Note: Descriptions are shown in the official language in which they were submitted.
Background of the Invention
The invention relates to measurement of the suscep-
tibility of bacteria to different antimicrobic drugs, with
automatic quantification of the susceptibility to each drug,
so that a physician may select a drug that will most effectively
treat an infecting bacterium and choose the appropriate dosage
for effective treatment.
The physician usually has a choice of about twelve
to fifteen types of antimicrobial agents for treating the forty
to sixty groups of pathogenic bacteria. Many of these agents
are ineffective against a given bacterial strain, but normally
some of them will be appropriate for treatment. In order for
the physician to choose the best antimicrobic, it is necessary
to isolate the pathogenic organism in the laboratory and then
test it against a panel of drugs to determine which drugs
inhibit growth and which do not. Ideally the doctor should
receive susceptibility information the same day the culture
is taken, since it is usually necessary to initiate therapy
immediately. Unfortunately, it currently takes one day to isolate
an organism, and it has required another day to test the
susceptibility of the organism to the antimicrobics. Therefore,
it has been customary for the physician to institute therapy
based on an educated guess at the time the patient is first
seen. If the sensitivity studies completed two days later
indicate that the guess was incorrect, therapy is changed to
the proper drug.
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Clearly an important goal in automating antimicrobic
testing would be to diminish the time lag between the initial
culture and the obtaining of sensitivity information. An
estimated 30 million antimicrobic susceptibility tests are
performed annually in the United States by labor in~ensive
manual methods. In addition to the potential economic advantages
of automation and obvious advantages ~o the patient in receiving
only the proper treatment, one could also anticipate better
precision, quality control and objectivity.
The most frequently used technique to measure
antimicrobial susceptibility has been the standardized
disc-diffusion method described by Kirby and Bauer (Bauer, Kirby
et al., "Antibiotic Susceptibility Testing by a Standardized
Single Disk Method", American Journal of Clinical Pathology,
1966, Vol. 45, No. 4, p. 493). By this method, isolates of
bacteria are grown in suspension to a standardized concentra-
tion (usually determined by visual turbidity) and streaked
onto nutrient agar (culture medium) in a flat glass Petri dish.
Paper discs impregnated with different antim~c^Lobial materials
~a are placed upon the agar streaked with bacteria, and the drug
is allowed to diffuse through the agar, forming a gradient
halo around the disc. As the bacteria replicate, they form
a visible film on the surface of the agar, but in the zones
surrounding the antibiotic-impregnated discs, growth is inhibited
if the organism is susceptible to that particular antimicrobial
agent. Since a concentration gradient has been established,
the zone of inhibition around the disc is roughly proporitonal
to the degree of susceptibility. Typically, the laboratory
classifies an organism as "sensitive'l', "intermediate", or
3Q "resistant" to each drug in the test panel. Thus the results
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establish a characteristic profile or "antibiogram" for that
organism.
The Kirby-Bauer disc-diffusion method has the
advantage of simplicity, but it suffers from several drawbacks.
One problem is that of time efficiency. In order that the
initial inoculum become visible on the Petri dish so that
zones of growth can be distinguished from zones of inhibition,
the bacteria numbers must increase by several orders of
magnitude over the original number. However, for determination
of whether or not the organism is growing in the antimicrobial
milieu, which is the only information required, a period that
would allow doubling of all the organisms should be theore-
tically sufficient with suitable detection equipment. For
most Gram-negative organisms, the doubling period is between
twenty and thirty minutes, following a lag phase. Therefore,
an automated system should be able to distinguish growth
within a thirty-minute period.
Another difficulty with the Kirby-~auer disc method
is that of standardization. If an organism is "resistant",
does that mean that it cannot be treated with higher than
normal doses of the microbial agent? Also, how does this
information relate to a site in the body where the antimicrobic
is concentrated (such as bile) or decreased in amount (such
as cerebrospinal fluid)?
To answer these questions, quantitative data are
necessary. To obtain quantitative results, it must be deter-
mined that minimum concentration of a drug will inhibit the
organism's growth. This quantitation of susceptibility is
~Z;~Ç6Z9
known as minimum inhibitory concentration or MIC. The
MIC may be determined by making serial dilutions of the drug
in agar or broth, and then inoculating each dilution of each
drug with a standardized suspension of bacteria. Since the
test procedure may involve as many as 70 to 80 individual tubes,
it can become a formidable task if the test is performed in
individual test tubes on a macroscale. Systems are available
in which the individual dilutions of antimicrobics are made in
plastic trays containing small micro-tubes. (Marsh and MacLowry,
"Semiautomatic Serial-Dilution Test for Antibiotic Suscepti-
bility", Automation and Data Processing in the Clinical Laboratory,
Springfield, Illinois, C. C. Thomas 1970). Organisms can be
inoculated in a single step using a multi-pronged template.
Thus, setting up the test is simplified, and it takes slightly
less time to provide quantitative data than qualitative
Kirby-Bauer information. There are now semiautomated devices
that dispense antimicrobial solutions into the microtubes.
Trays of microtubes are also commercially available with
frozen solutions in the tubes, and the Gram-negative anti-
microbial panels have been combined with biochemical tests
to identify enteric bacteria as well as to determine their
antimicrobic susceptibility.
Although MIC results give quantitative information
~hich allows consideration of multiple doses and multiple
sites, the MIC numbers in themselves can be confusing to the
clinician. To use MIC data correctly, a physician must refer
to tables of achievable antimicrobic levels as a function of
dosage and body site. Therapy will be effective if the
achievable drug level for a particular dose and site in the
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body is two to four times the MIC. With the present invention
described below, such interpretation of MIC data is accomplished
by a computer, which compares the MIC with a table of achievable
drug levels at different body sites and different doses.
Optical detection methods have been suggested and
have proven to be powerful tools to measure bacterial growth.
A laser light-scattering system can have the sensitivity to
detect a single bacterium. Optical methods measure the presence
of bacteria either by nephelometry or turbidity measurements.
I Nephelometry measures the ability of the bacteria particles
to scatter light, and the detector is aligned at an angle to
the axis of ~he light source. Turbidity measures the net
effect of absorbance and scatter~ and the transducer is placed
on the axis of the radiation source. Nephelometry measurements
are significantly more sensitive than turbidity measurements~
but since the nephelometer measures only that fraction of light
scattered by bacteria, the signal to the detector is small, and
both light source and transducer amplification must be corre-
spondingly large.
Automation in microbiology has lagged far behind
chemistry and hematology in the clinical laboratory. However,
there is presently an intensive effort by industry to develop
this field. The best publicized devices for performing automated
antimicrobic susceptibility testing use optical detection
methods. A continuous flow device fo~ detecting particles
0.5 micron or less has been commercially available since 1970;
however, probably due to its great expense, it has not been
widely used in the laboratory. Other devices using laser
.,
light sources have been suggested but have not proven commercially
practicable. Recently, the most attention has been directed to
three devices discussed below.
The Pfizer Autobac 1 system (United States Patent No.
RE. 28,801; "AUTOBAC" is a registered trademark of Pfizer, Inc.)
measures relative bacterial growth by light scatter at a fixed
35 angle. It includes twelve test chambers and one control
chamber in a plastic device that forms multiple contiguous
cuvettes. Antibiotics are introduced to the chambers via
impregnated paper discs. The antimicrobic sensitivity reader
comes with an incubator, shaker, and disc dispenser. Results
are expressed as a light scattering index ~LSI), and these
numbers are related to the Kirby-Bauer "sensitive, intermediate
and resistant". MIC measurements are not available routinely
with this instrument. In a comparison with susceptibilities
of clinical isolates measured by the Kirby-Bauer method, there
was 91% agreement. However, with this system some bacteria
strain-drug combinations have been found to produce a resistant
Kirby-Bauer zone diameter and at the same time a sensitive LSI.
The Auto Microbic System has been developed by
McDonnell-Douglas to perform identification, enumeration and
susceptibility studies on nine urinary tract pathogens using
a plastic plate containing a 4 x 5 array of wells. The
specimen is drawn into the small wells by negative pressure and
the instrument monitors the change in optical absorbance and
scatter with light-emitting diodes and an array of optical
sensors. A mechanical device moves each plate into a sensing
slot in a continuous succession so that each plate is scanned
once an hour, and an onboard digital computer stores the optical
data. The system will process either 120 or 2~0 specimens at a time. One
can query the status of each test via a CRT-keyboard console, and hard copy
can be made from any display. When the system detects sufficient bacterial
growth to permit a valid result~ it automatically triggers a print-out. Fol-
lowing identification in four to thirteen hours, a technologist transfers pos-
itive cultures to another system which tests for antimicrobic susceptibility.
The results are expressed as "R" (resistant) and "S" (susceptible); no quan-
titative MIC data are provided.
The Abbot MS-2 system consists of chambers composed of eleven con-
tiguous cuvettes. Similar to the Pfizer Autobac 1, the antimicrobial com-
pounds are introduced by way of impregnated paper discs. An inoculum con-
sisting of a suspension of organisms from several colonies is introduced into
the culture medium, and the cuvette cartridge is filled with this suspension.
The operator inserts the cuvette cartridge into an analysis module which will
handle eight cartridges ~additional modules can be added to the system). Fol-
lowing agitation of the cartridge, the instrument monitors the growth rate by
turbidimetry. I~hen the log growth phase occurs, the system automatically
transfers the broth solution to the eleven cuvette chambers; ten of these
chambers contain ~ntimicrobial discs, and the eleventh is a growth control.
70 The device performs readings at five-minute intervals, and stores the data in
a microprocessor. Following a pre-cut increase of turbidity of the growth
control, the processor establishes a growth rate constant for each chamber.
A comparison of the antimicrobic growth rate cons*ant and control growth rate
constant forms the basis of susceptibility calculations. The printout pres-
ents resuits as either resistant or susceptible; if intermediate, susceptibil-
ity information is expressed as an MIC.
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Non-optical methods have also been used or suggested
for measuring antimicrobic sensitivity in susceptibility testing.
These have included radiorespirometry, electrical, impedance,
bioluminescence and microcalorimetry. Radiorespirometry, based
on the principle that bacteria metabolized carbohydrate and the
carbohydrate carbon may be detected following its release as
CO2, involves the incorporation of the isotope C 4 into
carbohydrates. Released C1402 gas is trapped and beta counting
techniques are used to detect the isotope. The major difficulty
in applying the isotope detection system to susceptibility
testing, however, is that an antimicrobic agent may be able to
stop growth of a species of bacteria, yet metabolism of carbo-
hydrate may continue. Less likely, a given drug may turn off
the metabolic machinery that metabolizes certain carbohydrates,
but growth may continue. This dissociation between metabolism
; and cell growth emphasizes the fact that measurements for
detecting antimicrobic susceptibility should depend upon a
determination of cell mass or cell number rather than metabolism.
The electrical impedance system is based on the fact
that bacterial cells have a low net charge and higher electrical
impedance than the surrounding electrolytic bacterial growth
media. A pulse impedance cell-counting device can be used to
count the cells; however, available counting devices are not
designed to handle batches of samples automatically, and
generally do not have the capacity to distinguish between live
and dead bacterial cells. Another approach with electrical
impedance has been to monitor the change in the conductivity
of the media during the growth phase of bacteria. As bacteria
utilize the nutrients, they produce metabolites which have a
,
,
greater deg~ee o~ elec~rical condu,ctance th~n the native broth,
so that as metabolis,m occursr impedence decreases. ~owever,
since this technique measures cell metabolism rather ~han cell
mass, its applicability to antimicrobic susceptibility detection
suffers ~rom the same drawback as radiorespirometry.
Bioluminescence has also been suggested for the
detection of micro organisms. It is based on the principle
that a nearly universal property of living organisms is the
storage of energ~ in the form of high energy phosphates
(adenosine triphosphate, ATP), which can be detected through
reaction with firefly luciferase. The reaction results in the
emission of light energy which can be detected with great
sensitivity by electronic light transducers. Although a clinical
laboratory may obtain a bioluminescence system to detect the
presence of bacteria in urine~ the technique is expensive due
to the limited availability of firefly luciferase, and problems
have been encountered in standardizing the system.
Microalorimetry is the measurement o~ minute amounts
of heat generated by bacterial metabolism. The principle
exhibits certain advantages, but laboratories have not adopted
such a system, one serious drawback being that the system
measures metabolic activity rather than bacterial mass or number.
S_mmary of the Invention
According to the present invention there is provided
a method for performing optical density tests, employing a
sample tray having a series o~ wells containing liquid samples,
said wells having translucent bottoms, one said well being a
control well, comprising: holding said tray accurately in a
single predetermined stationary reading position without blocking
of,~ ht paths throu~h said wells~ sending light down through
all said weIls at roughly the same intensity to an array of
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light_intensity~diet~cti~g ph~toce~ls, there ~eing one photocell
ad~acent to each ~e~l, including a control photocell adjacent to
said control well~ electronically sequentially comparing the
signal from each said photocell o~ said ~rray with the signal
from said control photocell and developing a related signal
therefrom for each well.
~ ccording to another aspect of the present invention
there is provided an automatic scanning apparatus for performing
optical density tests, employing a sample tray having a series
of wells containing liquid samples, said wells having trans-
lucent bottoms, comprising: tray holding means for holding said
tray accurately in a single predetermined reading position
without blocking off light paths through said wells, a single
dif~used light source positioned above the sample tray, for
sending light down through all said wells at roughly the same
intensity, an array of light-intensity-detecting photcells on
the opposite side of the tray holding means from light source
means, one photocell adjacent to each well and positioned to .:
receive light from the light source which has been transmitted
through the well and its contents, sequential signal receiving
means connected to all the photocells for receiving sequentially
a signal from each said photocell in a prescribed order, without
any physical movement of the tray or photocells, each signal
corresponding to the intensity of light received by a said
photocell, and electronic sequencing means connected to said
signal receiving means for electronically causing it to receive
its signals in order.
The present invention provides an optical method and
~ apparatus which can be used for automatically determining
bacterial susceptibility to a number of di~erent antimicrobic
drugs, utilizing turbidimetry. The preferred system uses broth-
dilution
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to determine susceptibility. Serial dilutions of the anti-
microbic agent are inoculated with the organism and incubated
for a period sufficient to allow detectable growth. The disclosed
appàratus determines minimu~ inhibitory concentration (MIC) of
a particular antimicrobic drwg, which is the lowest concentration
of that drug that results in no detectable bacterial growth. Typ-
ically, ten antimicrobic drugs are evaluated, with seven different
dilutions of each drug being tested. Therefore, to obtain an MIC
determination for ten drugs, seventy tubes or wells must be inoc-
ulated and examined. In contrast to previows methods using individ-
ual full-sized test tubes, which were cumbersome and expensive, the
present system utilizes "micro-tubes", which are presently available
as disposable, molded plastic trays, each well of which holds approx-
imately 0.5 milliliter.
For measurement of the MIC values in these trays,
appropriate dilutions of each antibiotic must be placed in
the wells or micro-tubes. Semiautomated devices for making
the dilutions and filling the trays in large batches are
available commercially. Alternatively, a laboratory may
~0 obtain trays that are already filled with antibiotic dilutions
and kept frozen until use. To prepare the bacteria culture
for inoculation into the wells, a suspension of the bacterial
organisms in water is made in a container. By means of a
multiple-pronged device, a technician is able to inoculate a
uniform drop of bacterial suspension into each of the large
plurality (e.g., seventy) of wells with a single motion.
The bacteria and the various dilutions of the antimicrobic
agents are incubated for a time period sufficient to produce
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detectable bacterial growth, and the MIC may then be determined
as the lowest concentration of the effective antimicrobic agents
in which there is no evidence of growth.
Previously, the reading of such an MIC tray was done
by manual viewing performed by a technician, and was a
laborious procedure. An overnight incubation period was
generally required in order to produce visually detectable
patterns of growth. However, the disclosed apparatus and method
provide for the performance of the reading and interpretive task
automatically. Moreover, the device has the capability of inter-
polating the MIC between twofold dilutions, whereas by visual
reading a technician can only detect the difference between growth
and no growth and thus can only read MIC to the nearest twofold
dilution. With the sensitive photoelectric apparatus described
herein, together with the capabilities of a microcomputer the
different gradations of growth can be measured even after a rel-
atively short incubation period, and a precise MIC can be calculated
and displayed on a screen or printed out. Thus, the device makes
available continuous numerical data that improves accuracy and
allows quantitative quality-control techniques.
Photodetection of bacterial growth is accomplished
by passage of uniform intensity light through each of the wells
and through the translucent well bottoms following the incubation
period. The uniform light may be obtained from plural uniform
sources, one at each well, or by a single source of uniform,
diffused light over the entire tray. At the opposite side of
the tray, preferably below the tray, are an array of sequentially-
scanned photoelectric cells, one associated with each well.
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The sensed light intensity level at each well is compared by
computer with a light level corresponding to zero bacteria growth
to determine a relative value of turbidity. The reference value
may be obtained by the reading of a sterile control well.
In addition to the quantitative MIC data, the disclosed
apparatus and method provide a graphic interpretive printout to
guide the physician's therapy. The computer is programmed to
translate the MIC value into dosage ranges that would be necessary
to achieve blood levels of the antimicrobic drug effective to in-
hibit growth of the organism at a particular site. Por example, a
printout of "-" might be used to indicate that the organism is re-
sistant and no dosage of a drug can effect the organism. A print-
out of "+" may be used to mean that the organism is resistant and
may respond to high intramuscular or intravenous doses, with "++"
indicating intermediate sensitivity and that the organism may respond
to higher than recommended doses. A printout of "+++" would indicate
that the organism may be sensitive to the usual doses of an anti-
biotic, and "++++" would indicate a high degree of sensitivity and
thus an optimal drug with which to treat the infectious agent.
In one embodiment, a method according to the invention
for determining susceptibility of a bacteria culture to various
antimicrobic drugs and of determining the minimum inhibitory
concentration (MIC) of the bacteria culture to those drugs to
which it is susceptible comprises the steps of placing the
plurality of different antimicrobic drugs in a plurality of
wells in a light-transmissive tray, each drug being included
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in a series of wells in serially-diluted known concentrations;
establishing a known uniform concentration of the bacteria and
placing the uniform concentration in equal volumes of the wells;
following an incubation period, passing ligh~ in substantially
equal intensity through each well and determining a turbidity
value for the bacterial suspension of each well by sequentially
sensing the intensity of light transmitted through the bacterial
suspensions of the wells by means of photodetectors adjacent to
the wells opposite the light source; and in a computer, comparing
turbidity values with a turbidity value corresponding to zero
bacterial growth, thereby determining which antimicrobic drugs
have inhibited bacterial growth and the minimum concentration
of each inhibitory drug required to inhibit growth, and displaying
the determined information. The concentration of the bacteria
culture may itself be initially determined by turbidimetric
measurement utilizing a light source and at least one photo-
detector. The antimicrobic drugs may be placed in the tray
in a rectangular matrix of wells, with each column of wells
containing incrementally varying concentrations of a single
drug. Of course, any arrangement of the-wells or of the anti-
microbics in the wells is suitable, so long as the computer
has the proper information as to what is being tested in each
well. Control wells containing only the bacterial suspension,
as well as sterile control wells, may be included for self-
checking of the system and/or providing a transmitted light
value corresponding to zero bacteria growth. The system may,
as explained above, provide for translation of the MIC values
to dosage ranges necessary to establish the required antimicrobic
concentration at the body sites involved.
.. . .. .
36;~
Accordingly, it is among the objects of the invention
to provide an automated method and apparatus for quantitative
antimicrobic susceptibility testing, capable of providing
complete MIC data for a plurality of antimicrobics within a
very short period of time. An associated objective is to provide
in such a system a means for translating MIC results into a
complete interpretive therapeutic guide for the patient in
question. These and other objects, advantages and features of
the invention will be apparent from the following description
of the preferred embodiments, taken in conjunction with the
accompanying drawings.
Description of the Drawings
In the drawings:
Figure 1 is a perspective view showing an automated
antibiotic susceptibility apparatus according to the invention.
Figure 2 is an exploded perspective view showing a
sample tray and optical detection equipment forming a part
of the apparatus of Figure 1.
Figure 3 is a sectional elevational view showing a
portion of the apparatus.
Figure 4 is a diagram indicating the operation of
the system of the invention.
Figure 5 is a block diagram of an analog to digital
converter subsystem of the invention.
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23~29
~ Figure 6 is a block diagram of a microcomputer portion
'~ of the apparatus.
Figures 7A, 7B, and 7C are flow charts of operational
steps involved in the method of the invention.
Figure 8 is a schematic elevation view showing an
alternative form of optical detection apparatus which may be
included in the apparatus of the invention.
Figure 9 shows a form of printout which may be
utilized in connection with the apparatus of the invention.
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Description of the Preferred Embodiments
In the drawings, Figure 1 shows one example of an
external configuration which the susceptibility testing
apparatus 10 of the invention may take. The unit 10 com-
prises a photo unit or optical detection unit 11 and a
processor unit 12. The optical detec~ion unit 11 preferably
includes a drawer 13 for receiving and supporting a sample
tray 14 whic}l is examined by detection apparatus of the unit
ll when the drawer is closed and the testing operation is
begun. The detection unit 11 may also include a patient identifica-
tion input switch 16, a run switch 17 and a calibrate switch 18.
The processor unit 12 may include a readout display 19, an on/off
power switch 21, printer control buttons 22, and a printout exit
23 which dispenses a printed "ticket" 24 bearing the desired sus-
ceptibility information.
Figure 2 somewhat schematically represents the con-
figuration of the detection apparatus associated with the
optical detection unit 11 of the apparatus 10. Within the
detection unit 11 above the drawer 13 is a source of uniform,
diffuse light which may comprise, for example, a fluorescent
light bulb 26, a parabolic reflector 27 positioned thereabove
such that the lamp 26 is at the focal point of the reflec~or
27, and a diffuser 28 just below the lamp and reflector. The
arrangement of the lamp 26 and the reflector 27 provides a
nearly uniform distribution of light over the surface of the
diffuser 28, and the diffuser improves uniformity and re-
duces intensity to the desired level.
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~Z36~9
Within the drawer 13 are a sample tray holder 29
having a matrix of openings 31, and an array of photocells
32 therebelow in a matrix conforming to the position of the
openings 31 above. The openings 31 and the photocells 32
also correspond precisely to the position of sample testing
wells 33 of a sample tray 14 which is received in registry
above the tray holder 29 when a test is to be conducted. The
sample tray 14, or at least the bottom of each well 33, is
translucent so that light passing through the diffuser 28
penetrates the wells and their contents, passes through the
openings 31 in the tray holder 29, and reaches the photocells
32 below, which individually sense thé intensity of the light
passing through each well. The photocells may be of the type
manufactured by Clairex Electronics of Mt. Vernon, N.Y. as
Model CL702L. The tray holder 29 is preferably of a dark,
light-absorbing color such as black to reduce light trans-
mission between the wells and reflection of diffracted light
within any one well. The tray holder arrangement assures that
all light passing through the openings 31 is from the wells
33 rather than through other areas of the translucent sample
tray 14.
The sample tray 14 is preferably a disposable,
molded plastic tray, each well of which holds approximately
0.5 milliliter. Trays of this type are commercially avail-
able and have been used previously for simple visual type
"reading" techniques as discussed above. The wells 33 are
often referred to as "microtubes", since they replace cumber-
some full-sized test tubes which were used in the past
for this type testing.
Figure 3 shows a portion of the internal apparatus
of the optical detection unit 11 in cross section. The
drawing is somewhat schematic, without details of the
structural supporting arrangement within the unit 11 and the
drawer 13, but shows the relationship of the lighting com-
ponents 26, 27 and 28 to one another and to the sample tray
14, the tray holder 29 and the matrix of photocells 32. The
bottom of a supporting surface of the drawer 13 is shown in
this schematic view, with the photocells 32 mounted on that
10surface and the tray holder 29 surrounding and extending above
the array of photocells 32. The sample tray 14, several wells
33 of which are indicated in Figure 3 fits snugly over the tray
holder 29 with the wells 33 extending down into the openings
31 of the tray holder with little side-to-side tolerance
so that registry of the sample wells with the photocells is
assured.
The light source illustrated is a convenient and
preferred form; however, any light source or a plurality of
light sources which will provide light of equal intensity
~0directed into each well 33 of the sample tray 14 is suf-
ficient. In this regard, an alternative form of light source
and detection system is described below in connection with
Figure 8.
As discussed above, the sample tray 14 is pref- I -
erably laid out in a rectangular matrix, which may comprise
for example eight rows and ten columns. Other arrangements
would be adequate, but a rectangular matrix is space-efficient
and convenient. The wells 33 contain various dilutions of
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different antibiotics, and these may be arranged such that
each of the ten columns of wells contains a single anti-
biotic in a series of different dilutions. There may be
seven different concentrations of each antibiotic, with the
eighth well of at least several of the columns used for con-
trol purposes. For example, one control well might be used
for unrestricted growth of bacteria, and another well used
to represent no growth, with no bacteria inoculated into the
well.
Into the wells containing the various dilutions
of different antibiotics is introduced the patient bacteria
sample borne within a culture medium. This bacteria culture
is uniformly inoculated into each well, and this may be
accomplished by commercially available devices having a
matrix of prongs (not shown) arranged to register with each
well to be inoculated in the commercially available sample
tray 14. Of course, the antibiotics and the bacteria cul-
ture may be introduced to the wells in the reverse order,
but for convenience, efficiency and reliability it is pre-
~0 ferred that the antibiotic be introduced first.
Figure 4 indicates diagrammatically the operation
of the susceptibility testing apparatus 10. The lamp 26,
reflector 27 and diffuser 28 are shown transmitting uniform
diffuse light through a sample well 33a of the matrix of
wells of the sample tray 14. The well 33a contains one dil-
ution of one of the antibiotics being tested, inoculated
with a controlled volume and known concentration of the
bacteria in a culture sample. The same uniform diffuse
.~ - 19 -
light is also transmitted through a well 33n containing no
bacteria for providing a light intensity reading correspond-
ing to zero bacteria growth.
After an incubation period sufficient to allow
some detectible growth of the bacteria in the well 33a in
the event that growth is not prevented by the particular
antibiotic in the particular concentration being tested, a
growth culture 36 results therein. The light from the dif-
fuser passes through this culture 36 and through the bottom
of the well to a photocell 32a of the photocell matrix.
Here the intensity of the light is sensed and converted into
an electricàl analog value corresponding to the opacity of
the culture 36. This opacity value represents the turbidity
of the culture, stemming from the net effect of light ab-
sorption and scatter in the well 33a. At the same time, the
diffuse light passes through the sterile control well 33n
to a photocell 32n of the photocell matrix. Again, the
sensed light intensity is converted into an electrical analog
reference value.
The photocell 32a is connected to a plus input of
a differential amplifier 37 through a noise filter 38 and a
multiplexer 39 which functions to select each photocell 33
of the matrix of photocells in a prescribed sequence under
direction of a microcomputer 41. Thus, the photocell 32a
shown in Figure 4 is connected to the differential amplifier
37 only when the multiplexer 39 momentarily selects that
particular photocell. The reference photocell 32n is connected
to the minus input of the differential amplifier 37 and
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provides a reference voltage which is subtracted from the
plus input to provide an analog differential output. Thus,
the light intensity or turbidity value signal emanating from
the differential amplifier 37 is in the form of a reference
voltage which varies according to turbidity of the sample
being sensed, representing the increase in turbidity of that
sample since inoculation. Each analog signal is transmitted
in its turn to an analog to digital converter 42 which con-
verts the analog to a digital signal and sends it to the
microcomputer 41.
The microcomputer 41 and its operation are de-
scribed below with reference to Figures 5, 6 and 7. It functions
to correlate differential digital values ~from the ADC 42)
representing bacterial growth for the various wells with the
particular drug and its concentration in the subject well.
From this correlation, the microcomputer selects the ~ero
growth indication stemming from the weakest concentration of
each drug, and this concentration becomes the MIC for that
particular drug. If none of the wells containing a particu-
lar drug indicates inhibition of growth, the microcomputer
prints out the fact that the infectious organism is resistant
to that particular drug.
The remaining apparatus indicated in Figure 4 is
described below with reference to the other figures.
The analog circuitry associated with this system
10, including the analog to digital converter, is set forth
in the detailed block diagram of Figure 5. Figure 5 includes
~3~
the noise filter circuit 38, the multiplexer circuits 39
which are included within the dashed line box, the differ-
ential amplifier 37, and the analog to digital converter 42
along with its related supporting circuitry. The ten by
eight photocell matrix is also shown in Figure 5 for clarity
of understanding of this part of the system 10.
The multiplexer 39 includes a binary coded decimal
(BCD) to decimal decoder 101 driving column drivers lQ3,
and another BCD to decimal decoder 113 controlling FET
switches lll. A four bit digital line 100 from the micro-
computer 41 is connected to the binary coded decimal input
of the binary coded decimal to decimal decoder circuit 101
~which may be preferably implemented as a type 7442 TTL
integrated circuit or equivalent). Ten output lines 102
from the decoder lOl are connected to ten driver circuits
103. The driver circuits are preferably implemented as
operational amplifiers type LM 324 or equivalent.
As already explained above, the photocell matrix
is arranged as a rectangle with ten columns and eight rows.
Thus, the outputs from the ten driver circuits 103 are ap~
plied to the ten columns respectively via a bus 104 such
that when one driver is excited by operation of the decoder
101~ an excitation voltage is provided to one of the column
drive lines corresponding to the binary coded decimal column
select information input to the decoder 101 via the data
line 100 from the microcomputer 41. An eleventh of the
drivers 103 applies voltage continuously through a drive
line 105 to the reference cell 33n.
- 22 -
36~2~
Eight row lines 106 and one line 107 from the
reference cell 32n are applied as inputs to nine active
filter circuits within the filter 38. Each filter circuit
is preferably implemented by an operational amplifier, type
LM 324 or equivalent. The filters 38 function to remove
power line ripple so that the eight row output lines 108
and a reference output line 109 carry DC voltage levels only.
The eight output lines 108 are applied to eight field effect
transistor switches 111, respectively. The switches are
preferably implemented as integrated circuits type CD4016
CMOS quad bilateral switch gate chips or equivalent. An
output line 110 from the switches 111 is connected directly
to the plus input of the differential amplifier 37. The
ninth line 109 is applied directly to the minus input of
the logarithmic differential amplifier 37.
A three bit digital line 112 from the micro-
computer 41 is connected to the input of a second binary
coded decimal to decimal decoder 113 which is also preferably
implemented as a type 7442 TTL integrated circuit or equiv-
alent. The decoder 113 functions to selèct one of eight
output control lines 114 which in turn select one of the
eight field effect transistor switches 111 to connect one of
the filtered row lines to the plus input of the logarithmic
differential amplifier 37, in accordance with digital row
select information received from the microcomputer 41.
The logarithmic differential amplifier 37 is
preferably implemented as an Analog Devices type 756 or equiv-
alent, and the purpose of the amplifier 37 is to correct for
- 23 -
~ , ~
~3~
variations in light intensity from the light source 26. The
light-variation-corrected analog voltage output from the
amplifier 37 is supplied as an input to an operation ampli-
fier 116 which is provided with external potentiometers to
control gain and DC offset of the incoming signal from the
amplifier 37.
An output line 117 from the amplifier 116 is
supplied as an analog input to the analog to digital converter
42 which is preferably implemented with a National Semicon-
ductor M~15357 integrated circuit or equivalent. A digital
control line 118 from the microcomputer 41 is connected as
a trigger input to a monostable multivibrator one shot 119,
preferably implemented as a type 74121 TTL integrated circuit
or the equivàIent. An output pulse from the one shot 119 of
appropriate amplitude and duration is supplied via a line
121 to the analog to digital converter 42 to start the con-
version process. A timing generator (e.g. type 555) 122
applies timing pulses via a iine 123 to the analog to digital
converter 42 to control the sequence of operations thereof.
The analog to digital converter 42 utilizes the timing pulses
supplied on the line 123 during a conversion cycle to digitize
the analog information on the line 117 and provide an eight
bit digital output via an eight bit output bus 124 which is
supplied to an input port of the microcomputer 41.
The microcomputer 41 forms the central portion of
the system 10. The microcomputer includes a single chip
monolithic microprocessing unit (MPU) 140, which is pref-
erably implemented as a type 6800 manufactured by Motorola
_ 24 -
~. ,.
36zg
Semiconductor, American Microsystems, and other suppliers.
Although this particular microprocessor was chosen for the
described preferred embodiment of the present invention,
other types of microprocessors would function equally as
well, for example the Intel 8080, the Mostee 6502, the
Zilog Z80, the Fairchild F-8, etc. A suitable two-phase
clock 141 provides the necessary clock signals to the micro-
processing unit 140.
The main system program which is set forth in
hexadecimal code in the table following the specification
of the present invention is loaded into one and a half kilo-
bytes of programmable read only memory 142. The read only
memory 142 is preferably implemented with 2708 programmable
read only memories produced by Intel and other suppliers.
Other PROMs would be well suited for the program memory 142.
The microcomputer 41 also includes one kilobyte of random
access memory ~R~M) 143 which provides volatile storage of
data to be processed as well as a stack for the micropro-
cessing unit 140. The microprocessing unit 140, the clock
` 141 through the microprocessing unit 140, the program memory
142 and the data storage memory 143 are connected in parallel
to the system bus 144 which includes an eight bit data bus,
an eight bit control bus, and a sixteen bit address bus.
Input output interface is accomplished with three
peripheral interface adapters (PIA) 146, 147 and 148 which
are connected to the system bus 144. The interface adapters
146, 147 and 148 are preferably implemented as type 6820
integrated circuits produced by Motorola Semiconductor,
- 25 -
~Z3~2~
American Microsystems, and other suppliers. These inte-
grated circuits contain two ports apiece. Each port may be
used either to input data to the microprocessing unit 140
or to output data to output devices, as will be explained
hereinafter.
The first interface adapter 146 has its first port
connected to receive the eight bit digitized information via
the bus 124 from the analog-to-digital converter 42, as shown
in Figure 5. The first port of the interface adapter 146 also
provides the control signal line 118 which is connected to
the one shot 119 which functions to start the analog to digital
conversion process of the converter 42. The line 118 will
be further explained hereinafter. The second port of the
interface adapter 146 is connected to the multiplexer 39
with four bits provided for the column select control signal
via the bus 100, and the three remaining bits provide for the
row select control signal via the bus 112.
The second peripheral interface adapter 147
includes a first port which controls the printer 20. Two
~0 bits of data are input from status indicators in the printer
20 via a line 149. One of these bit positions is from a
microswitch which indicates that the paper form has been
properly inserted and that a printout can be made. The other
bit is a signal from the printer electronics which indicates
that the printer is either in a "print" or a "wait" opera-
tional mode. Four bits of the first port of the interface
adapter 147 are also used to control the printer and shift
data to be printed into the printer 20. The data is entered
- 26 -
3~iX9
serially via a line 151 from the first port of the adapter
147 to the printer 20. Other control functions carried out
by the four bits on the line 151 include line feed (advance
the paper one line), print (cause the print solenoid to make
an impression on the paper), and shift (move the next data
bit into position for printing). The second port of the
interface adapter 147 is not used in the present embodiment.
The third peripheral interface adapter 148 in-
cludes a first port which reads the thumbwheel switch 16
for patient identification information via a four-bit line
153. The upper four bit positions of this first port of
the adapter 148 are used to select and enable one of the
four thumbwheel positions via a four bit line 152. One bit
position of the line 152 is low to enable one of the four
switching positions. The lower four bits of the first port
of the adapter 148 are used to read data via a bus 153 from
the switch position selected by the upper four bits. The
data from the switch represent a binary number between zero
and nine. The second port of the interface adapter 148 is
used to supply data to the alpha-numeric display readout 19.
The display 19 is the Burroughs model SSD0132-0070 self-scan
display unit with built-in electronics. As explained, it is
controlled via a line 154 from the second port of the third
peripheral interface adapter 148. Data to be displayed on
the display 19 are entered into the unit via a line 156 in
a six bit code for all alpha-numeric'characters as well as
some special symbols.,,The data are read in from left to
right and appear on the display until new data are entered.
Thus, the upper two bits are provided via the line 154 to
_ 27 -
~Z3~i2~
control the display, wi~h one of the bits being a clear line
and the other being an enable line. The lower six bits are
provided via the line 156 for the purpose of sending parallel
data to the display presented to the user in accordance with
the operation of the system 10.
In addition to the characteristics of the inter-
face adapters 146, 147 and 148 described hereinabove, each
adapter also has an interrupt function. The interrupt is
an additional line which is available for monitoring the
status of external devices. In the presently described
system 10, the interrupts are used to monitor operator
actions of several types. Interrupt capabili~y which re-
sults in an output rather than an input is termed a strobe.
Strobes are utilized in the system 10 as well as interrupts.
Thus, the first peripheral interface adapter 146 controls
the conversion of data from analog to digital format via
the analog to digital converter 42 by utilizing a strobe
line 118 which is connected to the one shot 119 (Figure 5) to
start the analog to digital conversion operation.
The second peripheral interface adapter utilizes
an interrupt from the printer 20 via a line 155 and utilizes
one interrupt each from the run switch 17 via a line 158
and calibrate switch 18 via a line 159. The second port of
the second adàpter 147 utilizes the output strobes via a
line 157 to cause the printer 20 to execute a print cycle.
A third peripheral interface adapter 148 has two
interrupt inputs: one from a microswitch indicating that
- 28 -
~3~Z~
the photo uni~ drawer is open via a line 161 and one in-
dicating that the drawer is closed via a line 162.
The printer 20 is preferably implemented as an
MFE model TKllE with built-in electronics package. Data
is fed from the microcomputer 41 via the line 151 which
generates the proper control signals to enable the printer
electronics to cause the printer 20 to print, line feed or
shift data into internal registers. The data is fed to the
printer 20 in serial format, stored in buffers in the printer
electronics, and is then printed in parallel. The command
to print is generated as a strobe output of the second port
of the second peripheral inter~ace adapter 147 via the line
157. The printer is a commercially available unit presently
being sold for the original equipment manufacturer (OEM) market.
The operation of the system 10 is explicated by
the flowchart set forth in Figure 7. Therein, at a power on
step 166, the operator turns the power on to the system 10.
At that point, the display 19 informs the operator to insert
the calibration tray. At insertion step 168, the operator
inserts the tray, and at step 169, the operator closes the
drawer. At a logical step 170, thè system checks the identifica-
tion of the tray in the drawer. For this purpose a binary code
is implemented using the uppermost right two wells of the tray,
either of these wells belng either opaque or transparent, thus
providing identification of four possible types of trays. This
code is made to correspond to the combination antibiotics which
the tray contains.
- 29 -
~36~3
In the event that the type of tray is not iden-
tified at step 171, the system asks whether the tray is
inserted backwards at step 172. If so, the readout 19 dis-
plays a tray backwards indication at step 173, and the oper-
ator opens tile drawer at a step 174 and removes the tray,
orients it correctly, and reinserts it, then repeats steps
168, 169, 170 and 171.
Once the tray is identified at step 171, the
readout 19 displays the tray type at step 175, and directs
the operator to press the calibration switch 18 at a step
176. At step 177, the operator presses the calibration switch
18 whereupon the system tells the operator to wait at step
178. The wait signal remains until the system informs the
operator to remove the tray at step 179. The operator opens
the drawer at step 180. In the event that the tray is not
in backwards, and yet the tray remains unidentified at step
181, the operator is then instructed to open the drawer to
manually inspect the tray to find out why the system 10 is
unable to identify it.
At step 182, the readout 19 tells the operator to
close the drawer, and at step 183 the operator removes the
tray and closes the drawer. The readout 19 then tells the
operator that if a next test is desired, he should press
the run or calibrate button at step 18~. At a step 185,
the operator actually presses the run or the calibrate switch.
If the system has been previously calibrated at step 186,
then the readout 19 directs the operator to insert the test
tray at step 187. However, if the system 10 has not been
- 30 -
,
~Z3~
calibrated at step 186, the program returns to step 167 and
the calibration procedure is carried out as set forth in
steps 167 through 185.
At step 188, the operator opens the drawer and
inserts the test tray. The display 19 than tells the operator
to close the drawer at step 189. The operator closes the
drawer at step 190 and the tray identification is determined
at step 191. In the event that the tray is not identified,
the system then determines whether the tray is in backwards
at step 192. If so, the system informs the operator that the
tray is in backwards by a readout display at step 193. In
the event that the tray remains unidentified and it is not
in backwards, then at step 194, the operator is informed
that the tray is unidentified and the program loops back
to step 180 whereupon the operator opens the drawer and
repeats steps 180 through 191.
Once the identification of the tray has been
determined at logical step 191, the system 10 displays the
type of tray at the readout with step 195. Then the oper- :
ator is informed to set the patient identification informa-
tion into the identification switch 16 and insert the form
to be printed into the printer`20 at step 196. The operator
performs these operations at step 197 and when they are
completed, the display 19 tells the operator to press the
run switch 17 at step 198. The operator presses the run
switch 17 at step 199 and the patient identification infor-
mation is displayed at step 200. Then, the patient identi-
fication is printed on the form at a step 201 and then the
- 31 -
'' '
l~Z3Çi~9
MIC values and interpretive information are printed on the
form in step 202 to produce the form 203.
Once the form is printed with the patient identi-
fication MIC values and interpretive information the dis-
play tells the operator to remove the tray at step 204.
The operator opens the drawer and actually removes the tray
at step 205 whereupon the display 19 tells the operator to
close the drawer at step 206. The operator closes the
drawer at step 207 and the apparatus 10 then instructs the
operator to perform the next operation of either "run" or
"calibrate" at step 208 whereupon the program loops back to
step 185 where the run or calibration switches are operated
and the program is repeated as heretofore described until
all of the samples have been evaluated by the system lO.
Figure 8 shows schematically an alternative arrange-
ment for passing light through the wells 33 of the sample
tray 14 and detecting the resultant light intensity passing
through each well. The apparatus of Figure 8, which utilizes
fiber optics to transmit light, would replace the form of
light source and diffuser 26, 27 and 28 shown in Figures 2, 3
and 4. Tt would also eliminate the need for a large plurality
of photocells 32 in a matrix as shown in Figure 2, and would
replace the multiplexing unit 39 (Figure 4) with a substitute
arrangement which selects one cell at a time for receipt of
a penetrating quantum of light.
The apparatus of Figure 8 includes a light source
221 and a reflector 222, directing light through a lens 223
llZ36Z~
toward a rotatable selector plate 224 driven by a stepper
motor 225. The selector plate 224 has a single opening 226
(dashed lines) which sequentially directs light to different
fiber optic fibers 228 of a fiber optic bundle 229. The
stepper motor 225 is under the control of the microcomputer 41
via the lines 100 and 112 ~Figures 4 and 6), in lieu of and
to perform the same function as the multiplexer 39 indicated
in Figure 4 and 6. The fiber optic fibers 228 of the bundle
229 each go to individual testing wells 33 of the tray 14.
The fibers are indicated only schematically, as is the bundle
229.
Below the wells 33 are a second plurality of
fiber optic fibers 230 of a second bundle 231. Transmitted
light from each well is collected by a fiber 230 of the
bundle 231 and fed via a lens 232 to a single photocell
detector 233. A value corresponding to the intensity of
incident light is then fed to the filter 38, then to the plus
input of the differential amplifier 37, as in the apparatus
of the other embodiment described above.
In order to provide a control or reference value
which may be fed into the minus input of the differential
amplifier 37 to represent a base iight intensity corre-
sponding to zero bacterial growth, there must be one optical
fiber which always carries light through a reference sterile
control well, i.e. the well 33n of Figure 4, also indicated
in the schematic representation of Figure 8. Accordingly, a
single optical fiber 228n is positioned at the lens 223 in
such a way that it receives and carries light continuously
- 33 -
.~
3~2~
whenever the lamp 221 is energized, i.e. whenever any of the
wells 33 is being tested. The fiber 228n extends to a
position adjacent to the sterile control well 33n as shown,
and a receiving fiber 230n carries the transmitted light
to second lens 232n. The resultant analog light intensity
value for the control well is fed through the filter 38
to the minus input of the differential amplifier 37~ so that
the differential amplifier yields a differential analog
signal corresponding to increased turbitity in the tested
well from bacterial growth.
The remainder of the system remains the same as
described above. The principal advantage of the form illus-
trated in Figure 8 is the use of a single light source focused
on the fiber optic bundle and a single detector for all test
wells of the sample tray, providing a more uniform measure-
ment over the matrix of test wells in the tray. Light is
transmitted through only two wells of the tray at any given
time: the well currently being tested for turbidity, and the
sterile reference well 33n. The subsystem of~Figure 8 allows
for close standardization and easy calibration and checking.
Figure 9 shows a form of printout ticket 24 which
may be used in conjunction with the present invention,
with exemplary MIC susceptibility information and therapy
information. As discussed above, the apparatus of the
invention provides a graphic interpretive printout to guide
the physician's therapy, an example of this type information
being located in the right column of the ticket 24. The
computer algorithm translates the MIC values (left column)
.
~ 34 -
3'~
to dosage ranges that would be necessary to achieve blood
levels of each antimicrobic drug to effectively inhibit
growth of the organism. Figure 9 indicates one form that the
"therapy guide" information may take. With this format,
"-" indicates that the organism tested is resistant to that
particular antimicrobic, and that no dosage of the ~nti-
microbic can affect the organism. "+" indicates resistance
but that the organism may respond to high intra-muscular
intra-venous doses. "++" indicates that the organism is
intermediate in sensitivity to the particular antimicrobic,
and may respond to higher than recommended doses. A printout
of "+++~' indicates sensitivity to the usual recommended doses
of the antibiotic, and "++++" means that the organism ex-
hibits a high degree of sensitivity and thus is an optimum
drug with which to treat the infectious organism. A print-
out of "****" tells the physician that a dosage of that
particular antibiotic necessary for therapy may be toxic to
the patient.
- 35 -
~Z36~
TABLE OF P~ROGRAM FOR ANTIBIOTIC SUSCEPTIBILITY TESTING
The follo~ing listing cons~itutes the program for
antibiotic susceptibility testing in hexadecimal code
for direct loading into the programmable read only memory
; 5 142.
Address Program Instructions
0100 CE 80 00 6F 05 6F 04 86 2C A7 05 6F 07 ~6 7F A7
0110 06 C6 04 E7 07 6F 09 86 OF A7 08 86 OD A7 09 86
0120 2D A7 08 6F OD 86 FO A7 0C 86 OD A7 OD 6F OF 86
0130 FF A7 OE C6 2C E7 OF 7F 87 E~ 7F 87 E9 01 01 0`1
0140 7E Fl 3B;01 01 01 01 01 01 01 01 01 01 01 01 01
0150 01 01 01 01 01 01 7F 87 Dl 7F ~7 EO 7F 87 El 4F
0160 3D 3D 43 84 FO 26 06 CE F5 74 43 20 0C 86 4F 8D
0170 2E 43 84 FO 27 07 CE F5 61 B7 ~7 Dl 39 86 47 8D
0180 lE 43 84 FO 27 08 CE F6 10 B7 ~7 EO 20 07 CE F6
0190 lF 43 B7 87 El BD F3 36 BD F2 F5 BD F2 CF 39 B7
01AO 80 06 C6 60 BD F2 B9 B6 80 04 BD F2 F5 B6 80 04
OlBO 39 01 01 01 BD FO 56 B6 ~7 Dl 26 6C CE F5 D4 BD
OlCO F2 4A 20 03 BD F2 50 B6 ~7 D5 27 03 7E F2 98 B6
OlDO 87 D6 27 FO BD F3 36 BD F2 F5 CE F6 2E BD F2 CF
OlEO 86 OA BD F2 BD CE 84 00 B6 87 BO 26 03 CE 84 AO
01FO B6 87 D5 27 03 7E F2 98 BD F3 60 B6 87 D5 27 15
0200 B6 87 E0 27 03 7F 87 E8 B6 87 El 27 03 7F 87 E9
0210 7E F2 98 01 01 B6 87 EO 27 03 B7 ~7 E~'B6 87 El
0220 27 03 B7 87 E9 CE F5 8E BD F2 47 B6 87 D5 27 F8
0230 CE F5 9B BD F2 47 B6 %7 D4 27 F8 CE F5 29 BD F2
0240 47 B6 87 D6 27 03 7E FO Bl B6 37 D7 26 03 7E Fl
0250 3B 01 01 01 BD-FO 43 B6 87 Dl 27 03 7E Fl 28 B6
0260`~7 EO 27 OB B6 87 E8 26 10 CE F5 4B 7E Fl 28 B6
0270 87 El 27 F5 B6 87 E9 27 FOi~6 03 BD F2 BD CE F5
0280 A9 BD F2 47 B6 87 D5 27 03 7E F2 98 B6 87 D8 27
0290 FO CE F5 C9 BD F2 47 B6 87 D5 27 03 7E F2 98 B6
02A0 87 D7 27 FO 86 FF B7~7 AO B7 87 A3 B7;~7 A4 BD
02BO F2 FA BD F3 47 7E F6 FE 01 01 01 01 01 01 01 01
02C0 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
02DO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
02EO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
02FO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
~3~
0300 CE ~7 A4 B6 ~0 08 85 20 26 F9 B6 80 08 85 10 27
0310 FO C6 08 64 oa 25 04 ~6 OE 20 02 36 OF B7 ~ a8
0320 B6 80 OA B7 80 OA 4F 4C 26 FD 5A ~6 E6 09 8C 37
0330 9F 27 09 8C 87 AO 26 D9 C6 04 20 D7 86 02 B7 30
C340 08 86 OE B7 80 08 39 BD F3 36 BD F2 F5 BD F2 CF
0350 FF 87 D2 CE 87 D4 6F 00 0~ 3C 37 D9 26 F6 FE 87
0360 D2 OE 3E 39 CE ~0 00 A6 CD 35 30 27 03 B7 87 D4
0370 85 40 27 03 B7 87 D5 E6 OC A6 09 ~5 ~0 27 03 B7
0380 87 D6 85 40 27 03 B7 ~7 D7 E6 08 A6 OB 85 80 27
0390 03 B7 87 D5 E6 OA OE 3B 86 04 CE F5 87 36 7F 80
03AO OE C,6 ~0 FD F2 B9 BD F3 36 BD F2 CF C6 ~0 BD F2
03BO B9 32 4A 26 E5 7E Fl 25 5F B6 01 20 01 5F 37 5F
03CO 5A 01 01 01 01 26 F9 33 5A 26 F3 4A 26 FO 39 FF
03DO ~7 D2 E6 OO 27 OA 86 AA B7 80 OE gD 1~ SA 26 F6
03EO E6 Ol 08 A6 Cl 84 3F 8B ~O B7 80 OE ~D 07 5A 26
03FO Fl FE 87 D2 39 4F 4A 26 FD 39 BD F3 36 g6 F7 CE
0400 87 Al OD 49 25 OC BD F3 47 BD F2 00 BD F3 47 01
0410 01 39 B7 80 OC F6 30 OC C4 OF 37 43 85 50 26 07
0420 EB 00 E7 00 08 20 06 58 58 58 58 E7 00 33 43 GB
0430 BO F7 80 OE 20 CC C6 20 7F 80 OE BD-F2 F5 86 AA
0440 B7 8O OE 5A 26 F5 39 B6 80 08 85 20 26 F9 B6 80
0450 08 85 10 27 F9 86 04 B7 80 08 86 OE B7 80 03 39
0460 7F 80 06 C6 60 BD F2 B9 BD F2 F5 B6 80 04 BD F2
0470 F5 B6 80 04 43 A7 00 08 7C 80 06 B6 80 06 &4 o
0480 26 E6 B6 80 06 84 78 81 50 26 D8 39 FE 87 D2 0
0490 BD F3 47 09 FF 87 D2 A6 50 AO 00 2B lE BD F3 C
04AO B6 37 D3 84 F8 27 OE 81 AO 27 OA B7 87 D3 20 DC
04BO 01 01 01 01 01 39 01 01 01 01 01 86 07 16 B4 87
04CO D3 26 ~2 F7 87 AO 01 01 20 D3 FF 87 EE 5F CE F6
04DO AE B6 87 EO 26 05 C6 AO CE F6 5E B6 87 EF 10 FF
04EO 87 EE BB 87 EF 24 03 7C 87 EE B7 87 EF FE 37 EE
04FO A6 00 01 01 CE OF FF 85 07 27 lF CE 4F FF 85 06
0500 27 18 CE 44 FF 85 05 27 11 CE 44 4F 85 04 2~ OA
0510 CE 44 44 85 03 27 03 CE 38 ~ FF 87 A3 84 F8 44
0520 44 CE F6 34 FF 87 EE BB 87 EF 24 03 7G 87 EE B7
0530 87 EF FE 87 EE EE 00 FF 87 Al B6 87 AC 26 03 7A
0540 87 AO BD F2 00 39 01 01 01 01 01 01 01 01 01 01
0550 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0560 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0570 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0580 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0590 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05AO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05BO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05CO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05D0 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05EO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
05FO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
- 37 -
36~9
0600 08 10 49 4E 53 45 52 54 20 54 45 53 54 20 54 52
0610 41 59 05 15 49 4E ~3 45 52 54 20 43 41 4C 49 42
0620 52 41 54 45 20 34 52 41 59~ 0~) 20 4E 45 5~` 54 20
0630 54 45 53 54 20 50 52 45 53 53 20 52 55 4E 20 4F
0640 52 20 43 41 4C 49 42 52 41 54 45 06 14 43 41 4C
0650 49 42 52 41 54 49 4F 4E 20 52 45 51 55 49 52 45
0660 44 07 11 55 4E 49 44 45 4E 54 49 46 49 45 44 20
0670 54 52 41 59 07 11 54 52 41 59 20 49 4E 20 42 41
06g0 43 4B 57 41 52 44 53 OD 05 45 52 52 4F 52 OA OB
0690 52 45 4B 4F 56 45 20 54 52 41 59 OA OC 43 4C 4F
06A0 53 45 20 44 52 41 57 45 52 01 lE 53 45 54 20 50
06BO 41 54 4q 45 4E 54 20 49 44 20 41 4E 44 20 49 4E
06CO 5~ 45 52 54 20 46 4F 52 4D OB 09 50 52 45 53 53
06DO 20 52 55 4E 02 OF 50 52 45 53 53 20 43 41 4C 49
06EO ~2 52 41 54 45 06 13 49 4E 53 55 46 46 49 43 49
06FO 45 4E 54 20 47 52 4F 57 54 4~ 06 14 53 54 45 52
0700 49 4C 45 20 43 4F 4E 54 41 4C~ 49 4E 41 54 45 44
0710 00 OD 47 52 41 4D 20 50 4E 53 20 54 52 41 59 00
0720 OD 47 52 41 4D 20 4E 45 47 20 54 52 41 59 OE 04
0730 57 41 49 54 51 2A 25 6A 12 8A 64 AO 32 AO 16 AO
0740 SA 00 4A 00 2A 00 lA 00 OA 50 OA 25 OA 12 OA 06
0750 60 8A 30 4A 15 2A 76 AO 38 AO 19 AO 9A 50 28 31
0760 3A 43 4C 54 5C 05 18 21 29 32 3B 44 4C 05 2D 31
0770 3A 42 4B 54 5C 05 28 30 3A 43 4B 54 5C 05 00 09
0780 11 19 22 2A lB 05 20 29 32 3B 43 4C 54 05 lD 20
0790 29 32 3B 43 4C 05 2~ 30 39 42 4B 53 5C 05 00 08
07AO 10 lB 23l 2B 33 05 70 78 ~1 8A 93 9C A4 05 28 30
07BO 39 42 4B 54 5C 05 23 31 39 42 4B 54 5C 05 28 31
07CO 3A 43 4B 54 5C 05 39 42 4B 53 5C 64 6C 05 18 21
07DO 29 32 3B 44 4C 05 2D 31 3A 42 4B 54 5C 05 28 3C
07E0 3A 43 4B 54 5C 05 00 09 11 19 22 2A 33 05 00 08
07FO 10 lB 23 2B 33 05 70 78 81 3A 93 96 A4 05 CE 84
0800 5C B6 87 EO 26 03 CE 84 FO PF ~7 D2 BD F3 60 B6
0810 87 D5 27 03 7E F2 98 CE 84 00 B6 87 EO 26 03 CE
0820 84 AO A6 07 AO 57 2A 01 40 31 OA 2F 06 CE F5 FA
0830 7E Fl 28 A6 OF AO 5F 2A 01 40 CE F5 E5 81 OA 2D
0840 EF BD F3 8C 7E Fl 25 01 01 01 01 01 01 01 01 01
0850 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0860 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0870 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0880 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
0890 01 01 01 01 01 01 01 ûl 01 01 01 01 01 01 01 01
08AO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
08B0 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
08CO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
08DO, 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
08EO 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01
08FO 01 01 01 01 01 01 01 01 01 01 01 01 01 0]. 01 01
- 38 -
~ ~ ~ 36 Z~
1 The above described preferred embodiments provide
2 apparatus and a method for automatically determining the
3 minimum inhibitory concentration of a plurality of different
4 antibiotics necessary to stop growth of an infective orga-
nism being tested. Minimum inhibitory concentration infor-
6 mation is also transferred to dosage information by the
7 apparatus and method of the invention. The required time
8 to perform such a test is greatly reduced in comparison to
9 other methods, a great deal more information is provided,
10 and accuracy is improved. Various other embodiments and
11 variations to the preferred embodiments will be apparent to
~2 those skilled in the art and may be made without departing
13 from the spirit and scope of the following claims.
14
SUPPLEMENTARY DISCLOSURE
16 Collimation means, preferably beneath the tray
17 holding means, collimates the light from each well after it
18 has passed through the wells. For some tests there is
19 light filter means below the tray holding means, for
20 filtering the color values of the light passing through the
21 wells. The photocells may also be set at a,n inclination to
22 the vertical lines through the wells an~ operate as nephelo- '
23 meters.
24 The single diffuse source need not put out a
25 uniform light. The light need only be roughly even. Also,
26 the photodetectors may be inexpensive ones, providing
27 signals of different strengths for the same light intensity,
28 so long as the invention is practiced with an initial cali-
29 bration step. In this step, the light source directs
30 light over all the photode~ectors either without a tray
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,
~ ~ 3 ~
1 positioned above them or with an empty tray, to take any
2 variations in the plastic material of the tray into account
3 in the calibration. As another alternative, the tray wells
4 may be filled and then run through before any culture, at
S zero time relative to growth. In the calibration, a scan
6 is made and all values, i.e. photodetector output signal
7 values, are stored. When each actual test is run, a dif-
8 ference or ratio signal is created for each photocell, so
9 that only the difference in sensed light intensity is used,
disregarding effects of localized differences and intensi~y
11 and difference in the photocells themselves.
12 With the filters the invention makes it possible to
13 use an optical-electrical method for automatically reading
14 the color changes of a plurality of biochemical reactions in
small microtubes. As noted in the main disclosure the micro-
~6 tubes are, preferably, all part of a unitary sample tray, made
17 of suitable translucent material. Each microtube is a well of
18 this tray. In each well and in a standardized manner, is
19 placed a suitable chemical reagen~ or reagents; then each well
20 is inoculated with the sample. Photodetection of color
21 changes is accomplished by the passage of uniform intensity
22 light through each of the wells and through the translucent
23 well bottoms following an incubation period. At the opposite
2~ side of the tray, preferably below the tray, is an optical
25 filter designed to pass only certain wavelengths of light. ~e-
2~ neath the filter is an array of sequentially-scanned trans-
27 ducers such as photoelectric cells, one associated with each
28 well. The optical filter is designed so that a shift in color
29 in the wells will result in a predictably greater or lesser
30 amount of light passing through to the photoelectric cells.
4 0
~ 6 ~
1 The e~act filter used depends on the test con-
2 cerned. The filter may be made to be easily removable and
3 replaceable. For example, a large number o~ tests may be
4 run using only three filters one at a time; these three
being (for example) filters numbers 809, 863, and 878,
6 of Edmund Scientific Co., 785 Edscorp Building, Barrington,
7 New Jersey 08007.
~ The sequential signal-receiving means is con-
9 nected to all the photocells for receiving sequentially
10 a signal from each photocell in a prescribed order, each
11 signal corresponding to the intensity of light received
12 at the photocell. Electronic sequencing means is con-
13 nected to the signal-receiving means and electronically
14 causes it to receive its signals in order, all without
15 any mechanical movement of anything.
16 The sequencing, being automatic, is very fast,
17 going through 80 or 96 wells of a tray ill about five
1~ seconds or less. The automatic electronic scan has no
19 moving parts--an important feature.
Electronic sequencing is much more reliable than
~1 mechanical movement of a tray or o~her mechanical sequenc-
22 ing. Multiplexing has the advantages of speed, accuracy,
23 reliability and maintainability, i.e. easy maintenance.
24 ~or at least these reasons, the inven~ion is a significant
25 improvement over mechanical scanning.
26 As noted in the main disclosure, first comparator
27 means is connected to the signal-receiving means and
28 sequentially compares the signal from each photocell of
29 the array with the signal from the reference-detecting
30 photocell and then develops a different signal therefrom.
, ~ - 41 -
z~
1 The data storage means holds data values corresponding to
2 zero reaction or other base comparison values and holds
3 data relating to various organisms or tests. Second
comparator means is connected to the first comparator means
and to the data storage means, and sequentially makes a
6 comparison of each different signal value with a value
7 corresponding to that of the same well when empty or at
8 zero time or zero growth or reacts or develops at a result-
9 ant value from that comparison.
The third comparator means may be connected to
11 the second comparator means and to the data storage means
12 sequentially compares said resultant values with a large
3 number of stored values and for determining such conclusory
14 values as the probability values for the presence of
15 selected organisms in khe sample or the minimum inhibitory
16 concentratiOn desired.
17 Finally, the output means connected to the third
18 comparator means gives the results obtained, displaying
19 them or printing them out.
It will be apparent that ~he tray itself might
21 be a source of error. That is, its own light transmissivity
22 and opaqueness and f~aws can substantially affect the light
23 transmissivities received by the photocells, in addition to
24 the light transmissivity of the liquid in the wells. The
25 trays can vary from tray to tray, and they can also vary
26 in a tray from well to well. This could, of course, lead
27 to substantial errors that would give false impressions
28 and false results if not compensated or corrected.
29 The present invention accomplishes the needed
30 correction by the two different types of comparison stages
- 42 _
~ ~ ~ 3 ~ ~
1 already explained in the main disclosure.
2 First, for each reading in any sequence of wells
3 in the tray, each well is immediately compared with the
4 value obtained by direct light transmission to the ref-
S erence photocell. From this comparison, the device
6 provides an after culture value for each well, which is a
7 function of the after culture signal values (or amplifi-
8 cation thereof) for the tested well and for the reference
g photocell. This, of course, represents a comparison of
10 the light received at each photocell in the main array and
11 the intensity of the light received at the reference
12 photocell. The signal may be amplified and is used as
13 the operative signal, as shown in Fig. 4. The after
14 culture value for each well may be called a "difference"
15 signal value, regardless of the type of function which is
16 used in comparing the two values (tes~ well vs. reference
17 photocell) to produce this value. In the embodiment of
18 Fig. 4 the subtractive difference preferably is taken
19 between the two values, and the differential amplifier
20 amplifies the difference signal. However, the signal
21 value may instead be a ratio, and the signal from each
22 well compared with the reference photocell signal by
23 means of a log ratio module. In other words, there is
24 again a "difference" signal, but it is a difference in
~5 logarithms, so that the subtraction is really a division,
26 and a quotient or ratio is obtained instead of a difference
27 expressed as a logarithm.
28 Thus a first comparator may incorporate a log
29 ratio module and send out its related signal as an
30 amplitude ratio between each signal Sw obtained through a
.~ - 43 -
h ~ "
1 well and its photocell and a signal SR obtained from the
2 reference photocell. This related signal Sx = kl SW
3 R
4 where kl is a constant. This first comparator may also
incorporate a log ratio module and sends out its resultant
6 value Sv as a ratio k~ SX , where DV is the data reference
7 V
8 value and k2 a constant.
9 The first and second comparators may use the same
10 log ratio module. The second comparator may utilize as its
11 data reference value Dv, stored ratios read earlier from an
12 empty tray, so that DV = kl SWE for each well, where SwE
13 R
14 is the signal coming from an empty well.
The second comparator may utilize as its data
16 reference value Dv, stored ratios read earlier from a tray
17 containing the same liquid from which the signals Sw are
18 generated, but read at a time when there has been zero
19 growth, so that DV = kl SW for each well, where SwO is
n
1~
21 the signal coming from a well containing the liquid at zero
22 growth time.
23 Also the preliminary comparing means may include
24 a log ratio module for sending as its derived signal a
25 signal based on the ratio of the two signals it compares.
26 Thus, in the invention, each reading of each well,
27 at each stage where readings are taken, is compared by a
28 first comparator means with the reading at the reference
29 photocell, and a difference or ratio signal developed from
30 it. By this procedure, variations in light intensity from
~_ - 44 -
~ ~ ~ 3 ~ ~
1 the source over time, as would be induced by supply voltage
2 fluctuations, have no effect on the readings. Such varia-
3 tions will vary the reference and well photocells propor-
4 tionately, so that a ratio will cancel the errors out.
This is the purpose of the reference photocell.
6 Second, to further reduce the possibility of
7 error particularly due to flaws in the tray, and in view
8 of the fact that each tray is positively identified in
g the apparatus, as has already been described, a prior
10 reading may be taken through the tray before the reading
11 after bacterial culture; this prior reading is stored and
12 is later compared with the sample reading.
j 13 One way of taking the prior reading is to take
14 a reading of the tray in its empty state, before it is
15 filled with fluid, to compare the reading through each
16 empty well with the reading of the reference photocell,
17 as above, and to store ~he resulting difference signal or
18 ratio signal in the data storage portion of the microcom-
19 puter. Then the ratio signal (or difference signal)
20 derived from the liquid at the time of the after culture
21 reading is compared with the ratio signal (or difference
22 signal) of the empty wells. Thereby, each well is compared
23 with itself when full and when emp-ty, and errors due to the
24 wells are substantially eliminated.
Another way of taking this prior reading is to
26 take the prior reading, not of the empty tray but of the
27 tray just after its wells have been filled with the solu-
28 tion and prior to the culture; in other words, at sub-
29 stantially zero time so far as growth or culture is con-
30 cerned. This means that the reading is taken
~ .
~ - 45 -
~ Z 3 ~ 2~
1 through the actual solution~ and the ratio of that reading
2 to the reference electrode is stored in the data storage
3 bank for the later use.
4 With the zero based signal (however obtained)
in the data bank, and with the ratio or difference signal
6 provided for each well for the liquid after culture, then,
7 before proceeding further~ the next step is to compare by
8 a second comparator means the two ratio (or difference~
9 values, that is, to compare the ratio of the signal derived
10 from the light transmissivity of the specimen after culture
11 to the direct light reception by the reference cell, with
12 the ratio of the empty tray or tray with the same liquid
13 at zero time to the signal from the reference cell. This
14 second comparison may also be made by calculating a ratio
15 of the two ratios, which is preferably accomplished by ;~
16 taking the difference in logarithms of the two ratios,
17 resulting in another logarithm which is the log of the
18 comparison ratio, or of what may be called the comparison
l9 signal
In the next step, a third comparison depends
21 upon what test is being run. Basically, it is a compari-
22 son of the ratio signal obtained from the second comparator
23 means, which preferably is the logarithm of the comparison
24 signal, with values that are stored in the data storage
25 means to determine the final asked-for result.
26 For good results in this last step, especially
27 when applied to MIC procedure, a distinction is made
28 between a growth state and a no-growth state. The instru-
29 ment determines at the output from the second comparator
30 means, a voltage level or logarithm value that represents
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~ 2 ~ ~2 ~
1 the extent of bacterial growth, when that voltage level is
2 compared to voltages that are obtained from known sterile
3 and growth controls, these voltage values being stored in
4 the data bank of the microcomputer. A first step here is
to determine whether there is an adequate voltage (logarithm
6 value~ difference between the readings obtained from the
7 sterile and the growth control wells. This is done pre-
8 ferably by comparing the ratios for the two wells, i.e.
9 the products of the first comparator means for the two
10 wells, which are logarithms of ratios of well readings
11 vs. reference readings. The comparison of the two con~
12 trol values is done by taking a difference between the
13 two logarithms. The resulting difference is compared to
14 a predetermined, stored value representing adequate
15 growth-sterile difference for the test. If there is an
16 inadequate difference, this means either one of two things,
17 either that there had not been sufficient growth to pro-
18 vide an adequate difference, or that the sterile well had
19 been contaminated and that there had been growth there.
20 In either case, the instrument will display a reading such
21 as "insufficient growth-sterile difference", and the
22 computer returns to the beginning of the program. The
23 operator then checks to see which of these two possi-
24 bilities is the one that is present. If there is in-
25 sufficient growth, it may be due to a lack of time or
26 because there was nothing to grow. If there were con-
27 tamination, that would show and be readily detectable, and
28 the test must be re done.
29 Once the computer has established that there is
30 an adequate difference between the sterile condition and
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... . .
'-: ; .; '
l the expected growth condition from one well to another,
2 the calculated logarithm values and their difference are
3 used for computation of a break point, or a limit compari-
4 son signal value. Preferably, the break point is biased
toward the sterile value to achieve more sensitivity to
6 growth detection, via a preselected fraction of the sterile-
7 growth logarithm difference. The break point may, for
~ example, be placed at 25% of the determined sterile-
g growth difference (preferably a logarithm value as above),
10 added to the log value for sterility. For all wells where
ll there has been less growth than that represented by 25%
12 of the determined growth-sterile difference for the test
13 being conducted, then the concentration of those wells is
14 considered as inhibitory. For each drug being tested, the
15 concentration closest to the break point, but on the in-
16 hibitory side, is selected as the minimum inhibitory con-
17 centration value. Thus, supposing that there are a series
18 of wells of different dilutions and that the operation is
19 moving from wells of greater growth towards those of
20 lesser growth and toward the sterile condition, then the
~1 minimum inhibitory concentration is not found until the
22 irst well is reached which shows less than 25V/o of the
23 determined difference between the sterile and growth con-
24 trol wells. In this way, a "floating threshold" is
Z5 utilized, i.e. one which is calculated from controls in
26 the very test being conducted and with the same organism
27 being tested, rather than a fixed threshold which has been
~8 calculated based on prior information and stored.
29 Another important comparison which should be
30 performed preferably at least once a day, before series
8 -
'i
:~z~
1 of tests are performed, is an initial calibration step.
2 This initial calibration is in lieu of the empty tray
3 (or just filled tray~ reading procedure described above.
4 Like that procedure, this calibration procedure is impor-
5 tant in that it enables the use of a light source which
6 is not tatally uniform for each photodetector, but only
7 generally uniform, and also the use of ine~pensive photo-
8 detectors which may not be uniform or totally constant,
9 over a long period of time, in their sensitivity. By
10 this procedure the light is first passed directly (no
11 tray) to all photodetectors, including the reference
12 photocell, and ratio readings (preferably their logarithms)
13 are taken as above and recorded, These values are stored
14 and give a relative base line or initial calibration
15 value for each photocell. All subse~uent after culture
16 values (which are preferably logarithms of ratios as above)
17 are compared to these base line readings, and expressed
18 as "difference" (or log ratio) readings. Thus, any
19 differences in sensitivities of the various pho~ocells,
20 or differences in light intensity due to position, are
21 "zeroed out" by comparison of after culture ratios with
22 initial calibration ratios, the comparisons being sepa-
23 rate for each well.
24 As indicated above, the apparatus of this
25 invention is capable of performing various types of com-
26 parisons. Any specific comparison depends upon what
27 method is being used and which types of comparison are
28 appropriate.
29 In some instances, there may be only one com-
30 parison, the specific type of comparison depending on
- 49 -
.~
~ .
,
1 the particular apparatus or particular method belng used.
2 For example, it is advisable to relate for every sample
3 the signal received from each well with a signal of a
4 reference transducer. ~uch comparison negates the effect
of the variation of light intensity or power fluctuation
6 with time.
7 A second type of comparison is often made, in
8 addition to the first one. This may be considered as a
9 type of calibration procedure aimed at negating the vari-
ation of response of the different photosensors. The com-
11 parison may be achieved by storing the signals from each
12 of the photosensors before the tray is introdu~ed, and then
3 subtracting from this the corresponding signals generated
14 by each of the wells after the filled tray with its
cultured samples has been read. This technique of elimi-
16 nating transducer-to-transducer variation is important.
17 Further refinements, which are not necessarily
18 crucial, may be added to eliminate further well-to-well
19 variation. For example, variations in the plastic trays
20 or their contents may affect ~he accuracy of a reading.
2~ One way-to eliminate this problem is to calibrate with an
22 empty trày instead of calibrating without any tray in the
23 holder. A single empty tray may be used, assuming that all
24 trays to be used are substantially identical. Another
25 approach is to compare the wells of each individual tray
26 when empty with the results obtained a~ter filling them
27 with li~uid and culturing the liquid. This is more time
28 consuming and not usually necessary, but it is more accu-
29 rate. With suitable multiplexing wired into the device,
30 however, this becomes quite practical. Thus, it is possible
~ 5 ~ ~::
; ' ,
3~
1 to eliminate the variations in the signal fluctuating
2 with time, to eliminate the variation of one sensor versus
3 another, and also to compensate for tray-to-tray and well-
to-well variations.
A third type of comparison may be used ~or
6 certain tests, such as the MIC test, where the signal level
7 indicating bacterial growth is differentiated from the
8 signal level indicating no growth. This may be accom-
9 plished by comparison between various wells on the tray;
that is, some wells may be control wells or sterile no-
11 growth wells, in which there is no growth or which are
12 inoculated with suitable inhibitors. There is a possible
13 interpolation between the values of growth and no-growth,
14 as dîscussed above. Alternatively, by experimentation, one
15 can determine a signal value that differentiates between
16 growth and no-growth, and this-decision point may be used
17 instead of one derived by controls on board each tray.
18 We claim:
19
~0
21
22
23
24
26
27
28
29
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., .
