Sensitive and rapid determination of antimicrobial susceptibility

• US 20050048599A1
• Filed: 07/08/2004
• Published: 03/03/2005
• Est. Priority Date: 07/12/2003
• Status: Active Grant

• Current Assignee: Accelerate Diagnostics, Inc.
• Original Assignee: Accelr8 Technology Corporation
• Inventors: Goldberg, David A., Buttry, Daniel A., Saavedra, Steven Scott, Metzger, Steven W., Howson, David C.
• Granted Patent: US 7,687,239 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 20/00 Nanooptics, e.g. quantum optics or photonic crystals
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses

Abstract

The present invention relates to moving microorganisms to a surface, where they are grown in the presence and absence of antimicrobials, and by monitoring the growth of the microorganisms over time in the two conditions, their susceptibility to the antimicrobials can be determined. The microorganisms can be moved to the surface through electrophoresis, centrifugation or filtration. When the movement involves electrophoresis, the presence of oxidizing and reducing reagents lowers the voltage at which electrophoretic force can be generated and allows a broader range of means by which the target can be detected. Monitoring can comprise optical detection, and most conveniently includes the detection of individual microorganisms. The microorganisms can be stained in order to give information about their response to antimicrobials.

Claims

1. A system for the quantitation of microorganisms of a first type in a solution, comprising:

a chamber comprising a first electrode and a second electrode on opposing walls of the chamber;

an input port;

an output port;

a fluid transport means for transporting solution into the chamber through the input port and out of the chamber through the output port;

a first affinity component affixed to the first electrode, to which microorganisms of the first type adhere;

an electrical controller that controls the potential between the first electrode and the second electrode;

an automated detector that can detect the quantity of microorganisms of the first type adhered to the first affinity component; and

an information controller that stores the quantity of microorganisms of the first type as determined by the detector;

wherein the solution is introduced into the chamber through the input port, a potential is applied by the controller between the first and the second electrodes sufficient to cause electrophoresis to occur between the electrodes, causing movement of microorganisms of the first type towards the first electrode to occur, such that when the microorganisms are proximate to the first affinity component, they bind to the first affinity component and their quantity is determined by the detector and stored in the information controller.

2. The system of claim 1, wherein the microorganisms are selected from set of genera Pseudomonas, Stenotrophomonas, Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Haemophilus, Streptococcus, Staphylococcus, Enterococcus, Mycobacterium, Candida, Aspergillus and Neisseria.

3. The system of claim 1, wherein the oxidizing agent is selected from the set consisting of benzoquinone, a dithiol, a ketone, a ferrocinium, a ferricyanide, dihydroascorbate, oxidized glutathione, oxidized methyl viologen, and a halogen.

4. The system of claim 1, wherein the reducing agent is selected from the set consisting of dithiothreitol, dithioerythritol, a dithioalkane, a dithioalkene, a thioalkane, a thioalkene, a thiol, a hydroquinone, an alcohol, a ferrocene, a ferrocyanide, ascorbate, glutathione, methyl viologen, and a halide.

5. The system of claim 1, wherein the reduced product of the oxidizing reagent comprises the reducing agent.

6. The system of claim 1, wherein the conductivity of the solution is less than 100 microSiemens/cm.

7. The system of claim 1, wherein the conductivity of the solution is less than 10 microSiemens/cm.

8. The system of claim 1, wherein the solution comprises a zwitterionic buffer.

9. The system of claim 1, additionally comprising a concentrator that concentrates the microorganisms from a sample prior to introduction into the chamber through the input port.

10. The system of claim 9, wherein the concentrator comprises a centrifuge.

11. The system of claim 9, wherein the concentrator comprises ion exchange particles.

12. The system of claim 9, wherein the sample has a higher conductivity than the solution.

13. The system of claim 1, wherein the automated detector comprises an optical detector.

14. The system of claim 13, wherein the optical detector utilizes optical detection selected from set consisting of light scattering imaging, brightfield imaging, darkfield imaging, phase imaging, fluorescence imaging, upconverting phosphor imaging, quantum dot imaging, chemiluminescence imaging.

15. The system of claim 13, wherein an electrode selected from the set comprising the first electrode and second electrode is optically transparent.

16. The system of claim 13, wherein the target is illuminated by a laser.

17. The system of claim 13, wherein the detector additionally determines the position of each microorganism adhered to the affinity component, wherein the locations of the microorganisms are stored in the information controller along with the quantity of the microorganism at that location.

18. The system of claim 13, wherein the detector detects total amount of microorganisms of the first type through averaging of signal of a portion of the surface comprising substantially all of the microorganisms of the first type affixed to the first electrode.

19. The system of claim 13, wherein the first electrode is comprised of gold, and the detector utilizes surface plasmon resonance.

20. The system of claim 13, wherein the detector comprises a camera.

21. The system of claim 20, wherein the field of view corresponding to each pixel comprises a long axis that is less than 2 microns.

22. The system of claim 20, wherein the field of view corresponding to each pixel comprises a long axis that is less than 0.5 microns.

23. The system of claim 1, wherein the solution is in bulk movement during electrophoresis.

24. The system of claim 1, wherein two periods of electrophoresis are interspersed with a period in which the solution is in bulk movement.

25. The system of claim 1, wherein the solution additionally comprises microorganisms of a second type, wherein the detector can distinguish microorganisms of the first type from microorganisms of the second type.

26. The system of claim 25, wherein a first tag is comprised of a first binding agent linked to a first indicator that is detectable by the detector and a second tag is comprised of a second binding agent linked to a second indicator that is detectable by the detector and wherein the first indicator and the second indicator are distinguishable by the detector, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag and the second tag are reacted with microorganisms of the first type and microorganisms of the second type bound to the affinity component, and the detector substantially simultaneously detects the quantity of the microorganisms on the basis of the tags that are bound to the microorganisms.

27. The system of claim 25, wherein a first tag is comprised of a first binding agent linked to an indicator that is detectable by the detector and a second tag is comprised of a second binding agent linked to the indicator, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag is reacted with microorganisms of the first type bound to the affinity component and the detector detects the quantity and location of the microorganisms of the first type on the basis of the tags that are bound to the microorganisms, and subsequently, the second tag is reacted with microorganisms of the second type bound to the affinity component and the detector detects the quantity and location of the microorganisms of the second type on the basis of the tags that are bound to microorganisms that are in locations that were not previously detected by the detector.

28. The system of claim 25, additionally comprising a tag comprised of a binding agent linked to an indicator, wherein the binding agent comprises an antibody that binds to microorganisms of the first type.

29. The system of claim 25, wherein the detector distinguishes microorganisms of the first type from microorganisms of the second type on the basis of differing electrophoretic properties of the microorganisms.

30. The system of claim 1, wherein the first affinity component comprises a polyelectrolyte.

31. The system of claim 30, wherein the polyelectrolyte comprises a polycationic polymer.

32. The system of claim 31, wherein the polycationic polymer comprises amine moieties.

33. The system of claim 1, wherein the solution additionally comprises microorganisms of a second type, wherein microorganisms of the second type do not bind to the first affinity component.

34. The system of claim 33, wherein the affinity component is selected from the set consisting of antibody and aptamer.

35. The system of claim 33, additionally comprising a second affinity component bound to the first electrode, to which microorganisms of the second type adhere, wherein the detector can detect the quantity of microorganisms of the second type adhered to the second affinity component, wherein the system can distinguish microorganisms of the first type from microorganisms of the second type by whether the microorganisms adhere to the first affinity component or the second affinity component.

36. The system of claim 33, wherein the affinity component additionally comprises a polymer that has intrinsically low affinity for microorganisms, wherein the polymer is selected from a set consisting of polyethylene glycol or polyacrylamide.

37. The system of claim 33, additionally comprising a third electrode, co-planar with the first electrode, to which a second affinity component is bound and to which microorganisms of the second type adhere, wherein the potential on the first electrode and the third electrode can be independently controlled by the electrical controller.

38. The system of claim 1, wherein the detector can detect whether microorganisms of the first type are live or dead.

39. The system of claim 38, wherein the microorganisms are stained prior to being detected by the detector with a stain selected from the set consisting of mortal stain and vital stain.

40. The system of claim 1, wherein subsequent to the microorganisms of the first type adhering to the first affinity component, the microorganisms are placed in conditions conducive to growth.

41. The system of claim 40, wherein conditions comprise temperature between 34 and 40 degrees Centigrade.

42. The system of claim 40, wherein the solution is removed from the chamber via the output port and replaced by growth medium through the input port.

43. The system of claim 42, wherein the growth medium has a conductivity of less than 1 milliSiemens/cm, and the electrical controller maintains a potential of greater than 100 mV between the first electrode and the second electrode.

44. The system of claim 40, wherein microorganisms of the first type are detected by the detector at an initial time, and are detected at a second time after the microorganisms are allowed a predetermined growth time, wherein differences in the detected microorganisms of the first type at the two times provides evidence of the viability of the microorganisms of the first type in the growth conditions.

45. The system of claim 44, wherein an anti-microorganisms agent is added to the growth medium during the growth time.

46. The system of claim 45, wherein the detector detects if microorganisms of the first type are live or dead in response to the anti-microorganism agent, wherein prior to detection by the detector the microorganisms are stained with a stain selected from the set consisting of mortal stain and vital stain.

47. The system of claim 45, wherein the antimicroorganism agent is selected from the set consisting of cephalosporins, penicillins, carbapenems, monobactams, beta-lactams, beta-lactamase inhibitors, fluoroquinolones, macrolides, ketolides, glycopeptides, aminoglycosides, fluoroquinolones, and rifampin.

48. The system of claim 45, wherein the concentration of the anti-microorganism agent is changed over time to reflect the pharmacokinetics of the anti-microorganism agent in animal tissue.

49. The system of claim 44, wherein the microorganisms are reacted with a surplus of microorganism surface binding reactants at a first time period, after which the reactants are subsequently removed, and wherein at a second time period the microorganisms are reacted with a surplus of microorganism surface binding molecules modified by an indicator so as to be detectable by the detector, wherein the detection of the indicator by the detector indicates the growth of the microorganisms.

50. The system of claim 44, wherein the growth time is sufficient for at least a predetermined fraction of the microorganisms to double.

51. The system of claim 50, wherein the predetermined fraction is less than 10%.

52. The system of claim 1, wherein the solution additionally comprises a contaminant that binds to the first affinity component along with the microorganisms of the first type, wherein a condition is applied to the first affinity component which releases the contaminant without releasing the microorganism, whereas the contaminant is removed by application of the condition.

53. The system of claim 52, wherein the condition comprises temperature, magnetic field strength, electrophoretic force, dielectrophoretic force, shear fluid flow, ionic strength, pH, non-ionic surfactant concentration, ionic surfactant concentration, or competitor concentration.

54. The system of claim 1, wherein the solution additionally comprises a contaminant which binds to the first affinity component along with the microorganisms of the first type, wherein a condition is applied to the first affinity component which releases the microorganisms without releasing the contaminant, whereas the microorganisms are subsequently bound to a second affinity component affixed to the first electrode

55. The system of claim 54, wherein the condition comprises temperature, magnetic field strength, electrophoretic force, dielectrophoretic force, shear fluid flow, ionic strength, pH, non-ionic surfactant concentration, ionic surfactant concentration, or competitor concentration.

56. The system of claim 1. wherein the microorganisms of the first type are concentrated in the solution prior to being bound to the first affinity component, wherein the microorganisms are present in a first salt buffer of relatively low ionic strength, and the first salt buffer is proximal to a second salt buffer of relatively higher ionic strength and the first salt buffer and the second salt buffer adjoin at an interface, and wherein a first concentration electrode is located proximal to the interface and a second concentration electrode is located distal to the interface, wherein the placement of a potential between the first concentration electrode and the second concentration electrode causes the microorganisms to migrate through the first salt buffer by electrophoresis and their migration is reduced more than X fold upon meeting the interface.

57. The system of claim 56, wherein the second concentration electrode comprises the first electrode.

58. The system of claim 56, wherein the interface is located substantially at the input port.

59. The system of claim 56, wherein the ratio of conductivity between the first salt buffer and the second salt buffer is less than 1:

50.

60. A system for the quantitation of microorganisms of a first type in the presence of microorganisms of a second type, with both microorganisms present initially in a sample solution, comprising:

a centrifuge;

a detection chamber comprising a detection surface that has an affinity for microorganisms of the first type and microorganisms of the second type, wherein the detection surface can be placed into the centrifuge while it is in contact with the sample solution, and upon centrifugation, the microorganisms of the first type and the microorganisms of the second type are deposited onto the detection surface;

a growth medium for the microorganisms of the first type and the second type;

an automatic detector interfacing with the detection chamber that can detect the quantity of microorganisms of the first type adhered to the detection surface in the presence of the growth medium; and

an information controller that stores the quantity of microorganisms of the first type;

wherein the sample solution is centrifuged in the centrifuge onto the detection surface wherein they are bound to the surface, and wherein the microorganisms are cultured in the growth medium and detected by the detector such the that quantity of microorganisms of the first type are stored in the information controller.

61. The system of claim 60, wherein the microorganisms are bacteria selected from set of genera Pseudomonas, Stenotrophomonas, Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Haemophilus, Streptococcus, Staphylococcus, Enterococcus, Mycobacterium, Candida, Aspergillus, and Neisseria.

62. The system of claim 60, wherein the detection chamber additionally comprises a chamber tube, which reversibly joins with the detection surface to form a water-tight assembly into which the sample solution can be held during centrifugation, after which the chamber tube detaches from the detection surface to allow for detection by the detector.

63. The system of claim 62, wherein the cross-section is selected from square and rectangular.

64. The system of claim 62, additionally comprising a fluid assembly that joins with the detection surface subsequent to its detachment from the chamber tube, wherein the fluid assembly comprises a transparent window, an input port and an output port, wherein growth medium enters the fluid assembly through the input port, and wherein the transparent window is situated parallel and opposite to the detection surface, and the detector detects the microorganisms of the first type through the transparent window.

65. The system of claim 60, wherein the detector comprises an optical detector.

66. The system of claim 65, wherein the optical detector utilizes optical detection selected from set consisting of light scattering imaging, brightfield imaging, darkfield imaging, phase imaging, fluorescence imaging, upconverting phosphor imaging, quantum dot imaging, chemiluminescence imaging.

67. The system of claim 65, wherein the detector utilizes laser illumination.

68. The system of claim 65, wherein the surface is coated with gold and wherein the detector utilizes surface plasmon resonance.

69. The system of claim 65, wherein the detector additionally determines the position of each bacterium adhered to the detection surface, wherein the location of the microorganism is stored in the information storage along with the quantity of the microorganism at that location.

70. The system of claim 65, wherein the detector detects total amount of microorganisms of the first type through averaging of signal over a portion of the detection surface comprising substantially all of the microorganisms of the first type affixed to the detection surface.

71. The system of claim 65, wherein the detector comprises a camera.

72. The system of claim 71, wherein the field of view corresponding to each pixel comprises a long axis that is less than 2 microns.

73. The system of claim 60, wherein the detector can distinguish microorganisms of the first type from microorganisms of the second type.

74. The system of claim 73, additionally comprising a first tag comprised of a first binding agent linked to a first indicator that is detectable by the detector and a second tag comprised of a second binding agent linked to a second indicator that is detectable by the detector, wherein the first indicator and the second indicator are distinguishable by the detector, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag and the second tag are reacted with microorganisms of the first type and microorganisms of the second type bound to the detection surface, and the detector substantially simultaneously detects the quantity of the microorganisms on the basis of the tags that are bound to the microorganisms.

75. The system of claim 73, wherein a first tag is comprised of a first binding agent linked to an indicator that is detectable by the detector and a second tag is comprised of a second binding agent linked to the indicator, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag is reacted with microorganisms of the first type bound to the affinity component and the detector detects the quantity and location of the microorganisms of the first type on the basis of the tags that are bound to the microorganisms, and subsequently, the second tag is reacted with microorganisms of the second type bound to the detection surface and the detector detects the quantity and location of the microorganisms of the second type on the basis of the tags that are bound to microorganisms that are in locations that were not previously detected by the detector.

76. The system of claim 73, additionally comprising a tag comprised of a binding agent and an indicator that is detectable by the detector, wherein the binding agent binds to microorganisms of the first type and does not react with microorganisms of the second type.

77. The system of claim 60, wherein the affinity of the detection surface for microorganisms of the first type and microorganisms of the second type is conferred by a polycationic polymer.

78. The system of claim 77, wherein the polymer comprises amine moieties.

79. The system of claim 60, wherein the detector can detect whether the microorganisms of the first type are live or dead.

80. The system of claim 79, wherein the microorganisms are stained prior to being detected by the detector with a stain selected from the set consisting of mortal stain and vital stain.

81. The system of claim 60, wherein microorganisms of the first type are detected by the detector at an initial time, and are detected at a second time after the microorganisms are allowed a predetermined growth time, wherein differences in the detected microorganisms of the first type at the two times provides evidence of the viability of the microorganisms of the first type in the growth conditions.

82. The system of claim 81, wherein an anti-microorganisms agent is added to the growth medium during the growth time.

83. The system of claim 82, wherein the detector detects if microorganisms of the first type are live or dead in response to the anti-microorganism agent, wherein prior to detection, the microorganisms of the first type are treated with a stain selected from the group consisting of mortal stain and vital stain.

84. The system of claim 82, wherein the antimicroorganism agent is selected from the set consisting of cephalosporins, penicillins, carbapenems, monobactams, beta-lactams, beta-lactamase inhibitors, fluoroquinolones, macrolides, ketolides, glycopeptides, aminoglycosides, fluoroquinolones, and rifampin.

85. The system of claim 82, wherein the concentration of the anti-microorganism agent is changed over time to reflect the pharmacokinetics of the anti-microorganism agent in animal tissue.

86. The system of claim 81, wherein the growth time is sufficient to allow a predetermined fraction of the microorganisms to double.

87. The system of claim 86, wherein the fraction is less than 10%.

88. The system of claim 81, wherein the microorganisms are reacted with a surplus of microorganism surface binding reactants at a first time period, after which the surface binding reactants are subsequently removed, and wherein at a second time period the microorganisms are reacted with a surplus of microorganism surface binding molecules modified by an indicator so as to be detectable by the detector, wherein the detection of the indicator by the detector indicates the growth of the microorganisms.

89. A system for the quantitation of microorganisms of a first type in the presence of microorganisms of a second type, with both microorganisms present initially in a sample solution, comprising:

a substantially planar filter that separates an entry chamber from an exit chamber, an affinity surface of which resides in the entry chamber and has an affinity for microorganisms of the first type and microorganisms of the second type, wherein microorganisms of the first type and microorganisms of the second type are retained on the filter during filtration;

a transparent cover to the entry chamber opposite to the filter;

an input port connecting to the entry chamber, an output port connecting to the exit chamber;

a growth medium for the microorganisms of the first type and the second type;

an automatic optical detector interfacing with the transparent cover that can detect the quantity of microorganisms of the first type adhered to the affinity surface in the presence of the growth medium; and

an information controller that stores the quantity of microorganisms of the first type;

wherein the sample solution enters the entry chamber from the input port, is filtered through filter, and microorganisms adhered onto the filter are then cultured in the growth medium and detected by the detector such the that quantity of microorganisms of the first type are stored in the information controller.

90. The system of claim 89, wherein the microorganisms are bacteria selected from set of genera Pseudomonas, Stenotrophomonas, Acinetobacter, Enterobacter, Escherichia, Klebsiella, Proteus, Serratia, Haemophilus, Streptococcus, Staphylococcus, Enterococcus, Mycobacterium, Candida, Aspergillus, and Neisseria.

91. The system of claim 89, wherein the optical detector utilizes optical detection selected from set consisting of light scattering imaging, brightfield imaging, darkfield imaging, phase imaging, fluorescence imaging, upconverting phosphor imaging, quantum dot imaging, chemiluminescence imaging.

92. The system of claim 89, wherein the detector utilizes laser illumination.

93. The system of claim 89, wherein the detector additionally determines the position of each bacterium adhered to the detection surface, wherein the location of the microorganism is stored in the information storage along with the quantity of the microorganism at that location.

94. The system of claim 89, wherein the detector detects total amount of microorganisms of the first type through averaging of signal over a portion of the detection surface comprising substantially all of the microorganisms of the first type affixed to the detection surface.

95. The system of claim 89, wherein the detector comprises a camera.

96. The system of claim 95, wherein the field of view corresponding to each pixel comprises a long axis that is less than 2 microns.

97. The system of claim 89, wherein the detector can distinguish microorganisms of the first type from microorganisms of the second type.

98. The system of claim 97, additionally comprising a first tag comprised of a first binding agent linked to a first indicator that is detectable by the detector and a second tag comprised of a second binding agent linked to a second indicator that is detectable by the detector and wherein the first indicator and the second indicator are distinguishable by the detector, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag and the second tag are reacted with microorganisms of the first type and microorganisms of the second type bound to the detection surface, and the detector substantially simultaneously detects the quantity of the microorganisms on the basis of the tags that are bound to the microorganisms.

99. The system of claim 97, additionally comprising a first tag comprised of a first binding agent linked to an indicator that is detectable by the detector and a second tag comprised of a second binding agent linked to the indicator, wherein the first binding agent binds to microorganisms of the first type, and the second binding agent binds to microorganisms of the second type, wherein the first tag is reacted with microorganisms of the first type bound to the affinity component and the detector detects the quantity and location of the microorganisms of the first type on the basis of the tags that are bound to the microorganisms, and subsequently, the second tag is reacted with microorganisms of the second type bound to the detection surface and the detector detects the quantity and location of the microorganisms of the second type on the basis of the tags that are bound to microorganisms that are in locations that were not previously detected by the detector.

100. The system of claim 97, additionally comprising a tag comprised of a binding agent and an indicator that is detectable by the detector, wherein the binding agent binds to microorganisms of the first type and does not react with microorganisms of the second type.

101. The system of claim 89, wherein the affinity of the affinity surface for microorganisms of the first type is conferred by a polycationic polymer.

102. The system of claim 101, wherein the polymer comprises amine moieties.

103. The system of claim 89, wherein the detector can detect whether the microorganisms of the first type are live or dead.

104. The system of claim 103, wherein the microorganisms are stained prior to being detected by the detector with a stain selected from the set consisting of mortal stain and vital stain.

105. The system of claim 89, wherein microorganisms of the first type are detected by the detector at an initial time, and are detected at a second time after the microorganisms are allowed a predetermined growth time, wherein differences in the detected microorganisms of the first type at the two times provides evidence of the viability of the microorganisms of the first type in the growth conditions.

106. The system of claim 105, wherein an anti-microorganisms agent is added to the growth medium during the growth time.

107. The system of claim 106, wherein the detector detects if microorganisms of the first type are live or dead in response to the anti-microorganism agent, wherein prior to detection, the microorganisms of the first type are treated with a stain selected from the group consisting of mortal stain and vital stain.

108. The system of claim 106, wherein the antimicroorganism agent is selected from the set consisting of cephalosporins, penicillins, carbapenems, monobactams, beta-lactam antibiotics, beta-lactamase inhibitors, fluoroquinolones, macrolides, ketolides, glycopeptides, aminoglycosides, fluoroquinolones, and rifampin.

109. The system of claim 106, wherein the concentration of the anti-microorganism agent is changed over time to reflect the pharmacokinetics of the anti-microorganism agent in animal tissue.

110. The system of claim 105, wherein the microorganisms are reacted with a surplus of microorganism surface binding reactants at a first time period, after which the surface binding reactants are subsequently removed, and wherein at a second time period the microorganisms are reacted with a surplus of microorganism surface binding molecules modified by an indicator so as to be detectable by the detector, wherein the detection of the indicator by the detector indicates the growth of the microorganisms.

111. The system of claim 105, wherein the growth time is sufficient for a predetermined fraction of the microorganisms to double.

112. The system of claim 111, wherein the fraction is less than 10%.

113. The system of claim 89, wherein the filter is comprised of material selected from the set consisting of polycarbonate, polyethylene terephthalate, glass, aluminum, aluminum oxide, Teflon®

, or nitrocellulose.

114. The system of claim 89, wherein the filter comprises a track-etched filter.

115. The system of claim 89, wherein the filter is comprised of pores and the median filter pores are less than 250 micron in diameter.

116. The system of claim 89, additionally comprising a first electrode and a second electrode in the entry chamber and positioned on opposite sides of the filter wherein during the filtering of the microorganisms of the first type and microorganisms of the second type, a potential is applied between the first and the second electrodes to create an electrophoretic field that causes a force on the microorganisms that separates the microorganisms across the filter on the basis of their respective electrophoretic properties.

117. The system of claim 116, wherein the solution comprises a reducing agent and an oxidizing agent, wherein the presence of the reducing and oxidizing agents in the solution allows the electrophoretic force to be created with a lower potential difference than in the absence of the reducing and oxidizing agents.

118. A method of detecting the response of a test microorganism to a condition, comprising:

capturing the microorganism on a surface of a substrate using an applied force, wherein the surface has an affinity for the microorganism;

detecting at a first time a property of the microorganism on the surface with a camera;

placing the microorganism in a condition;

detecting at a second time the property of the microorganism of the surface with the camera; and

determining the response of the microorganism to the condition by the amount of difference of the property of the microorganism between the first time and the second time.

119. The method of claim 118, wherein the microorganism is selected from the set consisting of bacterium, mycoplasma, protozoan, fungus, and plant cell and animal cell.

120. The method of claim 118, wherein the affinity of the surface is conferred by a cationic polymer.

121. The method of claim 118, wherein the applied force comprises electrophoresis.

122. The method of claim 121, wherein the electrolyte in which electrophoresis occurs comprises an oxidizing agent and a reducing agent, wherein the potential at which the electrophoresis occurs is below 2 volts.

123. The method of claim 118, wherein the applied force comprises filtration.

124. The method of claim 118, wherein the applied force comprises magnetic force, and a paramagnetic tag is bound to the microorganism in order to confer responsiveness of the microorganism to a magnetic field.

125. The method of claim 118, wherein the force comprises centrifugation.

126. The method of claim 118, wherein the force comprises dielectrophoresis.

127. The method of claim 118, wherein the property comprises the amount of stain that is detected.

128. The method of claim 127, wherein the stain is selected from the set consisting of vital stain and mortal stain.

129. The method of claim 118, wherein the property comprises the size of the organism.

130. The method of claim 118, wherein the property comprises the presence of a second microorganism at the second time adjacent to the location of the test microorganism, wherein the second microorganism is not present at the first time.

131. The method of claim 118, wherein the condition comprises the presence of an agent selected from the set consisting of antibiotic, bacteriophage, animal virus, double-stranded RNA, DNA, anticancer drug, antifungal drug, antimalarial drug, and antiprotozoal drug.

132. The method of claim 118, wherein determining at the first time additionally comprises locating the microorganism on the surface with the camera and storing the location of the microorganism in a storage medium, whereby the microorganism is located at the second time by retrieving the location from the storage medium.

133. A system for determining the susceptibility of microorganisms in a sample to an anti-organism agent in a growth medium, comprising:

a first chamber and a second chamber, each chamber comprising an input and an output port and an affinity surface;

a fluid handler, connected to each input and output port of each chamber, wherein the fluid handler can deliver a portion of the sample to the first and second chambers and wherein the fluid handler can supply the anti-organism agent in the growth medium to the first chamber and the growth medium in the absence of the anti-organism agent to the second chamber;

a concentrator which concentrates the microorganisms that have been delivered by the fluid handler in each chamber onto the affinity surface of that chamber and wherein the microorganisms that are so concentrated are attached to the surface in the respective chamber; and

a detector which can detect a condition of the microorganisms attached to the affinity surfaces in response to the anti-organism agent in the growth medium;

wherein the fluid handler delivers a portion of the sample to the first and second chambers, the concentrator concentrates the microorganisms wherein they are attached to the affinity surfaces, the fluid handler supplies the anti-organism agent in the growth medium to the first chamber and the growth medium in the absence of the anti-organism agent to the second chamber, and the detector detects the condition of the microorganisms in each of the chambers, wherein the susceptibility of the microorganisms to the anti-organism agent is evidenced by a predetermined disparity in the condition displayed by the microorganisms in the first chamber and the second chamber.

134. The system of claim 133, wherein the detector comprises an optical detector.

135. The system of claim 134, wherein the optical detector comprises a camera.

136. The system of claim 134, wherein the microorganisms of the first type are treated with a stain prior to their detection by the detector.

137. The system of claim 133, wherein the concentrator comprises a first electrode and a second electrode, wherein the affinity surface is affixed to the first electrode.

138. The system of claim 137, wherein a potential applied between the first and second electrode causes microorganisms to move to the first electrode by electrophoresis.

139. The system of claim 138, wherein the sample comprises a reducing agent and an oxidizing agent.

140. The system of claim 137, wherein a potential applied between the first and second electrode causes microorganisms to move to the first electrode by electrophoresis.

141. The system of claim 133, wherein the concentrator comprises a filter.

142. The system of claim 133, wherein the concentrator comprises a centrifuge.

143. The system of claim 133, wherein the anti-organism agent comprises an antibiotic.

144. The system of claim 133, wherein the chamber comprises a volume of less than 10 microliters.

145. The system of claim 133, wherein the condition is the viability of the microorganisms.

146. The system of claim 133, wherein the condition is the presence of daughter microorganisms.

147. The system of claim 133, wherein the detector detects the condition for individual microorganisms.

148. The system of claim 133, wherein the predetermined disparity is the ratio between fraction of individual microorganisms in the first chamber displaying the condition to the fraction of individual microorganisms in the second chamber displaying the condition.

149. The system of claim 133, additionally comprising a microorganism of a second type in the sample, wherein the concentrator additionally concentrates the microorganisms of the second type that have been delivered into the chamber by the fluid handler in each chamber onto the affinity surface of that chamber and wherein the microorganisms of the second type that are so concentrated are attached to the surface, and wherein the detector which can detect the response of the microorganisms of the second type to the first anti-organism agent in the growth medium;

and wherein the detector can distinguish microorganisms of the first type from microorganisms of the second type.

150. A method of rating the susceptibility of microorganisms in a sample to a concentration of a lethal chemical agent in a growth medium, comprising the steps of:

partitioning the sample of microorganisms into a first portion and a second portion;

incubating the first portion in the growth medium lacking the lethal chemical agent;

culturing the second portion in the growth medium with the lethal chemical agent at the concentration;

detecting the number of microorganisms in the first portion that exhibit a viability condition at a first time and a second time;

counting the number of microorganisms in the second portion that exhibit the viability condition at the first time and the second time;

rating the microorganisms as being susceptible to the concentration of the lethal chemical agent if the change in the number of microorganisms in the second portion exhibiting the viability condition exceeds the change in the number of microorganisms in the first portion exhibiting the viability condition in a predetermined manner.

151. The method of claim 150, wherein the viability condition is death.

152. The method of claim 151, wherein death is evidenced by mortal staining of the microorganisms.

153. The method of claim 151, wherein death is evidenced by a lack of vital stain of the microorganisms.

154. The method of claim 151, wherein death is evidenced by a lack of daughter microorganisms.

155. The method of claim 151, wherein death is evidenced by a lack of increase in cell size.

156. The method of claim 150, wherein the predetermined manner comprises determining the change in the number of microorganisms in the second portion that is statistically significant, by a predetermined amount and the change in the number of microorganisms in the first portion that is not statistically significant by the predetermined probability.

157. The method of claim 156, wherein the predetermined probability is a chi-squared probability of less than 0.05.

158. The method of claim 157, wherein the predetermined probability is a chi-squared probability of less than 0.01.

159. The method of claim 150, wherein detecting comprises counting the number of microorganisms.

160. The method of claim 150, wherein the difference between the first time and the second time is less than the doubling time of the microorganisms.

161. The method of claim 150, wherein the change in the number of microorganisms detected at the first time and the second time is less than a predetermined fraction of the number of microorganisms detected at the first time.

162. The method of claim 161, wherein the predetermined fraction is 10%.

163. The method of claim 150, wherein the change in the number of microorganisms detected at the first time and the second time is less than a predetermined number.

164. The method of claim 163, wherein the predetermined number is 10.