log ratio module and sends out its out its
14 resultant value Sv as a ratio k2 SX , where DV is the
DV
16 data reference value and k2 a constant.
17 The first and second comparators may use the
18 same log ratio module. The second comparator may utilize
19 as its data reference value Dv, stored ratios read earlier
20 from an empty tray, so that DV = kl SWE for each well,
21 SR
22 where SwE is the signal coming from an empty well.
23 The second comparator may utilize as its
24 data reference value Dv, stored ratios read earlier from
25 a tray containing the same liquid from which the signal
26 Sw are generated, but read at a time when there has been
27 zero growth, so that DV = 1 SWO for each well, where
28 SR
29 sWO is the signal coming from a well containing the liquid
30 at zero growth time.
31 Also, the preliminary comparing means may include
32 a log ratio module for sending as its derived signal a
33 signal based on the ratio of the two signals it compares.
34 Thus, in the invention, each reading of each
35 well, at each stage where readings are taken, is compared
36 by a first comparator means with the reading at the refer-
37 ence photocell, and a difference or ratio signal developed
38 ~`~
~L~L3~
1 from it. By this procedure, variations in light intensity
2 from the source over time, as would be induced by supply
3 voltage fluctuations, have no effect on the readings.
Such variations will vary the reference and well photo-
5 cells proportionately, so that a ratio will cancel the
6 errors out. This is the purpose of the reference photo-
7 cell.
8 Second, to further reduce the possibility of
9 error particularly due to flaws in the tray, and in view
10 of the fact that each tray 14 is positively identified
11 in the apparatus, as has already been descrlbed, a prior
12 reading may be taken through the tray before the reading
13 after bacterial culture; this prior reading is stored
14 and is later compared with the sample reading.
One way of taking the prior reading is to take
16 a reading of the tray 14 in its empty state, before it
17 is filled with fluid, to compare the reading through each
18 empty well with the reading of the reference photocell,
19 as above, and to store the resulting difference signal or
20 ratio signal in the data storage portion of the micro-
21 computer 41. Then the ratio signal (or difference signal)
22 derived from the liquid at the time of the after culture
23 reading is compared with the ratio signal (or difference
24 signal) of the empty wells. Thereby, each well is compared
25 with itself when full and when empty, and errors due to
26 the wells are substantially eliminated.
27 Ano~her way of taking this prior reading is to
28 take the prior reading, not of the empty tray but of the
29 tray just after its wells have been filled with the solu-
30 tion and prior to the culture; in other words, at sub-
31 stantially zero time so far as growth or culture is
32 concerned. This means that the reading is taken through
33 the actual solution, and the ratio of that reading to
34 the reference electrode is stored in the data storage
35 bank for the later use.
36 With the zero based signal (however obtained)
37 in the data bank, and with the ratio or difference signal
38 provided for each well for the liquid after culture, then,
,.3~t~6
- 42 -
1 before proceeding further, the ne.~t step is to compare by
2 a second comparator rneans the two ratio (or difference)
3 values, that is, to compare the ratio of the signal
4 derived from the light transmissivity of the specimen
5 after culture to the direct light recep-tion by the refer-
6 ence cell, with the ratio of the empty tray or tray with
7 the same liquid at zero time to the signal from the ref-
8 erence cell. This second comparison may also be made by
9 calculating a ratio of the two ratios, which is prefer-
10 ably accomplished by taking the difference in logarithms
11 of the two ratios, resulting in another logarithm which i9
12 the log of the comparison ratio, or of what may be called
13 the comparison signal.
14 In the next step, a third comparison depends
15 upon what test is being run. Basically, it is a com~
16 parison of the ratio signal obtained from the second
17 comparator means, which preferably is the logarithm of
18 the comparison signal, with values that are stored in the
19 data storage means to determine the final asked-for result.
For good results in this last step, especially
21 when applied to MIC procedure, a distinction is made
22 between a growth state and a no-growth state. The
23 instrument determines at the output from the second
24 comparator means, a voltage level or logarithm value that
25 represents the extent of bacterial growth, when that
26 voltage level is compared to voltages that are obtained
27 from known sterile and growth controls, these voltage
28 values being stored in the data bank of the microcomputer
29 41. A first step here is to determine whether there is
30 an adequate voltage (logarithm value) difference between
31 the readings obtained from the sterile and the growth -~
32 control wells. This is done preferably by comparing the
33 ratios for the two wells, i.e. the products of the first
34 comparator means for the two wells, which are logarithms
35 of ratios of well readings vs. reference readings. The
36 comparison of the two control values is done by taking
37 a difference between the two logarithms. The resulting
38 difference is compared to a predetermined, stored value
- ~3 -
1 representing adequate growth-sterile difference for the
2 test. If there is an inadequate difference, this means
3 either one of two things, either that there had not been
4 sufficient growth to provide an adequate difference,
5 or that the sterile well had been contaminated and that
6 there had been growth there. In either case, the instru-
7 ment will display a reading such as "insufficient growth-
8 sterile difference", and the computer returns to the
9 beginning of the program. The operator then checks to
lO see which of these two possibilities is the one that is
11 present. If ~here is insufficient growth, it may be due
12 to a lack of time or because there was nothing to grow.
13 If there were contamination, that would show and be
14 readily detectable, and the test must be re-done.
Once the computer has established that there is
16 an adequate difference between the sterile condition and
17 the expected growth condition from one well to another,
18 the calculated logarithm values and their difference are
19 used for computation of a break point, or a limit com-
20 parison signal value. Preferably, the break point is
21 biased toward the sterile value to achieve more sensitivity
22 to growth detection, via a preselected fraction of the
23 sterile-growth logarithm difference. The break point may,
24 for example, be placed at 25% of the determined sterile-
25 growth difference (preferably a logarithm value as above),
26 added to the log value for sterility. For all wells
27 where there has been less growth than that r~presented
28 by 25% of the determined growth-sterile difference for
29 the test being conducted, then the concentration of
30 those wells is considered as inhibitory. For each drug
31 being tested, the concentration closest to the break
32 point, but on the inhibitory side, is selected as the
33 minimum inhibitory concentration value. Thus, supposing
34 that there are a series of wells of different dilutions
35 and that the operation is moving from wells of greater
36 growth towards those of lesser growth and toward the
37 sterile condition, then the minimum inhibitory concentration
38
,...... " . .. .. .. . . .
3~
; 1 is not found until the first well is reached which shows
2 less than 25~/o of the determined difference between the
3 sterile and growth control wells. In this way, a
4 "floating threshold" is utilized, i.e. one which is calcu-
5 lated from controls in the very test being conducted and
6 with the same organism being tested, rather than a fixed
7 threshold which has been calculated based on prior infor-
8 mation and stored.
9 Another important comparison which should be
10 performed preferably at least once a day, before series
11 of tests are performed, is an initial calibration step.
12 This initial calibration is in lieu of the empty tray
13 (or just filled tray) reading procedure described above.
14 Like that procedure, this calibration procedure is
important in that it enables the use of a light source
]6 which is not totally uniform Eor each photodetector,
17 but only generally uniform, and also the use of inexpen-
18 sive photodetectors which may not be uniform or totally
19 constant, over a long period of time, in their sensi-
20 tivity. By this procedure the light is first passed
21 directly (no tray) to all photodetectors) including
22 the reference photocell, and ratio readings (preferably
23 their logarithms) are taken as above and recorded. These
24 values are stored and give a relative base line or initial
25 calibration value for each photocell. All subsequent
26 after culture values (which are pre~erably logarithms
27 of ratios as above) are compared to these base line
28 readings, and expressed as "difference" (or log ratio)
29 readings. Thus, any differences in sensitivities of the
30 various photocells, or differences in light intensity due
31 to position, are "zeroed out" by comparison of after
32 culture ratios with initial calibration ratios, the
33 comparisons being separate for each well.
34
35 An alternate type of light source (Fi~. 8~:
36 Fig. 8 shows schematically an alternative
37 arrangement for passing light th~ough the wells 33 of the
38 sample tray 14 and detecting the resultant light intensity
7~36
- ~5 -
1 passing through each well. The apparatus of Fig. 8, which
2 utilizes fiber optics to transmit light,would replace the
3 form of light source and diffuser 26, 27 and 28 shown in
4 Fig.s. 2, 3 and ~. It would also eliminate the need for
5 a large plurality of photocells 32 in a matrix as shown
6 in Fig. 2, and would replace the multiplexing unit 39
7 (Fig. 4) with a substitute arrangement which selects one
8 cell at a time for receipt of a penetrating quantum of
9 light.
The apparatus of Fig. 8 includes a light source
11 221 and a reflector 222, directing light through a lens
12 223 toward a rotatable selector plate 224 driven by a
13 stepper motor 225. The selector plate 224 has a single
14 opening 226 (dashed lines) which sequentially directs
15 light to different fiber optic fibers 228 o~ a fiber
16 optic bundle 229. The stepper motor 225 is under the
17 control of the microcomputer 41 via the lines 100 and
18 112 (Figs. 4 and 6), in lieu of an to perform the same
19 function as the multiplexer 39 indicated in Figs. 4 and
20 6. The fiber optic fibers 228 of the bundle 229 each go
21 to individual testing wells 33 of the tray 14. The
22 fibers are indicated only schematically, as is the bundle
23 229.
24 Below the wells 33 are a second plurality of
25 fiber optic fibers 230 of a second bundle 231. Trans-
26 mitted light from each well is collected by a fiber 230
27 Of the bundle 231 and fed via a lens 232 to a single
28 photocell detector 233. A value corresponding to the
29 intensity of incident light is then fed to the filter 38,
30 then to the plus input of the differential amplifier 37,
31 as in the apparatus of the other embodiment described
32 above.
33 In order to provide a control or reference
34 value which may be fed into the minus input of the dif-
35 ferential amplifier 37 to represent a base light inten-
36 sity corresponding to zero bacterial growth, there must
37 be an optical fiber which always carries light through
38
- 46 -
1 a reference sterile control well, i.e. the well 33n of
2 Fig. 4, also indicated in the schematic representation
3 of Fig. 8. Accordingly, a single optical fiber 228n is
4 positioned at the lens 223 in such a way that it receives
S and carries llght continuously whenever the lamp 221 is
6 energized, i.e. whenever any of the wells 33 is being
7 tested. The fiber 228n extends to a position adjacent to
8 the sterile control well 33n as shown, and a receiving
9 fiber 230n carriesthe transmitted light to second lens
10 232n. The resultant analog light intensity value or
11 the control well is fed through the filter 38 to the
12 minus input of the differential amplifier 37, so that
13 the di~ferential ampliier yields a differential analog
14 signal corresponding to increaæed turbidity in the tested
15 well from bacterial growth.
16 The remainder of the system remains the same as
17 described above. The principal advantage of the form
18 illustrated in Fig. 8 is the use of a single light source ;
19 focused on the fiber optic bundle and a single detector
20 for all test wells of the sample tray, providing a more
21 uniform measurement over the matrix of test wells in ~he
22 tray. Light is transmitted through only two wells of
23 the tray at any given time: the well currently being
24 tested for turbidity, and the sterile reference well 33n.
25 The subsystem of Fig. 8 allows for close standardization
26 and easy calibration and checking.
27
28 A printout ticket for MIC (Fig. 9):
29 Fig. 9 shows a form of printout ticket 24 which
30 may be used in conjunction with the present invention,
31 with exemplary MIC susceptibility information and therapy
32 information. As discussed above, the apparatus of the
33 invention provides a graphic interpretive printout to
34 guide the physician's therapy, an example of this type
35 information being located in the right column of the
36 ticket 24. The computer algorithm translates the MIC
37 values (left column) to dosage ranges that would be
38 -
7~7~t~
- 47 -
1 necessary to achieve blood levels of each antimicrobic
2 drug to effectively inhibit growth of the organism. Fig.
3 9 indicates one form that the "therapy guide" information
4 may take. With this format, "-" indicates that the
5 organism tested is resistant to that particular anti-
~ microbic, and that no dosage of the antimicrobic can
7 affect the organism. "~" indicates resistance but that
8 the organism may respond to high intra-muscular intra-
9 venous doses. "++" indicates that the organism is
10 intermediate in sensitivity to the particular antimicrobic,
11 and may respond to higher than recommended doses. A
12 printout of "~" indicates sensitivity to the usual
13 recommended doses of the antibiotic, and "~ " means that
14 the organism exhibits a high degree o:~ sensitivity and
thus is an optimum drug with which to treat the infectious
16 organism. A printout of "****" tells the physician that
17 a dosage of that particular antibiotic necessary for
18 therapy may be toxic to the patient.
19
20 Use of the apparatus of Figs. 2-4 in bacterial identifi-
21 cation:
22 As discussed above, the sample tray14is prefer-
23 ably laid out in a rectangular matrix, which may comprise,
24 for example, eight rows and ten columns. Other arrange-
25 ments would be adequate, but a rectangular matrix is
26 space-efficient and convenient. In this method, the wells
27 33 contain various reagents.
28 Into the wells containing the various reagents
29 is introduced the patient bacteria sample borne within
30 a culture medium. This bacteria culture is uniformly
31 inoculated into each well, and this may be accomplished
32 by commercially available devices having a matrix of
33 prongs (not shown) arranged to register with each well
34 to be inoculated in the commercially available sample
35 tray 14 Of course, the reagents and the bacteria
36 culture may be introduced to the wells in the reverse
37 order, but for convenience, efficiency and reliability
38
~: ~ .. . . - , . .
: , . - , , . .: : . ::
~ ~ ~ 377~i
- 4~3 -
1 it is preferred that the reagents be introduced first.
2 Fig. 4 indicates diagrammatically the operation.
3 The lamp 26, reflector 27, and diffuser 28 are shown
4 transmitting uniform diffuse light through a sample
5 well 33a of the matrig of wells of the sample tr~y 14.
6 The well 33a contains one reagent, or group of reagents,
7 and one sensor (e.g.~ one reagent and one pH indicator),
8 inoculated with a controlled volume and known concentra-
9 tion of the microorganisms in a culture sample. The same
10 uniform dif~use light is also directly transmitted through
11 a filter 35 to a reference photocell 32n.
12 After an incubation period sufficient,to allow
13 some detectible reaction of the microorganism with the
14 reagent in the well 33a, in the event that there is a
15 reaction, a reaction product 36 results therein. The
16 light from the diffuser passes through this reaction
17 product 36 and through the bottom of the well and through
18 the filter 35 to a photocell 32a of the photocell matrix.
19 Here the intensity of the light is sensed and converted
20 into an electrical analog value corresponding to the
21 opacity of the reaction product 36. This opacity value
22 represents the intensity of color of the culture and its
23 reaction with the reagent, stemming from the net effect
24 of light absorption and scatter in the well 33_. At the
25 same time, the diffuse light passes to a photocell 32n
26 of the photocell matrix. Again, ~he sensed light intensity
27 is converted into an electrical analog reference value.
28 The photocell 32a is connected to a plus input
29 of a log ratio module 37 through a noise filter 38 and a
30 multiplexer 39 which functions to select each photocell
31 33 of the matrix of photocells in a prescribed sequence
32 under direction of a microcomputer 41. Thus, the pho~ocell
33 32a shown in Fig. 4 is connected to the log ratio module
34 37 only when the multiplexer 39 momentarily selects that
35 particular photocell. The reference photocell 32n is
36 connected to the minus input of the log ratio module 37
-~ 37 and provides a reference voltage which is subtracted from
38
~13~
_ ~9 _
1 the plus input to provide an analog differential output.
2 Thus, the light intensity signal emanating from the log
3 ratio module 37 is in the form of a reference voltage
4 which varies according to the opacity of the sample being
5 sensed, representing the increase in opacity of that
6 sample since inoculation. Each analog signal is trans-
7 mitted in its turn to an analog-to~digital converter 42
8 which converts the analog to a digital ~ignal and sends
9 it to the microcomputer 41.
The microcomputer 41 functions to correlate
11 differential digital values (from the ADC 42) represent-
12 ing bacterial reaction with the reagent for the various
13 wells.
14 The analog circuitry associated with the system
15 of Fig. 4 includes the noise filter circuit 38, the multi-
16 plexer circuits 39 which are included within the dashed-
17 line box, the log ratio module 237, and the analog-to-
18 digital converter 42 along with its related supporting
19 circuitry, all as described before in connection with
20 Figs. 1-6.
21 The logarithmic log ratio module 237 is prefer-
22 ably implemented as an Analog Devices type 756 or equiva-
23 lent, and the purpose of the log ratio module 37 is to
24 correct for variations in light intensity from the light
25 source 26. The light-variation-corrected analog voltage
26 output from the log ratio module 37 is supplied as an
27 output line 117.
28
29 Example of bacteria identification:
Bacteria that must be identified in the clinical
31 laboratory may be taken from a large number of body sites.
32 Wounds suspected of being infected, nose and throat cul-
33 tures, aspirates from abscesses, feces, and sputum speci-
34 mens are some of the more common sites from which bacteria
35 may be cultured. Normally sterile body fluids are also
36 frequently investigated for the presence of bacteria. In
37 suspected cases of septicemia, blood may be sent to the
38
~ ~ ~.3~f'7~i
- 50 -
l laboratory, and urine is frequently cultured ~or the
2 diagnosis of urinary tract infections. Additionally,
3 cerebrospinal fluid for suspected meningitis, pleural
4 fluid for suspected pleuritis, pericardial fluid for
5 pericarditis, and ascitic fluid Eor suspected peritonitis
6 may be sent to the laboratory for the culture, identi-
7 fication, and susceptibility testing of bacteria.
8 A nurse, physician, or technologist may obtain
9 cultures by either using cotton swabs, or directly
10 inoculating the specimen into a :Liquid medium. In the
ll case of a liquid specimen, such as blood or a body fluid,
12 the original material is introduced into a bottle or
13 tube of sterile nutrient broth media. For cultures of
14 solid structures, such as a wound, skin, eye, etc., it is
15 necessary to collect a specimen with a swab. Once
16 transported to the laboratory, the swab is streaked over
17 the surface of nutrient agar. Agar is a seaweed deriva-
18 tive that forms a gel. Molten agar may be poured into
19 a shallow, cylindrical glass dish (Petri dish) to form
20 a layer appro~imately five millimeters deep. When
21 bacteria grow on the surface of the agar, individual
22 organisms which are originally invisible to the naked
23 eye multiply to become colonies that are easily visible.
24 Each isolated colony is the aggregate "off-spring" of
25 a single bacterial progenitor. Thus, by utilizing a
26 single colony or a group of similar colonies, a pure
27 culture of bacteria may be obtained for susceptibility
28 testing or identification. In the case of bacteria
29 that were originally isolated in a liquid nutrient
30 broth, it is necessary to subculture these organisms
31 on agar plates in order to obtain pure cultures. It is
32 these isolated colonies that are subjected to identifi-
33 cation testing by the present invention. A detailed
34 discussion of specimen collection and preparation is
35 described in the American Soc~e~y o~ Microbiology Manual
36 of Clinical Microbiology, 2nd Ed. Lennette, E.H., Spaulding,
37 E.H., and Truant, J.P., Editors, ASM, Washington, D.C. 1974.
38
8~3
- 51 -
l In time sequence, once a culture is obtained,
2 brought to the laboratory, and plated on agar plates
3 for isolation, it is usually necessary to wait twelve
4 to eighteen hours for there to be sufficient colonial
5 growth for further testing. Once pure colonies have been
6 isolated, the medical technologist makes a preliminary
7 identification based on the colonial morphology and the
8 microscopic appearance of the bacteria. To assist in
9 this classification, the bacteria are stained with a
10 dye and iodine mordant together with a red counterstain.
11 If the bacterial walls have affinity for the stain, they
12 will appear blue and are referred to as "Gram Positive".
13 If the bacteria do not stain positively, the red counter-
14 stain will prevail; and these organisms are classified as
15 being "Gram Negative". A well-isolated colony is -trans-
16 ferred into eight ml. sterile saline which is supple-
17 mented with 0.02% Tween 80.
18 The saline suspension of bacteria is trans-
19 ferred into a plastic seed tray, and a transfer lid is
20 placed over the tray. The transfer lid contains plastic
21 prongs that are spaced in such a way that each prong will
22 pick up a small but uniform drop of bacteria suspension
23 and mate with the wells 33 in another plastic tray 14
24 that contain biochemical reagents. After the bacteria
25 have been introduced to the biochemical microtubes 33,
26 certain tubes (H S, lysine, arginine, and ornithine)
27 are overlayed with mineral oil to seal the reaction
28 mixtures from a~mospheric oxygen. The biochemicals
29 containing bacteria are incubated in a non-CO incubator
3Q at 35C. for 18-24 hours. The reactions may then be
31 read by the instrument 10, presently described.
32 The biochemical tests which are read and
33 interpreted by this invention span a wide range of
34 fermentative reactions. The following list expands
35 in detail these bioc~emical reactions.
36
37
38
. : . ,,: : ~ , . , . - ,.- :,. .. ... - .. . ~ : : ,
i``
3~
5~
1 Carbohyd ate Fermentation
2 The carbohydrates used with this invention are
3 dextrose, sucrose, raffinose, rhamnose, arabinose,
4 inositol, adonitol, and cellobiose. The fermentation of
S a specific carbohydrate results in acid formation. The
6 resulting drop in pH is detected by a phenol red indicator
7 changing the color from red to yellow.
9 Urea
Bacteria which produce urease split urea forming
11 two molecules of ammonia. Since ammonia is basic, the
12 resulting rise in pH can be detected by a phenol red pH
13 indicator changing the color from orange to red.
14
Indole
16 The metabolism of the amino acid tryptophane
17 results in the formation of indole which is detected by the
18 addition of Kovac's reagent. If indole is present, a red
19 color develops.
21 Lysine, Arginine, Ornithine
22 Decarboxylation of these compounds results in
23 an alkalization of the media which is detected by the pH
24 indicator bromscresol purple. A positive reaction is
brown and a negative reaction is colorless to gray.
26
27 Tryptophane Deaminase
28 Bacteria capable of deaminating tryptophane
29 produce phenyl pyruvic acid. In the presence of ferric
ammonium citrate, this reaction product produces a brown
31 color, whereas a negative reaction is clear.
32
33 Esculin Hydrolysis
34 The ability of an organism to hydrolyze esculin
35 is de~ected by ferric ammonium citrate in the medium, which -
36 reacts with the hydrolysis products to form a black pre-
37 cipitate.
38
,':' ' , ~ ' , ,` ' ., . '.' ' ! ' ' ' ' . ' ' ,' ' ', ,' ' '
~L~L3~7~;
- S3 -
1 Vo~es Proskauer
2 Acetoin is produced from sodium pyruvate and
3 indicated by the formation of a red color after addition
4 of KOH and alpha-naphthol.
6 O.N.P.G.
7 Beta galactosidase hydrolizes orthonitrophenyl-
8 beta-galactose, which liberates the yellow colored
9 orthonitrophenyl.
;''
11 Citrate, Malonate, Acetamide, Tartrate
12 The utilization of these substrates as the sole
13 source of car-bon for metabolism results in a rise in pH
14 that is detec-ted as a s~ift of green to blue by the pH
15 indicator bromthymol blue.
16
17 O.F. Carbohydrates
18 Oxidation or fermentation of a carbohydrate
19 results in acid formation. The consequent drop in pH
20 is detected as a shiftfrom blue or dark green to yellow
21 or light green by the pH indicator bromthymol blue.
22
23 Nitrate
24 The ability of an organism to reduce nitrate
25 to nitrite is detected by the addition of alpha-
26 naphthylamine and sulfanilic acid, which produce a red
27 color in the presence of nitrite. To confirm that nitrate
28 has not been reduced to nitrogen gas, zinc powder is added
29 to all negative tests to detec~ the pr~sence of unreduced
30 nitrate. This test is performed before the plate is read
31 by the instrument, and -the results are manually entered
32 when the instrument's display ~ueries the operator.
33
34 Starch Hydrolysis
Starch reacts with Gram's iodine to produce a
36 blue-black color. If an organism hydrolyzes starch, the
37 absence of starch is detected by the iodine yielding a
38 brown rather than blue-black color.
~3~36
- 54 -
1 Oxidase
2 Like nitrate, this test is performed "off-line"
3 and manually entered into the instrument on command. The
4 recommended oxidase test is the tetramethyl-p-phenylene-
5 diaminedihydro-chloride procedure described on page 679
6 of the second edition of the ASM Manual of Clinical
7 Microbiology cited above
9 MacConkey
MacConkey's agar is a selective medium that is
11 used to differentiate major groups of gram negative micro-
12 organisms from one another. Growth or no-growth on this
13 medium is manually entered into the instrument on command.
14 The organisms presently identified by this
15 system are gram negative bacilli. These fall into two
16 major classifications: enteric, or dex~rose fermentors;
17 and non-enteric, or dextrose non-fermentors. The follow-
18 ing list includes a number of organisms that are identi-
19 fied by the present system.
21
22
23
24
26
27
28
29
31
32
33
34
36
37
38
~7~;
- 55 -
1 D~ OS~ 9~ K~S DE~TROSE NON-EF,~ TEP~S
3 Escherichia coli Pseudomonas aeruginosa
4 E. coli indole neg. Ps. fluorescens
E. coli H2S pos. Ps. putida
6 E. coli urea pos. Ps. cepacia
7 E. coli adecarboxylata Ps. maltophilia
8 Shi~ella dysenteriae Ps. stutzeri
g Sh. flexneri Ps. putrefaciens
Sh. boydii Ps. pickettii
11 Sh. sonnei Flavobacterium meningosept.
12 Ed~ardsie~la tarda Flavo. species
13 Sal~onella enteriditis Acinetobacter anitratus
14 Sal. typhi Ac. lwofi
Sal. cholera-suis Achromobacter sp.
16 Sal. parat~Jphi A A. xyloso~idans
17 Arizona hinshawii Moraxella
18 Citrobacter freundii B. bronchiseptica
19 Ci. diversus Alkaligenes sp.
Ci. amalonaticus Eikenella corrodens
21 Klebsiella pneumoniae CDC Group II F
22 Kl. oxytoca CDC Group II J
23 Kl. ozaenae CDC Group II K-l
24 Kl. rhinoscleromatis CDC Group II K-2
Enterobacter aerogenes CDC Group IV C-2
26 Ent. cloacae CDC Group VE-l
27 Ent. agglomerans CDC Group VE-2
28 Ent. gergoviae
29 Ent. sakazakii
Haniae alviae
31 Serratîa marcescens
32 Ser. liquefaciens
33 Ser- rubidea - ~ -
34 Proteus w lgaris
Prot. mirabilis
.
36 Morganella morganii
37 Providencia rettgeri
J 38 Prov. stuartii
~ .
, : .
,. . ~ ;, . . "
, :.:~ .
: ~ .. , , , ,, ~, , , , , , :
- 5~ -
1 DE~TROSE FERMENTERS (continued)
3 Prov. alcalifaciens
4 Yersinia enterocolitica
Y- pestis
6 Y- pseudotuberculosis
7 Chromobacter violacium
8 Pasteurella sp.
g Past. multocida
Aeromonas hydrophilia
11 Vibrio cholera
12 Vibrio par-ahemolyticus
13 V. alginolyticus
14 Plesiomonas shigelloides
16 The data base in the present invention thus may 17 be the frequency of occurrence of twenty-one chemical
18 reac~ions with seventy-seven different organisms, or a total
19 cor.~bination of 1,617 probabilities.
21 The following three cases will be considered: -
22 1) A Pseudomonas aeruginosa infec~ion of the kidneys o~ a
23 patient with chronic pyelonephritis, 2) A case of Klebsiella
24 pneumoniae infection of the lungs in an alcoholic,
3) A case o~ Salmonella enteritis in a patient who owns a
26 pet turtle.
27
28 The first patient was a middle aged woman who has
29 had chronic urinary tract infections with flank pain and
fever for many years. She was sent to a laboratory where
31 a urine specimen was passed for bacterial analysis. The
32 urine was streaked onto an agar plate with a calibrated
33 platinum Ioop so that a quantitative estimate of bacteria
34 growth could be obtained. A growth of similar appearing
colonies were obtained that numbered over 100,000 per
36 milliliter. The organism grew on McConkey's agar, and was
37 both ni~rate and oxidase positive. Several simi.lar colonies
38
~ ~ .
~L~377
- 57 -
l were suspended in saline (salt solution) for evaluation b~
2 the ins~rument being described.
4 The second patient was an alcoholic who was found
unconscious and brought to a co~nty hospital. He subse-
6 quen~ly suffered pne~lmonia, and a sputum specimen was
7 obtained for bacterial evaluation. When the sputum cup
8 was sent to the laboratory, representative portions were
9 streaked onto various types of agar plates, and the next
day, colonies were noted that to the technologist did not
11 appear to be normal flora. A gram stain of these organisms
12 revealed gram negative bacilli, so the tec~nologist made the
13 decision for further evaluation. A suspension of the
14 bacteria was made in saline for further testing.
16 The third case was that of a grade school pupil
17 who had a sudden onset of diarrhea. Upon questioning, the
18 physician learned that the child had recently been given
19 a pet turtle. The mother was asked to send the child and
tur~le to a local laboratory so they could obtain stool
21 specimen from both the child and the turtle. The technolo-
22 gist plated representative parts of the stool on several
23 types of selective agar, and there were some suspicious
24 colonies that warranted further evaluation. These colonies
were placed into saline for further identification by the
26 present invention.
27
28 All of the three specimens had bacteria isolated
29 from them that contained bacteria suspicious for disease.
These bacterial colonies were placed into a saline suspension
31 and thoroughly agitated to obtain optimum dispersion. The
32 saline suspension was poured into a sterile plastic dish,
33 and a transfer lid containing a matrix o~ 96 prongs was
34 used to inoculate a drop of bacterial suspension into each
of 96 wells of a plastic tray. The wells contained anti-
36 microbic dilutions as well as biochemical substrates and
37 indicators. The trays were allowed to incubate o~ernight;
,A
' . ' .'
' ;: , " ' . ' ' ~ `.
- 5~ -
1 and the next day, following proper calibration, were placed
2 into the instrument for automatic identification. The
3 instr~mlent made a reading of each of the wells through
4 appropriate filters,and a table residing in computer memory
interpreted these digitized voltages as either a positive
6 reaction or a negative reaction,, The computer then went
7 through each of the seventy-seven possible organisms and
8 computed the probability of occurrence. With the three
9 present organisms, the frequencies and results obtained
by the instrument are summarized in the following table:
11
12
13.
14
16
17
18
'19
21
22
23
24
26
27
28
29
..
3~ ,
32
33
34
36
37
.., 38
., .
.377~
- 59 -
1 PS.AERUGI~IOS~ K.PNE~IONIAE S EIITERITIDIS
2 BI0C~MICAIJ Fl~Q. R~SULT ~ ~ . RESULT
3 De~trose 00.1 neg 99.9 pos 99.9 pos
4 Sucrose -- 99.0 pos 00.6 neg
5 Sorbitol -- 99.4 pos 95.0 pos
6 Raffinose -- 99.2 pos 3.0 neg
7 Rhamnose -- 99.3 pos 90.0 pos
8 Arabinose -- 99.9 pos 99.9 pos
9 Inositol -- 98.0 pos 30.0 pos
10 Adonitol -- 90.0 pos 00.1 neg
11 Cellobiose -- 99.O pos 5.0 neg
12 Urea 50.0 pos 90.0 pos 00.1 neg
13 H2S 00.1 neg O0.1 neg 95.0 pos
14 Indol 00.1 neg 6.0 neg 1.0 neg
15 Lysine 00.1 neg 98.0 pos 95.0 pos
16 Arginine 95.0 pos 1.0 neg 50.0 pos
17 Ornithine 00.1 neg 1.0 neg 97.0 pos
18 Tryptophane 00.1 neg 00.1 neg 00.1 neg
19 Esculin 00.1 neg 99.O pos 1.0 neg
20 V.P. 00.1 neg 90.0 pos 00.1 neg
21 O.N.P.G. 00.1 neg 99.0 pos 1.0 neg
22 Citrate 95.0 pos 98.0 pos 90.0 pos
23 Malonate 90.0 pos 94.0 pos 00.6 neg
24 OF Glucose 95.0 pos -- __
25 OF Maltose 00.1 neg
26 OF Xylose 85.0 pos -- __
27 Acetamide 90.0 pos
28 Tartrate 00.1 neg _ __
29 Starch 00.1 neg -- --
30 Nitrate 75.0 pos -- --
31 MacConkeY 85.0 pos
32 Oxidase 99.9 pos --
33
34
36
37
rJ
. .
~L3t^~7~6
- 60 -
1 The table o~ probabilities stated a~ove are
2 relevant only to positive reactions. If, in fact, the
3 reactions were negative, the result would be 1 0 minus this
4 probability. For instance, with the sucrose reaction of
S.enteritidis, the probability for a positive reac~ion would
6 be 00.6%, but since the reaction was negative, the actual
7 probability is 99.4%.
g Each of the actual probabilities of each biochemical
reaction are cumulatively multiplied for each of the se~enty-
11 seven organisms in the data base to obtain the net probability
12 for each organism. The organism with the highest net
13 probability is the most likely organism. If the net prob-
14 ability of the most likely organism is less than 1 x 10 6,
then the instr~ment flashes a warning to the operator that
16 the frequency is low, and possible technical errors should
17 be checked out. If the net probability is greater than this
18 value, then the instrument proceeds to normalize. This is
19 done by dividing each of the organisms' net probabilities
by the sum of all of the net probabilities. Thus, an
21 estimate of the probabilities relative to each of the
22 organisms is obtained. In the case of the three examples
23 described above, these normalized probabilities are greater
24 than 95%, so the instrument proceeds to display the most
likely organism's probability on a thirty-two character
26 Burroughs display and print the most probable organism's
27 genus and species on a Practical Automa~ion Model DMTP-9
28 alpha-~umeric ticket printer.
29
A specific example of procedure
31 (Figs. llA, llB, and llC):
32 The flow sheets llA, llB, and llC illustrate
33 procedure according to the present invention, after the
34 type of tray-checking etc. shown in Figs. 7A, 7B, and 7C.
36
37
38
~.
.3~'7~
- 61 -
1 Thus, when the device is instructed to commence,
2 at start 300, the apparatus determines at 301 whether there
3 is a tray 14 containing biochemicals in place or not. If
4 there is no such tray 14 the light transmission will be
the same for all wells and such a known transmission will
6 give the answer "No"; then the computer returns at 302 to
7 the main program. If the light transmissions result in the
8 answer "Yes", then the dextrose voltage is read at 303 and
9 the value compared at 304 with t:he stored positive-negative
table in the computer. If the answer is positive, the
11 organism is a dextrose fermenter and each of the biochemicals
12 in receptacles 1 through 21 are read in at 305.
13
14 If the answer is "No", the organism is a dextrose
non-fermenter, and the nex~ stage is for the operator to
16 enter manua~ly whether the organism grew or did not grow on
17 MacConkey's agar, at 306. This is an "off-line" test. If
18 it grew, the operator enters "1"; if not, he enters "0".
19 The processor waits for this input and stores the data at
307. Next, the operator manually enters "1" if oxidase is
21 present or "0" if it is not present, as determined by another
22 "off-line" test, queried and entered at 308; the processor
23 again waits for the data and stores it at 309. A third
24 "off-line" test used when the organism is not a dextrose
fermenter is the nitrate test, and the operator is queried
26 and enters the nitrate at 310 as either "1" or "0", depending
27 on whether it is present or not, and at 311 the processor
28 again waits for the data and stores it. After that, the
29 biochemicals receptacles 10-27 are read in at step 312.
31 Thus, if the dextrose test is positive, the results
32 for biochemicals 1 to 21 are read into the program at 305;
33 if the dextrose results are negative, the presence or absence
34 of MacConkey's growth, oxidase, and nitrate are determined
and ~hen the results for biochemicals 10 to 27 are read in.
36
37
38
. . , ~ , , ,
r --
~37~
- 62 ~
either event, the next step (after either 305
2 or 312, whichever is applicable) i5 the step 3~3 7 where the
3 stored data in the tables is used to determine for each
4 biochemical still pertinent (l to 21 or 10 to 27) whether
each is posi~ive or negative. Step 314 then stores the
6 positive and negative indications in a table or as packed
7 data, in terms of probabilities. If any reaction is negative,
g then the probability used is 1.000 minus the actual negative
9 probability, as box 315 shows. E.g., a negative probability
of 99.5% is stored as 0.005. At this point, in box 316,
11 the biotype is calculated and stored.
12
13 The next step 317 (Fig. 11B) multiplies the prob-
14 abilities of each taxon and accumulates the sum, and then
at step 318 sets up a table of non~normalized probabilities
16 for each ta~on. From this, the computer then sorts at 319
17 the three organisms with the highest probabilities.
18
19 If at step 320 the most frequent probability found
is less than 1 x 10~6, step 321 displays to the operator
21 "VERY RARE BIOTYPE" and instructs the operator to call the
22 company that provides the trays, and then the device returns
23 at 322 to the main program for this answer is unacceptable.
24 If the answer if "No" then the comparator determines at s~ep
323 whether the most frequent organism is greater than
26 1 x 10- 6 but less or equal to 1 x 10- 5 ~ If the answer is
27 "Yes" the display at 324 says "RARE BIOTYPE-PRINT?
28 (1 or 0)". If the operator wishes to go ahead and print
29 this information he presses "1" on the keyboard, steps 325
and 326. If he presses "O", the computer returns to the
31 main program at 327.
~- 32
33 If he presses "1"~ or if the most probable
34 organism has a probability greater than 1 x 10- 5 ~ then the
computer normalizes the three most probable organisms at
36 step 328, by dividing the three highest frequencies by the
37 sum of all the frequencies.
38
-
:ll13 77~ !r
~ 63
l In Fig. llC, the outpllt from step 328 is dealt2 with. If the most probable organism has a normalized
3 frequency between .950 and .999 (as asked at step 329), then
4 the machine prints that one (or ones) out at step 329 in
terms of probability percentage (e.g., 98.21%) and returns
6 to the dextrose positive flag at 331.
8 If the most probable organism has a normali~ed
9 frequency between 0.850 and 0.950 (step 332), that organism
and its percentage are printed out at step 333, and the
11 program goes to step 331 to determine again w~ether the
12 organism is a dextrose fermenter or not.
13
14 If the response to both steps 329 and 332 is
negative and if the most probable organism has a probability
16 bet~een .75-.85 at step 334, then it is printed out at the
17 step 335 and the percentage indicated, and the program at
18 that point goes to step 331 for dextrose determination.
l9 If the relative probability of the most probable organism
is less than 75%, the display first says "LOW SELECTIVITY
21 RECHECK" at step 336, followed one second later by
22 "OOOOOOOOOOOOO--XX.X%" at step 337, followed in ~urn one
23 more second later by "STILL WANT TO PRINT? (1 OR O)" at
24 step 338. If at step 33~ the operator does want to print,
he presses l'l't at step 240 and the information is printed,
26 followed by sending the program to step 331 for the question
27 of whether the dextrose is posi~ive or negative. If the
28 answer is "no", he presses "O" and returns to the main
29 program at 341.
31 The step 342 asks whether the dextrose is positive,
32 and if the answer is "yes", then the program goes via an
33 output line 342. If the answer is "no" then the org~anism
34 is evaluated for its sensitivity to the antibody Colistin.
Comparison of Colis~in with expected result for the organism
36 is made at step 343 and then at step 344. The program looks
37 up a ta~le to see whether the result is correct. If not,
38
- 6~ -
1 it displays "R~CHECK ID & COLISTIN DISAGl~E" and goes to
2 the output llne 342. If the answer is "Yes", a similar
3 procedure is performed at steps 346, 347, and 348 with the
4 antibiotic Nitrofurantoin.
6 A "Yes" result leads to step 349 where the most
7 probable organism and biotype number are printed. The output
8 from de~trose positive along line 342 and from the two
9 recheck steps 345 and 348, also go to this step 349.
After that has been printed, the computer returns to the main
ll program at 350.
12
13
14
16
17
18
19
21
22
23
24
26
27
28
2g
31
32
33
34
36
37
38
~3~7
- 65 -
]. TABIIE 0~ PROGP~M FOR ANTIEIOTIC SUSCEPTIBILIT~ TESTING
_
3 The following l.isting constitutes the program ~or
4 antibiotic susceptibility testing in hexadecimal code
~or direct loading into the programmable read only memory
6 142. -
8 Address _ro~ram Instructions
12 .: ol oo ~C,E :~O.CO'; Gr 'C5 61- ,C4 85 ,2C ~.7,.C5''6F, O,7,,~6 .?F ~.73
,~0110 06, C6'O4- E7 0.7- 6r 0~ -85.. OF...A7:'O0 8~, 0D.^.,7.,C;7 ',~
13 i 01?.0 2r)'A7 OV '.,r OD ~6' FO A7 OC .86 OD A7~ O"a,5F C~r' '3G I
14 , Ol3O~,Fr A7 CE .'C5 2C E7~ OF .7F~ 8? E;~ 7F ;~7 E9 Cl C~; 01l
..0l~:O 7E Fl 3,~ Cl Cl 01 Gl CI 01 01 Oi' OI 01 01 01 ~CI~
150l5~ bl Ol Ol Ol Ol Cl i~ 37,Dl '7F. ~7 EO 7F 87 ~l 4r-'
16 ~, 0150 ~? 3D ~:3 ~4 FO,,26 b5 .CE F5;,i,4 43 2C .CC ~5 ~,. '.~-
0l70 2~ 43'g4, FO 2? 07 C~ l~5.'.6l ?. ~? Dl,:39, r76 47 8D,
17 ' 0l:3O Ir, 43 B4~,F.O. 27,.0~ CE rG .IO'.. B7.. 5?.EO ,20 07lCE~I'6'
18 , Cl9O IF,~I3,,~,7~:3r E1,~ D,D F3 36 BD .F2 FS B.D.. ,r2, cr. 39 '37
,~ 51~0 rJO 06. C6, 5O'ED F2 V9 36 50 C4 BD. F2 F5.B5 ,C7O C4'
19 . 013,0 ,39 01 Cl C~ D .F,Q~ 56 36 37.~.DI,,2.G' 5C CE F5 D4 ~D
c I cn F2 4f~ 20 .C3 t;D F2 50 V6 57 D5 27 03 7E F2 90 j~36
OIDO ~7 DG 2; FO r;D r3 35 rV~D ~2.FS ,C~. F6' 2E V~ E2 CF
21 ; OIEO ,~6, Of~ BD r2 r3~ CE rS4 00 36. 57 EO 25, 03 C_ 8~ .0
. .'OIFO.36 j'7'r)5 27 b3 7E F2 .98 .3D F3 60 E;5 87 D5 27 IS'.:
23 :0200 ~0 ~7 EO 27 03 7F.:~7.. E8 B6 .3.?. ~ 1 .'2.7.03 7F j'37 E9
0210 7E F2 9~ 01 Ol,.E6i7 ~0 .2? Q3 B?: 5? E3 36 ~7 El
24 . 0220 27.. 03 ~7,:,87 E9 CE F5 ,'3E~'~33 r''2. ~1'7 'v~j. g7 '3:5 .27 .,.
2t 0230 CE.'F5. 9V,3D,F,2,,,47.:36; ~:7.D4 ~7` F8 ,C,E ,F5 29 Vj~ F2,
~:~.G24O 47 B~ 87 ~D6 27- 03 ~7E ~FO ~B.1 ~5 ~37 ~D7~26 03 ~7E Fl ~
26 l 02.50 3v, 01 01 .01. ~3D FO 43 æ6 57 :Dl 2? 03.7r Fl 28,B6.
!.~0260 !37 EO ,21,7,OOS v6 87 E8 26' lO CE FS JjI~ ~7~ F 1 ~ n8 E'~6
27 1 027 0 ~r37 .EI 27.. ''F5 v6 87 ~9 27 FO 86 C~3 P.D F2 vD Cl. F5
28 L ~O-n30 . A9 . '3D F2 47 B6 87 D5 27 b3 7E r 2 .9~ ~6 ~37 D~ 27.
029b FO CE~r5 C9 BD F.;2 47''.~6 ~37 D5 2.7; 03 7E F;2 .9S B6,
29 ~;. 0 2.~ 0 ~,7., D7 . ',27;.: F.0 ~ 6 ' FFr.~7 ~ i, AO, B7~8,7 . ,A3 ~7., ~ 7 A ! ~ n
:~ 02~30 F,2 Fl~ :~.D:~F3. J-17 7E F6 .FE. bi 0101: 01 01. 01 01 01
02CO Oi OI ;oi ol, oi o:i ol: bl: ol oi oi oi :ol :!Oi.;OI `01
31 C2DO of~:o~ ol .ol .oi :o.l ol :oi bl:,ol -oi OD.ol; bl oi ol .
32 02` 0`01 Oi 01^ Ol, OL. ~;1 01 jO 01 i I 1 O g
3~ .
36
37
38
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" . ' , ~ ' .'' . . :
:; ' ' ' ` : , ~ ' ' ' . '. ' ,
.
~ " ' ' ,' . ' : ' ' '
- 66 -
<IMG>
- ~13~'7~6
3 ~.~,,r~,~ ,.q~.~a~s~ ~r~ }-~r;-~s~ rq~ ~t~ f~
4 ~06CO:.,03 lO.`~i9 4E'.53,.45' S2 5'4'?0,.5il Z15,,,,53'5,4 2G 54'52'
oSlo~ili 5$.:.05..'.i'5,,.49,4E~53 45-52''54 20,~i3;41.'/1C 43.42
1.0520 52 ~1l 5~1 ~15 20 5~1 52 ~il 59 00 20 4~ ~1~ 5~ 5~1 20
~,06.30;54.45,53'54 20,50.52 45 53 53 20 52 55 4~ 20,4F
6 ;.C6'lO.S~ ~0.. ~13 ~ IC ~9,42 5~ 5~ ~15 O".'l~
7 ,,.065Q 4? 4~ 52, ~! ~ 54 49 4F 4F. 20 52 45 5.1 55 49 52 45
~.06~0 44 Q7 11 55 4E 4? 44'45 4E'54',~i~ 4G 49 45 44 2a
8 r:CG7,0 54 52 4:1 59 07 11 54'52 41'59 20'~19 4~ 20 42 41
9' 106~0 43 4~.57 ~1l 52 4~1l53 OD OS 45,:'52 52 4F 52 OA 0
,0~90,52,,,45:4D'4F:.55.il5 20' 54'52 41'59,0A OC 43 4C 41
~,'C5AO''53 ~S 20;~ 52'41 S7~ 45 52.01.IT' 53 45 54'20 50,
1.06~0 4~1 54'49,45.'4E 54 2U 49 44 20 41'4~ ~14 20 . 4~
11 ~OGCO.53 45 52-54 20 46 i1~ 5~ 4D'0~ 0~'50 52'45'5?,.53
12 ~.,C5nO'20.. 52:55.4~.,02.. 0~,.. 50.. 52-.45'53,,.53,20.. ~13.4;1 4C 49
-0,~'0 ~12 52',41`5~~'45 06 '13 4~ , 53.,55 4~'46 ~ 3'~
13 ~0~10 4~ 4T 54.,2,0.47.52 4~ 57 5~1 4~ Of~ 1~; 53~54 45_.52
1~ '0700.4~ 4C.45 20.~i3 4F .~ 54 41, 4D 4~ 4 r iii 5iJ ~;5
~0710 00 OD..~i.7..52....41.4D..20 50:4F,53 20 54 52",.4,1 59.CC
L0720-OD.47.52.41 4D ~'0 4T~. 45.4i 20 54 52 41.,59,, 0!; Q4
16 ~.0730 57 ~ i9 5~1 51 2~ 25 6A 12 ~,AL 64 AOl32.~LC 1~ r~C;
1.0740 RA 00 4^ ob 2A OG'lA oo ~A, 50 pA 25 OA 12 CP 05
17 r,o750 60 ~ 30,4A 15 2A.7G..... A0 35 AO:,19:A0 9A 5C 2~j.31
~75C 3A 43:4C 54' 5C .05 1~ 2i 29',32 3~ 44.,4C 05 23 31
18 1 0770 3A.42 ilB 5ij 5C 05 2~ 30 3.q 43 4~ 54,5C 05 00 0~
19 07~0'11 19:22'2~ 0~ 2C'29'32 :3~ 43 ~IC 5~1 OS,I~ 20
07~0,29 '3~'3~ '3'4'C 05'2r3 30' 39"4~ '5~'5C Q5 CC'CS
loi.~o 10 Ir.23 2~ 33 'C5 70 73 31 ~A ~3 9C'A4 05 2'i 30
21 1'0730 3~ ~12 4~ 5'4 5C 05 2r~ 3'1 3~ 42'4'~ 54 5C 05..... 2J ~l
107co 3.4 43'4Y..54 5C 05 39 42, 4B 53,5C 64 GC C~ .21
22 ~07~,0 2~ 32'3~, 4'h.'`4C.05 2D'31'3A 42 43 54 5C 05 2~.30
~Ci_C 3R 43,42 54 5C~5:0C~O~ .22~2i~33 05 OC'03
23 iO7F0 10~ 3.2B'33 05 70 '~ P. ~3,,?6 'A~l C5 C~ ~:
iO300 50 P6 ~7.E0 25 03 CE,.54'FO F~ ~7 D~ 9D F3 60 3G
!oZ10 ~7 ri5 27 03 7E F2 9~.CE ~4 00 D~ 8'7 EO 2G C3.C~
¦'0020.84 AO ~ oi.;.r.o.s7.21~ o i: ~o 31 0.~ 2~ 05 C~ F5 FA-
26 iC~qO 7~ Fl 2~ Cr AO 5F 2A-O'1 i~o CE F5 ~5 ~1 o.r~ 2D
27 ~OS40 EF:~ F3 ~C.7E Fi 25 01 01 C! 01 31 Cl OI 01 01
, I G~5C 01 Cl 01 01 01 Ol.Ol pl 0l 01,01 oi ol bl ol ol
28 ! 0~63 ! ol ~ol Ql .bl; o.l 01. ai .ol ol~ 31 ol Ql ol .ol pl.;
29 1'.0~7').01 01 0! C1.01'01.`01~01... 01 01 01 01 ~ 1.-C1 OI
'O~'.O 0,I,'C1,01"0.1,.01,;''01''01j'~01.01 01i''01 .01"'0,r 01 01,01
.G~?C 01.0,1. 01 01 OL Oi 0.1'01 01 01 .0l 01,GI, 01 01 'Cl.
O~RG 01,bl OI~O'i 01 bl.Ql 01 01'01 01 01 01~01 01 .01
. . . . O ~ i ~ O ~ O I O I , O I O 1 . O I O 1 0 1 0 1 . p 1 0 ,1 , o, i ~ ,o i i C) I O i ~ O I
32 ~.C~. 01.,.'01'0.1''01'01,-01' 01 01:01'.01.C1.01 ,0,1~CI Cl OI -, '-o.~Dn .oi ~o~i ol o.I :o.i..~i ol ol oi ol` ol; c! ol~ oi 'OI'''C,1
33 -OSEO 01'01,01 0'1'.Oi.b1 01:'01.... 0'i Oi.01:01~01 .,01 01"01
34 i'o~o Ol':Ol`'Oi':n'1.""01''01' 01 0'1:'01'01 nl 0.1-01,01'Gl 01
36
37
' 33
,, ' , : :i
~ . : . : .: .. . .
- 68 -
1 Concerning Comparisons
2 As indicated above, the apparatus of ~his inven-
3 tion is capable oE performing various types of compari-
4 sons. Any specific comparison cLepends upon what method is
5 being used and which types of comparison are appropriate.
6 In some instances, there may be only one compari-
7 son, the specific type of comparison depending on the
8 particular apparatus or particular method being used. For
9 example, it is advisable to relate or every sample the
si~nal received from each well with a signal of a reference
11 transducer. Such comparison negates the e~fect of the
12 variation of light intensity or power fluctuation with
13 time.
14 A second type of comparison is often made, in
15 addition to the first one. This may be considered as
16 a type of calibration procedure aimed at negating the
17 variation of response of the different photosensors. The
18 comparison may be achieved by storing the signals from
19 each of the photosensors before the tray is introduced,
20 and then subtracting from this the corresponding signals
21 generated by each of the wellsafter the filled tray with
22 its cultured samples has been read. This technique of
23 eliminating transducer-to-transducer variation is impor-
24 tant.
25 Further refinements, which are not necessarily ~!:
26 crucial, may be added to eliminate further well-to-well
27 variation. For e~ample, variations in the plastic trays
28 or their contents may affect the accuracy of a reading.
29 One way to eliminate this problem is to calibrate with
30 an empty tray instead of calibrating without any tray in
31 the holder. A single empty tray may be used, assuming
32 that all the trays to be used are substantially identical.
33 Another approach is to compare the wells of each individual
34 tray when empty with the results obtained after filling
35 them with liquid and culturing the liquid. This is more
36 time consuming and not usually necessary, but it is more
37 accurate. With suitable multiplexing wired into the
38
'.'
~3~B~
69 -
1 device, however, this becomes quite practical. rrhus,
2 it is possible to eliminate the variations in the
3 signal fluctuating with time, to eliminate the vari-
4 ation of one sensor versus another, and also to compensate
5 for tray-to-tray and well-to-welL variations.
6 A third type of compar:ison may be used for
7 certain tests, such as the MIC test, where the signal
8 level indicating bacterial growth is differentiated
9 from the signal level indicating no growth. This may be
10 accomplished by comparison between various wells on the
11 tray; that is, some wells may be control wells or sterile
12 no-growth wells, in which there is no growth or which
13 are inoculated with suitable inhibitors. There is a
14 possible interpolation between the values of growth and
15 no-growth, as discussed above. Alternatively, by experi-
16 mentation, one can determine a signal value that dif-
17 ferentiates between growth and no-growth, and this decision
18 point may be used instead of one derived by controls on
19 board each tray.
Some of the claims which follow specifically
21 identify the types of comparisons made, while others
22 merely call for suitable comparisons to be made or for
23 apparatus which make these comparisons possible.
24 The above described preferred embodiments provide
25 apparatus and a method for automatically determining the
26 minimum inhibitory concentration of a plurality of
27 different antibiotics necessary to stop growth of an
28 infective organism being tested. Minimum inhibitory
29 concentration information is also transferred to dosage
30 information by the apparatus and method of the invention.
31 The required time to perform such a test is greatly reduced
32 in comparison to other methods, a great deal more infor-
33 mation is provided, and accuracy is improved. ~arious other
34 embodiments and variations to the preferred embodiments
35 will be apparent to those skilled in the art and may be
36 made without departing from the spirit and scope of the
37 following claims.
38 We claim:
