Method and composition for the detection and study of cellular activity or the like and means for applying such method
First Claim
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.
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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.
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Citations
97 Claims
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1. A method for determining the presence or absence of cells, cellular fractions or organites in a liquid medium, said method comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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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:
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(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. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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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:
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(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. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70)
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71. A method for determining whether a predetermined acceptable level of bacterial contamination in a liquid sample has been exceeded, said method comprising:
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(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. - View Dependent Claims (72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91)
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92. A composition for monitoring aerobic or anaerobic cells, cellular fractions or organites in a medium, said composition comprising:
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(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. - View Dependent Claims (93, 94, 95, 96, 97)
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Specification