Method and composition for the detection and study of cellular activity or the like and means for applying such method

• US 4,390,620 A
• Filed: 12/16/1980
• Issued: 06/28/1983
• Est. Priority Date: 07/04/1977
• Status: Expired due to Fees

• Current Assignee: Agence Nationale de Valorisation de la Recherche (Bpifrance SA /Old/)
• Original Assignee: Agence Nationale de Valorisation de la Recherche (Bpifrance SA /Old/)
• Inventors: Selegny, Eric, Junter, Guy-Alain, Lemeland, Jean-Francois, Vingent, Jean-Claude
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US06/216,911
• Time in Patent Office: 924 Days
• Field of Search: 435/4, 435/5, 435/7, 435/8, 435/29, 435/34, 435/35, 435/36, 435/37, 435/38, 435/39, 435/40, 435/243, 435/808, 435/817, 204/195 B, 204/1 E, 23/230 B, 252/408
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

The invention pertains to a method for the study of the behaviour or detection of cells, organites or cellular fractions, possibly present in a medium.

It comprises providing said medium with the essential nutrients to promote the activity of the said cells, organites or cellular fractions, if any, as well as with an "electron transporter" such as lipoic acid which is liable of forming a redox system and of intervening in one of the metabolic routes involved by the above said activity, particularly growth of cells.

The method of the invention then further comprises measuring and detecting a possible modification of the relative proportions of the oxidized and reduced form of the "electron transporter". The time required for such modification to possibly take place, as well as other parameters of the reaction can then be correlated to the presence in the medium concerned of a cellular activity or analogous.

Claims

1. A method for determining the presence or absence of cells, cellular fractions or organites in a liquid medium, said method comprising:

(1) mixing

(A) a liquid medium having sufficient ionic conductivity to permit potentiometric measurements, a pH buffered about 7.0, and containing an energy substrate selected from those which cells, cellular fractions or organites are known to metabolize by at least one metabolic route with

(B) an electron transporter having an initial ratio of oxidized to reduced forms selected from those electron transporters which are a known part of at least one of said metabolic routes, whereby the metabolic activity of the cells, cellular fractions or organites induces a change in the initial ratio of oxidized to reduced form of said electron transporter causing a change in potential of said liquid medium, and

(2) measuring the potential of the medium as a function of time to determine the change in potential which resulted from said change in the ratio of oxidized to reduced form of the electron transporter, said change in ratio having resulted from the metabolic activity of cells, cellular fractions or organites during the time period over which measurements were made, and

(3) relating the change in potential to the presence or absence of cells, cellular fractions or organites in the liquid medium at the time when the measurements were commenced.

2. The method of claim 1 wherein a change in potential is observed, thereby indicating a presence, rather than absence of cells, cellular fractions or organites.

3. The method of claim 1 wherein no change in potential is observed, thereby indicating an absence rather than a presence of cells, cellular fractions or organites.

4. The method of claim 1 wherein the measurements of potential are continuous.

5. The method of claim 1 wherein the measurements are intermittent.

6. The method of claim 1 wherein a minimum of two measurements of the potential are made.

7. The method of claim 1 wherein the electron transporter is mixed with the liquid medium at a time no later than the time at which a change in potential would occur if the electron transporter had been mixed at the commencement of measurements.

8. The method of claim 1 wherein the cells are bacteria.

9. The method of claim 8 wherein the bacteria are aerobic.

10. The method of claim 8 wherein the bacteria are optionally aerobic.

11. The method of claim 8 wherein the bacteria are selected from the group consisting of E. Coli, Staphylococcus aureus, Staphylococcus epidermidis, and Salmonella typhimurium.

12. The method of claim 1 wherein the cells are eukaryotic.

13. The method of claim 1 wherein the cellular fractions are mitochondrial fractions.

14. The method of claim 1 wherein the organites are viruses.

15. The method of claim 1 wherein the metabolic route is the route by which alpha-ketonic acids are decarboxylated.

16. The method of claim 15 wherein the alpha-ketonic acid is pyruvic acid.

17. The method of claim 1 wherein the electron transporter is a coenzyme.

18. The method of claim 17 wherein the coenzyme is selected from those coenzymes which are a known part of the metabolic route by which alpha-ketonic acids are decarboxylated.

19. The method of claim 18 wherein the alpha-ketonic acid is pyruvic acid.

20. The method of claim 19 wherein the coenzyme is selected from the group consisting of lipoic acid, NAD and FAD.

21. The method of claim 17 wherein the electron transporter is in the oxidized form.

22. The method of claim 1 wherein the electron transporter is a cofactor.

23. The method of claim 1 wherein the energy substrate is a constituent sugar.

24. The method of claim 23 wherein the sugar is glucose.

25. The method of claim 23 wherein the sugar is lactose.

26. The method of claim 1 wherein the energy substrate is an adaptive sugar.

27. The method of claim 26 wherein the adaptive sugar is selected from the group consisting of xylose, maltose, galactose, and arabinose.

28. A method for determining the initial concentration of cells which are capable of cellular division in a sample containing an unknown concentration of a known type of said cell, said method comprising:

(1) preparing several aliquots of a liquid medium having a pH buffered about 7.0, a sufficiently high ionic conductivity to permit potentiometric measurements, and comprising an energy substrate selected for its ability to promote metabolic activity of said cells by at least one metabolic route, and an electron transporter selected from those electron transporters which are a known part of said metabolic route;

(2) preparing several inocula comprising the known type of cell, each of the several inocula containing a different but known concentration of said cells;

(3) inoculating the several aliquots of liquid medium with one of the inocula containing a known concentration of said cells;

(4) inoculating at least one aliquot with the sample whose concentration is unknown;

(5) measuring the potential of each aliquot as a function of time;

(6) determining the time at which a potentiometric wave occurs in each aliquot, thereby determining the latent time of each aliquot;

(7) constructing a standard curve showing concentration of cells in the inocula containing known cell concentrations as a function of the latent time of the liquid medium into which these inocula were inoculated;

(8) determining from the standard curve the concentration of cells in the sample whose concentration of cells was unknown using the latent time of the aliquot into which this sample was introduced, thereby determining the concentration of cells which are capable of cellular division in the sample containing an unknown concentration of a known type of cell.

29. The method of claim 28 wherein the cells are bacteria.

30. The method of claim 29 wherein the bacteria are E. Coli.

31. The method of claim 28 wherein the measurements of potential are continuous.

32. The method of claim 28 wherein the measurements are intermittent.

33. The method of claim 28 wherein the metabolic route is the route by which alpha-ketonic acids are decarboxylated.

34. The method of claim 33 wherein the alpha-ketonic acid is pyruvic acid.

35. The method of claim 28 wherein the electron transporter is a coenzyme.

36. The method of claim 35 wherein the coenzyme is selected from those coenzymes which are a known part of the metabolic route by which alpha-ketonic acids are decarboxylated.

37. The method of claim 36 wherein the alpha-ketonic acid is pyruvic acid.

38. The method of claim 37 wherein the coenzyme is selected from the group consisting of lipoic acid, NAD and FAD.

39. The method of claim 35 wherein the electron transporter is in the oxidized form.

40. The method of claim 28 wherein the electron transporter is a cofactor.

41. The method of claim 28 wherein the energy substrate is a constituent sugar.

42. The method of claim 41 wherein the sugar is glucose.

43. The method of claim 41 wherein the sugar is lactose.

44. The method of claim 28 wherein the energy substrate is an adaptive sugar.

45. The method of claim 44 wherein the adaptive sugar is selected from the group consisting of xylose, maltose, galactose, and arabinose.

46. A method for determining the behavior of cells or cellular fractions in a liquid medium in response to a selected constituent introduced into the medium, said method comprising:

(1) preparing at least two aliquots of a liquid medium containing said cells and having a pH buffered about 7.0, a sufficiently high ionic conductivity to permit potentiometric measurements, and comprising an energy substrate selected for its ability to promote metabolic activity of said cells or cellular fractions by at least one metabolic route, and an electron transporter selected from those electron transporters which are a known part of said metabolic route;

(2) introducing a selected constituent into at least one of said aliquots, keeping at least one aliquot devoid of the selected constituent;

(3) measuring the potential of each aliquot as a function of time;

(4) determining the time at which a potentiometric wave occurs in each aliquot, thereby determining the latent time;

(5) determining the slope of the variation of potential of each aliquot;

(6) comparing the amplitude of potential, latent time and slope of the aliquots containing the selected constituent with the amplitude of potential, latent time and slope of the aliquots devoid of said constituent, thereby determining the behaviour of the cells in the liquid medium in response to the selected constituent.

47. The method of claim 46 wherein the cells are bacteria.

48. The method of claim 46 wherein the selected constituent is a drug.

49. The method of claim 46 wherein the selected constituent is a cellular regulator.

50. The method of claim 49 wherein the cellular regulator is a metabolic activator or metabolic inhibitor.

51. The method of claim 46 wherein the selected constituent is at least one antibiotic.

52. The method of claim 46, 47 or 51 wherein the comparison of amplitudes of potential reveals a lower amplitude of potential drop, followed by a rise in potential in the aliquot containing the antibiotic, thereby indicating that the antibiotic blocks for a short term the microbial activity of the bacteria.

53. The method of claims 46, 47 or 51 wherein the comparison of latent times and slopes reveals either a reduced slope or increased latent time in the aliquot containing the antibiotic, thereby indicating that the antibiotic partially inhibits metabolism of the bacteria or reduces the growth rate.

54. The method of claims 46 or 49 wherein the response of said cells to more than one regulator is determined, thereby allowing for a study of the combined effects of the regulators.

55. The method of claims 46, 47 or 51 wherein the response of more than one type of cell to a single antibiotic is determined.

56. The method of claim 46 wherein the measurements of potential are continuous.

57. The method of claim 46 wherein the measurements are intermittent.

58. The method of claim 46 wherein the metabolic route is the route by which alpha-ketonic acids are decarboxylated.

59. The method of claim 58 wherein the alpha-ketonic acid is pyruvic acid.

60. The method of claim 46 wherein the electron transporter is a coenzyme.

61. The method of claim 60 wherein the coenzyme is selected from those coenzymes which are a known part of the metabolic route by which alpha-ketonic acids are decarboxylated.

62. The method of claim 61 wherein the alpha-ketonic acid is pyruvic acid.

63. The method of claim 62 wherein the coenzyme is selected from the group consisting of lipoic acid, NAD and FAD.

64. The method of claim 60 wherein the electron transporter is in the oxidized form.

65. The method of claim 46 wherein the electron transporter is a cofactor.

66. The method of claim 46 wherein the energy substrate is a constituent sugar.

67. The method of claim 66 wherein the sugar is glucose.

68. The method of claim 66 wherein the sugar is lactose.

69. The method of claim 46 wherein the energy substrate is an adaptive sugar.

70. The method of claim 69 wherein the adaptive sugar is selected from the group consisting of xylose, maltose, galactose, and arabinose.

71. A method for determining whether a predetermined acceptable level of bacterial contamination in a liquid sample has been exceeded, said method comprising:

(1) mixing

(A) a liquid medium having sufficient ionic conductivity to permit potentiometric measurements, a pH buffered about 7.0, and containing an energy substrate selected from those which bacteria are known to metabolize by at least one metabolic route, and containing the contents of a liquid sample, itself containing a predetermined acceptable level of bacterial contaminants with

(B) an electron transporter having an initial ratio of oxidized to reduced forms selected from those electron transporters which are a known part of at least one of said metabolic routes, whereby the metabolic activity of the bacterial contaminants induces a charge in the initial ratio of oxidized to reduced form of said electron transporter causing a change in potential of said medium, and

(2) measuring the potential of the medium as a function of time to determine the change in potential which resulted from said change in the ratio of oxidized to reduced form of the electron transporter, said change in ratio having resulted from the metabolic activity of said bacterial contaminants during the time period over which measurements were made, and

(3) selecting a time between the time at which a change in potential first occurs and the time at which the potential reaches a minimum value, and

(4) calculating the difference between the potential of the medium at this preselected time and the potential at the commencement of measurements, thereby determining a threshold potential difference corresponding to a predetermined acceptable level of bacterial contamination, and

(5) repeating step (1) under identical physico-chemical conditions with the exception that the medium comprises the contents of a liquid sample whose level of bacterial contamination is unknown, and

(6) measuring the potential of the medium of step (5) at least twice, as soon as mixing is complete, and, again, at the time selected in step (3), and

(7) calculating the difference between the two potentials measured in step (6), and

(8) comparing the difference in potential calculated in step (7) with the threshold potential difference calculated in step (4),

whereby a difference in potential as measured in step (7) greater than the threshold potential difference is indicative of a level of bacterial contamination in the liquid sample of step (5) greater than the predetermined acceptable level and a difference in potential as measured in step (7) less than the threshold potential difference is indicative of a level of bacterial contamination in the liquid sample of step (5) less than the predetermined acceptable level.

72. The method of claim 71 wherein the measurements of step (6) are continuous.

73. The method of claim 72 wherein a potential difference equal to the threshold potential difference is measured in the sample of step (5) earlier than the preselected time of step (3), thereby indicating a level of bacterial contamination in the liquid sample of step (5) greater than the predetermined acceptable level.

74. The method of claim 71 wherein the liquid sample is selected from the group consisting of city water, river water, milk, suspensions of food solids, solutions of food solids, blood, urine, and industrial liquids.

75. The method of claims 71, 72 or 73 wherein the electron transporter is mixed with the liquid medium at a time no later than the time at which a change in potential would occur if the electron transporter had been mixed at the commencement of measurements.

76. The method of claims 71, 72 or 73 wherein the bacteria are aerobic.

77. The method of claims 71, 72 or 73 wherein the bacteria are optionally aerobic.

78. The method of claims 71, 72 or 73 wherein the bacteria are selected from the group consisting of E. Coli, Staphylococcus aureus, Staphylococcus epidermidis, and Salmonella typhimurium.

79. The method of claims 71, 72 or 73 wherein the metabolic route is the route by which alpha-ketonic acids are decarboxylated.

80. The method of claim 79 wherein the alpha-ketonic acid is pyruvic acid.

81. The method of claims 71, 72 or 73 wherein the electron transporter is a coenzyme.

82. The method of claim 81 wherein the coenzyme is selected from those coenzymes which are a known part of the metabolic route by which alpha-ketonic acids are decarboxylated.

83. The method of claim 82 wherein the alpha-ketonic acid is pyruvic acid.

84. The method of claim 83 wherein the coenzyme is selected from the group consisting of lipoic acid, NAD and FAD.

85. The method of claim 81 wherein the electron transporter is in the oxidized form.

86. The method of claims 71, 72 or 73 wherein the electron transporter is a cofactor.

87. The method of claims 71, 72 or 73 wherein the energy substrate is a constituent sugar.

88. The method of claim 87 wherein the sugar is glucose.

89. The method of claim 87 wherein the sugar is lactose.

90. The method of claims 71, 72 or 73 wherein the energy substrate is an adaptive sugar.

91. The method of claim 90 wherein the adaptive sugar is selected from the group consisting of xylose, maltose, galactose, and arabinose.

92. A composition for monitoring aerobic or anaerobic cells, cellular fractions or organites in a medium, said composition comprising:

(a) an energy substrate capable of promoting the growth of said cells, and

(b) lipoic acid as an electron transporter and oxidoreduction indicator, whereby cellular activity is determined by the proportion of the oxidized and reduced forms of the lipoic acid.

93. The composition of claim 92 wherein the composition has sufficient ionic conductivy to permit potentiometric measurements and a pH buffered about 7.0.

94. The composition of claim 92 wherein the energy substrate is a sugar.

95. The composition of claim 94 wherein the sugar is glucose.

96. The composition of claim 92 which is liquid.

97. The composition of claim 96 which is concentrated, thereby allowing for dilution for use.