Antibiotic susceptibility testing

• US 4,448,534 A
• Filed: 10/05/1979
• Issued: 05/15/1984
• Est. Priority Date: 03/30/1978
• Status: Expired due to Term

• Current Assignee: Dade Microscan, Inc.
• Original Assignee: American Hospital Corporation
• Inventors: Hathaway, James C., Wertz, Richard K., Cook, Albert M.
• Primary Examiner(s): Rosenberger, R. A.
• Application Number:
  US06/082,228
• Time in Patent Office: 1,684 Days
• Field of Search: 356/432, 356/433, 356/434, 356/435, 356/436, 356/441, 356/442
• US Class Current: 356/435
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00

Abstract

Apparatus is provided for automatically scanning electronically each well of a multi-well tray containing many liquid samples. A light source, preferably a single source, is passed through the wells to an array of photosensitive cells, one for each well. There is also a calibrating or comparison cell receiving the light. Electronic apparatus reads each cell in sequence, quickly completing the scan without physical movement of any parts. The resultant signals are compared with the signal from the comparison cell and with other signals or stored data and determinations are made and displayed or printed out. Thereby, and by the methods of the invention, such matters as minimum inhibitory concentrations (MIC) of drugs and identification of microorganisms are achieved.

Claims

1. A method for performing simultaneously a plurality of interrelated optical density tests, employing a sample tray having a multiplicity of wells in rectangular grid arrangement containing liquid samples, said wells having translucent bottoms, comprising:

holding said tray accurately in a single predetermined stationary reading position without blocking off light paths through said wells,

sending light from a light source through all said wells at approximately the same intensity to an array of light-intensity-detecting well-associated photocells, there being one photocell adjacent to each well,

while simultaneously sending light directly from said light source to a reference detecting photocell without passing the light through a said sample,

electronically sequentially transmitting the signals from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell,

sequentially comparing the signal from each said well-associated photocell of said array with the simultaneous signal from said reference detecting photocell and developing a related signal therefrom for each well indicating a first adjusted value for each well,

sequentially making an electronic comparison of each said related signal value with an electronically stored data reference value corresponding to a preliminary value for each well derived like said first adjusted value without interposing the sample between said light source and any of said well-associated photocells, and developing a resultant value from that comparison,

sequentially comparing electronically said resultant values with one another for said interrelated optical density tests and with other stored values and determining a conclusion therefrom, and

reading out the desired results thereby obained.

2. The method of claim 1 wherein the step of developing a related signal comprises generating a signal as a ratio of each signal derived via a well to the signal derived from the reference photocell.

3. The method of claim 2 wherein the step of developing a resultant value comprises generating another ratio signal as the ratio of said related signal to a similarly derived ratio signal obtained by initially reading the photocells unobstructed by the tray.

4. The method of claim 2 wherein the step of developing a resultant value comprises generating another ratio signal as the ratio of said related signal to a similarly derived ratio signal obtained by reading the tray wells filled with the liquid but before any growth or culture thereof.

5. A method for performing simultaneously a plurality of interrelated optical density tests, employing a sample tray having a multiplicity of wells in a rectangular grid, said wells having translucent bottoms, comprising:

(a) sending light from a light source vertically at approximately the same intensity to an array of light-intensity-detecting well-associated photocells, there being one photocell corresponding to each well,

(b) while simultaneously sending light directly from said light source to a reference detecting photocell,

(c) electronically sequentially transmitting the signal from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell,

(d) sequentially comparing the signal from each said photocell of said array with the simultaneous signal from said reference detecting photocell and developing a first related signal therefrom for each well-associated photocell,

(e) storing said first related signal in a digital computer forming an integral part of the system,

(f) holding the tray having its wells filled with liquid samples, accurately in a single predetermined reading position without blocking off light paths through said wells,

(g) sending light from said light source vertically through all said filled wells at roughly the same intensity to the same said array of light-intensity-detecting well-associated photocells, there being one photocell for each well,

(h) while simultaneously sending light directly from said light source to said reference detecting photocell without passing the light through a said sample,

(i) electronically sequentially transmitting the signals for filled wells from all said photocells in a prescribed order, each signal corresponding to the intensity of light received by a said photocell,

(j) sequentially comparing electronically, in the digital computer, 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,

(k) sequentially making an electronic comparison of each said second related signal value with the corresponding stored first related signal value for the same well, and developing a resultant value from that comparison,

(l) sequentially comparing electronically said resultant values with one another for said interrelated optical density tests and with other stored values and determining a desired result from that comparison, and

reading out the desired results thereby obtained.

6. 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, utilizing a plurality of interrelated optical density tests, comprising:

providing a microtube tray having a rectangular grid array of many light-transmissive wells, and a series of photodetectors, including a photodetector associated with each said well and a reference photodetector not associated with a tray well, each photodetector being adapted to provide a signal corresponding to the sensed light intensity,

initially calibrating the photodetectors by passing light from a source of generally uniform intensity to the photodetectors and electronically sequencing the photodetectors to read a signal from each photodetector, comparing in a onboard computer the values of the signals obtained for each well-associated photodetector sequentially with the value of the reference signal obtained simultaneously for the reference 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 with the computer, storing and retaining the calibration value for each wel-associated photodetector,

placing in the wells a plurality of different antimicrobic drugs, each drug being included in a series of wells in serially diluted known concentration of the bacteria, and placing the wells adjacent to the well-associated photodetectors,

following a period for bacterial growth, passing light of generally uniform intensity simultaneously through each well and to the reference photodetector and reading the intensity of the transmitted light with the photodetectors by electronically sequencing the photodetectors to read an after-culture signal from each,

comparing in the computer the value of each after-culture signal obtained from the well-associated photodetectors sequentially with the value of the after-culture signal obtained substantially simultaneously from the reference photodetector and providing an after-culture value for each well-associated photodetector which is a function of the after-culture well photodetector signal and the after-culture reference signal,

comparing in the computer, 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 different wells and for variations in the sensitivities of the photocells,

automatically and sequentially comparing in the computer the comparison signal values with one another and with a limit comparison signal value which represents a cutoff between inhibition and growth,

correlating in the computer the comparisons with stored data identifying the antimicrobic drug and concentration in each well, and obtaining therefrom an indication of which antimicrobic drugs inhibit growth of the bacteria,

automatically selecting with the computer 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 in situ the minimum inhibitory concentration for each inhibitory drug, and, for each drug that does not inhibit growth, displaying that the bacteria is resistant to that drug.

7. The method of claim 6 wherein each of the first three comparing steps comprises calculating with the computer a value representing the ratio of the two involved values, so that, in effect, a value representing the ratio of the first two comparison ratios is obtained as said comparison signal for each well, whereby any variations in the light output of the light source over time are cancelled out through use of ratios with the reference photodetector signal values, and constancy of light intensity from the source is not critical over either time or location.

8. The method of claim 7 wherein the calculation of a value representing the ratio of the two involved ratios comprises calculating the logarithm of the ratio of the two involved values, so that the third comparison step, wherein after culture values are compared with initial calibration values, comprises calculating a difference in logarithms.

9. The method of claim 8 wherein the placing step includes providing a sterile control well with no bacteria culture and providing a growth control well with bacteria culture but no antimicrobic drug, and further includeing calculating in the computer said limit comparison signal value, including taking the difference between the after culture values of these two wells and calculating a threshold which is a preselected portion of such difference away from the sterile control well after culture value.

10. The method of claim 9 wherein said preselected portion is about 25%.

11. The method of claim 6 wherein the placing step includes providing a sterile control well with no bacterial culture and providing a growth control well with bacterial culture but no antimicrobic drug, and further includeing calculating in the computer said limit comparison signal value, including taking the difference between the after culture values of these two wells and calculating a threshold which is a preselected portion of such difference away from the sterile control well after culture value.

12. The method of claim 11 which further includes the step of comparing, prior to calculating the limit comparison signal value, the calculated difference between the after culture values for the sterile and growth control wells with a predetermined, stored value representing adequate growth-sterile difference for the test, and if the calculated difference is less than the predetermined, stored value, displaying that the calculated difference is inadequate, so that a check on the test is provided.

13. The method of claim 6 which further includes automatically comparing, in the computer, the minimum concentration information for each inhibitory drug with information relating to dosages required to achieve such minimum concentrations at the required body sites, and automatically indicating the dosage range required to control the bacteria for each inhibitory drug.

14. The method of claim 13 which further includes indicating if the required dosage range may be toxic to the patient.

15. A method of identifying microorganisms, comprising:

placing a series of different reagents in a large number of wells arranged in a rectangular grid in a light-transmissive sample tray held stationary throughout the method,

establishing a known uniform concentration of 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 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,

initiating an automatic sequence while the tray is held stationary of

(a) 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,

(b) automatically and electronically sequentially comparing, in a computer, 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 generating a signal from such comparison,

(c) automatically and sequentially comparing that signal with a value corresponding to inhibited reaction for each well,

(d) automatically and sequentially comparing the opacity values from different tests to obtain probability values for various suspected organisms,

(e) automatically and sequentially comparing these probability values with computer-stored information about the suspected organisms, and

(f) automatically and sequentially printing the names of the most probable organism and the computer probability values for each.

16. Apparatus for determining susceptibility of a bacterial culture to various antimicrobic drugs and for determining the minimum inhibitory concentration of the bacteria culture to those drugs to which it is susceptible, utilizing a plurality of interrelated optical density tests, said apparatus having a sample microtube tray with a large number of light-transmissive wells arranged in a rectangular matrix for containing uniform samples of the bacterial culture and series of varied concentrations of a plurality of antimicrobic drugs, comprising:

tray holding means for supporting the sample tray in a single accurate predetermined stationary position throughout the tests, assuring proper transmission of light through said wells,

light source means positioned in vertical relationship to the sample tray for sending light of generally uniform intensity generally vertically through all wells simultaneously,

a stationary array of light intensity detecting well-associated photocells opposite said light source means, one adjacent to each well and positioned to receive light from the light source which is transmitted through the well and its contents,

a reference light-intensity-detecting photocell for receiving light from said light source means without passing through a said sample,

sequential signal receiving means connected to all the photocells for receiving sequentially a signal from each said well-associated photocell in a prescribed order, and simultaneously with each said signal a signal from said reference photocell, each signal corresponding in amplitude to the intensity of light received by a said photocell and thus to the turbidity of the contents of the well,

electronic sequencing means for delivering the photocell signals to said signal receiving means in an automatic sequence, rapidly, one at a time,

data storage and recall means forming part of said apparatus for storing and recalling values relating to bacterial growth, including temporary storage means and permanent storage means,

first comparator means connected to said signal receiving means, for sequentially comparing the signal from each said photocell of said array, after a period for bacterial growths with the signal received simultaneously from said reference detecting photocell and developing a related signal from those two signals,

second data recall and comparator means connected to said first comparator means and to said data storage means for sequentially making a comparison of each said related signal value with a sequentially recalled data reference value corresponding to a temporarily stored pre-test value taken prior to any bacterial growth for each said well-associated photocell, and developing a resultant value from that comparison and storing the resultant values temporarily,

third data recall and comparator means connected to said second comparator means and to said data storage means for sequentially comparing said temporarily stored resultant values with one another and for making comparisons with values corresponding to inhibited bacterial growth,

determination means connected to said third data recall and comparator means for determining from said comparisons and from comparisons with pertinent permanently stored data, which antimicrobic drugs inhibit growth of the bacteria and for determining and indicating for each inhibitory drug the minimum concentration of that drug which will inhibit such growth.

17. The apparatus of claim 16, further including means associated with said third data recall and comparator means for calculating said values corresponding to inhibited bacterial growth, including calculating a threshold inhibited growth value which is a preselected portion of the difference between the after growth resultant values from a growth control well with bacteria culture but no antimicrobic drug and a sterile control well with no bacteria culture.