US4385113A
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Rapid, quantitative determination of bacteria in water
Assignee: National Aeronautics and Space Administration
•
Filing: 1978-03-20
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Granted: 1983-05-24
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Expires: 1998-03-20
Abstract
Abstract A bioluminescent assay for ATP in water borne bacteria is made by adding nitric acid to a water sample with concentrated bacteria to rupture the bacterial cells. The sample is diluted with sterile, deionized water, then mixed with a luciferase-luciferin mixture and the resulting light output of the bioluminescent reaction is measured and correlated with bacteria present. A standard and a blank also are processed so that the light output can be correlated to bacteria in the sample and system "noise" can be substracted from the readings. A chemiluminescent assay for iron porphyrins in water borne bacteria is made by adding luminol reagent to a water sample with concentrated bacteria and measuring the resulting light output of the chemiluminescent reaction. The light output is correlated with bacteria present. A standard and a blank are also processed so that the light output can be correlated to bacteria in the sample and system "noise" can be subtracted from the readings. Modifications may be made in the methodology to differentiate between live and dead bacteria. An automatic system automatically performs a biolumenscent ATP assay on a concentrated bacterial sample. Reservoirs are provided for the sample, standard and blank. These are sequentially mixed with nitric acid from a reservoir by using two channels of a peristaltic pump. This acid mixture is then mixed with sterile, dionized water from another reservoir using two additional channels of a peristaltic pump and the resulting mixture is then mixed with a luciferase-luciferin mixture from an additional reservoir by employing two more channels of a peristaltic pump. The resulting solution flows through a photometer which indicates the level of the bioluminescent light reaction.
Inventors
- 👤 with respect to an invention of Frosch Robert A. Administrator of the National Aeronautics and Space Administration
- 👤 Emmett W. Chappelle
- 👤 Grace L. Picciolo
- 👤 Richard R. Thomas
- 👤 Eldon L. Jeffers
- 👤 Jody W. Deming
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 21:43
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/66; C12Q1/02; C12Q1/04; C12Q1/06; G; G01; G01N; G01N21/00; G01N21/75; G01N21/76
Claims Analysis
Total Claims: 54
Claim 1
Independent Claim
135 words
A method for the rapid assay of bacteria containing iron porphyrins in a water sample comprising treating said sample by: a. bubbling carbon monoxide through a portion of said water sample; b. adding an aliquot of said sample portion to luminol reagent; c. measuring the emitted light level; d. adding another aliquot of said sample portion to luminol reagent; e. measuring the emitted light level; f. developing a blank by removing bacteria from yet another portion of said water sample; g. adding an aliquot of said blank to luminol reagent; h. measuring the emitted light level; i. developing a standard by a bacterial culture followed by a conventional bacterial count; j. adding an aliquot of said standard to luminol reagent; k. measuring the emitted light level; and l. calculating the bacteria concentration in the sample.
Claim 2
Independent Claim
135 words
A method for the rapid assay of bacteria containing iron porphyrins in a water sample comprising treating said sample by: a. bubbling carbon monoxide through a portion of said water sample; b. adding an aliquot of said sample portion to luminol reagent; c. measuring the emitted light level; d. adding another aliquot of said sample portion to luminol reagent; e. measuring the emitted light level; f. developing a blank by removing bacteria from yet another portion of said water sample; g. adding an aliquot of said blank to luminol reagent; h. measuring the emitted light level; i. developing a standard by a bacterial culture followed by a conventional bacterial count; j. adding an aliquot of said standard to luminol reagent; k. measuring the emitted light level; and l. calculating the bacteria concentration in the sample.
Claim 3
Dependent Claim
18 words
The method of claim 1 wherein said water sample undergoes bacterial concentration prior to said carbon monoxide bubbling.
Claim 4
Dependent Claim
16 words
The method of claim 2 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 5
Independent Claim
191 words
A method for the rapid assay of bacteria containing adenosine triphosphate in a water sample consisting essentially of treating said sample by: a. rupturing bacterial cells in a portion of said water sample, thereby releasing adenosine triphosphate; b. diluting the treated portion of said water sample by the addition of sterile, deionized water; c. adding an aliquot of the portion of said water sample to a luciferase-luciferin solution; d. measuring the emitted light level; e. developing a blank by removing bacteria from another portion of said water sample; f. treating said blank as if to rupture bacterial cells; g. diluting said blank by the addition of sterile, deionized water; h. adding an aliquot of said blank to a luciferase-luciferin solution; i. measuring the emitted light level; j. developing a standard by adding a known amount of ATP to sterile, deionized water; k. treating said standard as if to rupture bacterial cells; l. diluting said standard by the addition of sterile, deionized water; m. adding an aliquot of said standard to a luciferase-luciferin solution; n. measuring the emitted light level; o. calculating the adenosine triphosphate concentration; and p. calculating the bacteria concentration.
Claim 6
Independent Claim
191 words
A method for the rapid assay of bacteria containing adenosine triphosphate in a water sample consisting essentially of treating said sample by: a. rupturing bacterial cells in a portion of said water sample, thereby releasing adenosine triphosphate; b. diluting the treated portion of said water sample by the addition of sterile, deionized water; c. adding an aliquot of the portion of said water sample to a luciferase-luciferin solution; d. measuring the emitted light level; e. developing a blank by removing bacteria from another portion of said water sample; f. treating said blank as if to rupture bacterial cells; g. diluting said blank by the addition of sterile, deionized water; h. adding an aliquot of said blank to a luciferase-luciferin solution; i. measuring the emitted light level; j. developing a standard by adding a known amount of ATP to sterile, deionized water; k. treating said standard as if to rupture bacterial cells; l. diluting said standard by the addition of sterile, deionized water; m. adding an aliquot of said standard to a luciferase-luciferin solution; n. measuring the emitted light level; o. calculating the adenosine triphosphate concentration; and p. calculating the bacteria concentration.
Claim 7
Dependent Claim
17 words
The method of claim 4 wherein said water sample is prefiltered prior to said bacterial cell rupture.
Claim 8
Dependent Claim
18 words
The method of claim 4 wherein said water sample undergoes bacterial concentration prior to said bacterial cell rupture.
Claim 9
Dependent Claim
16 words
The method of claim 6 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 10
Dependent Claim
21 words
The method of claim 4 wherein said water sample is prefiltered and undergoes bacterial concentration prior to said bacterial cell rupture.
Claim 11
Dependent Claim
16 words
The method of claim 8 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 12
Independent Claim
203 words
A method for measuring adenosine triphosphate in a water sample and thereby determining bacterial concentration consisting essentially of a. forming reservoirs of a water sample, a blank, a standard, an acid, a luciferase-luciferin mixture, and sterile, deionized water; b. pumping said water sample and said acid and thereafter mixing same for a predetermined period of time; c. pumping said water sample--acid mixture and said sterile, deionized water and thereafter mixing same; d. pumping said water sample--acid--sterile, deionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; e. pumping said blank and said acid and thereafter mixing same for said predetermined period of time; f. pumping said blank--acid mixture and said sterile, deionized water and thereafter mixing same; g. pumping said blank--acid--sterile, dionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; h. pumping said standard and said acid and thereafter mixing same for said predetermined period of time; i. pumping said standard--acid mixture and said sterile, deionized water and thereafter mixing same; j. pumping said standard--acid--sterile, deionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; and k. calculating the bacterial concentration.
Claim 13
Independent Claim
203 words
A method for measuring adenosine triphosphate in a water sample and thereby determining bacterial concentration consisting essentially of a. forming reservoirs of a water sample, a blank, a standard, an acid, a luciferase-luciferin mixture, and sterile, deionized water; b. pumping said water sample and said acid and thereafter mixing same for a predetermined period of time; c. pumping said water sample--acid mixture and said sterile, deionized water and thereafter mixing same; d. pumping said water sample--acid--sterile, deionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; e. pumping said blank and said acid and thereafter mixing same for said predetermined period of time; f. pumping said blank--acid mixture and said sterile, deionized water and thereafter mixing same; g. pumping said blank--acid--sterile, dionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; h. pumping said standard and said acid and thereafter mixing same for said predetermined period of time; i. pumping said standard--acid mixture and said sterile, deionized water and thereafter mixing same; j. pumping said standard--acid--sterile, deionized water mixture and said luciferase-luciferin mixture and thereafter mixing same and measuring the emitted light level; and k. calculating the bacterial concentration.
Claim 14
Dependent Claim
18 words
The method of claim 10 wherein all said pumping is accomplished by a plurality of peristaltic pump channels.
Claim 15
Dependent Claim
16 words
The method of claim 10 wherein each of said predetermined periods of time is 60 seconds.
Claim 16
Dependent Claim
18 words
The method of claim 10 wherein said water sample is prefiltered prior to said water sample reservoir formation.
Claim 17
Dependent Claim
19 words
The method of claim 10 wherein said water sample undergoes bacterial concentration prior to said water sample reservoir formation.
Claim 18
Dependent Claim
16 words
The method of claim 14 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 19
Dependent Claim
22 words
The method of claim 10 wherein said water sample is prefiltered and undergoes bacterial concentration prior to said water sample reservoir formation.
Claim 20
Dependent Claim
16 words
The method of claim 16 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 21
Dependent Claim
96 words
A bioluminescent method for rapidly assaying bacteria in a water sample by detecting adenosine triphosphate in the water sample without first removing non-bacterial sources of adenosine triphosphate consisting essentially of treating the sample by: (a) rupturing bacterial cells in a portion of the sample, diluting the portion by the addition of a sterile water, adding an aliquot of the portion to a luciferase-luciferin mixture and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the bacteria concentration.
Claim 22
Dependent Claim
96 words
A bioluminescent method for rapidly assaying bacteria in a water sample by detecting adenosine triphosphate in the water sample without first removing non-bacterial sources of adenosine triphosphate consisting essentially of treating the sample by: (a) rupturing bacterial cells in a portion of the sample, diluting the portion by the addition of a sterile water, adding an aliquot of the portion to a luciferase-luciferin mixture and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the bacteria concentration.
Claim 23
Dependent Claim
14 words
The method of claim 18 wherein bacteria are concentrated prior to bacterial cell rupture.
Claim 24
Dependent Claim
14 words
The method of claim 18 wherein said blank employs sterile water and nitric acid.
Claim 25
Dependent Claim
14 words
The method of claim 18 wherein the standard employs pure ATP diluted in water.
Claim 26
Independent Claim
134 words
A method for the rapid assay of bacteria containing iron porphyrins in a water sample comprising treating said sample by: a. adding hydrogen peroxide to a portion of said water sample and allowing the mixture to stand; b. adding an aliquot of said mixture to luminol reagent; c. measuring the emitted light level; d. developing a blank by removing bacteria from another portion of said water sample; e. treating said blank by adding hydrogen peroxide; f. adding an aliquot of said blank to luminol reagent; g. measuring the emitted light level; h. developing a standard by a bacterial culture followed by a bacterial count; i. treating said standard by adding hydrogen peroxide; j. adding an aliquiot of said standard to luminol reagent; k. measuring the emitted light level; and l. calculating the bacteria concentration.
Claim 27
Independent Claim
134 words
A method for the rapid assay of bacteria containing iron porphyrins in a water sample comprising treating said sample by: a. adding hydrogen peroxide to a portion of said water sample and allowing the mixture to stand; b. adding an aliquot of said mixture to luminol reagent; c. measuring the emitted light level; d. developing a blank by removing bacteria from another portion of said water sample; e. treating said blank by adding hydrogen peroxide; f. adding an aliquot of said blank to luminol reagent; g. measuring the emitted light level; h. developing a standard by a bacterial culture followed by a bacterial count; i. treating said standard by adding hydrogen peroxide; j. adding an aliquiot of said standard to luminol reagent; k. measuring the emitted light level; and l. calculating the bacteria concentration.
Claim 28
Dependent Claim
23 words
The method of claim 22 wherein the emitted light level measurements of steps "c" and "g" are made after a 6 second delay.
Claim 29
Dependent Claim
21 words
The method of claim 22 wherein said water sample undergoes bacterial concentration prior to said hydrogen peroxide addition of step "a."
Claim 30
Dependent Claim
16 words
The method of claim 24 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 31
Independent Claim
200 words
A method for the rapid assay of iron porphyrins in microorganisms contained in a water sample comprising treating said sample by: a. concentrating the microorganisms on a filter and retaining the filtrate; b. adding a sodium hydroxide-ethanol mixture to said filter and allowing said mixture to stand for a period of time sufficient to extract iron porphyrins from the microorganisms; c. collecting said sodium hydroxide-ethanol-iron porphyrin extract d. assaying said extract by mixing a portion of said extract with luminol reagent and measuring the emitted light level; e. developing a blank by passing a portion of said filtrate through a new filter; f. adding a sodium hydroxide-ethanol mixture to said new filter and allowing it to stand for a period of time sufficient for extraction of iron porphyrins, thereby simulating an extract; g. collecting said blank; h. assaying said blank by mixing a portion of said simulated extract with luminol reagent and measuring the emitted light level; i. developing a standard by a bacterial culture followed by a conventional bacteria count; j. assaying said standard by mixing a portion of said standard with luminol reagent and measuring the emitted light level; and k. calculating the bacteria concentration in said sample.
Claim 32
Independent Claim
200 words
A method for the rapid assay of iron porphyrins in microorganisms contained in a water sample comprising treating said sample by: a. concentrating the microorganisms on a filter and retaining the filtrate; b. adding a sodium hydroxide-ethanol mixture to said filter and allowing said mixture to stand for a period of time sufficient to extract iron porphyrins from the microorganisms; c. collecting said sodium hydroxide-ethanol-iron porphyrin extract d. assaying said extract by mixing a portion of said extract with luminol reagent and measuring the emitted light level; e. developing a blank by passing a portion of said filtrate through a new filter; f. adding a sodium hydroxide-ethanol mixture to said new filter and allowing it to stand for a period of time sufficient for extraction of iron porphyrins, thereby simulating an extract; g. collecting said blank; h. assaying said blank by mixing a portion of said simulated extract with luminol reagent and measuring the emitted light level; i. developing a standard by a bacterial culture followed by a conventional bacteria count; j. assaying said standard by mixing a portion of said standard with luminol reagent and measuring the emitted light level; and k. calculating the bacteria concentration in said sample.
Claim 33
Dependent Claim
17 words
The method of claim 26 wherein said sodium hydroxide-ethanol mixture is a 0.1 N NaOH--50% ethanol solution.
Claim 34
Dependent Claim
22 words
The method of claim 26 wherein all the emitted light levels are measured about six seconds after injection into said luminol reagent.
Claim 35
Independent Claim
185 words
A method for measuring iron porphyrins in a water sample and thereby determining bacterial concentration, comprising: a. forming reservoirs of a water sample, a blank, a standard, a hydrogen peroxide solution and luminol reagent; b. pumping said water sample and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; c. pumping said water sample--hydrogen peroxide solution mixture and said luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; d. pumping said blank and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; e. pumping the blank--hydrogen peroxide solution mixture and luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; f. pumping said standard and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; g. pumping said standard--hydrogen peroxide solution mixture and said luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; and h. calculating the bacterial concentration.
Claim 36
Independent Claim
185 words
A method for measuring iron porphyrins in a water sample and thereby determining bacterial concentration, comprising: a. forming reservoirs of a water sample, a blank, a standard, a hydrogen peroxide solution and luminol reagent; b. pumping said water sample and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; c. pumping said water sample--hydrogen peroxide solution mixture and said luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; d. pumping said blank and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; e. pumping the blank--hydrogen peroxide solution mixture and luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; f. pumping said standard and said hydrogen peroxide solution and thereafter mixing same for a first predetermined period of time; g. pumping said standard--hydrogen peroxide solution mixture and said luminol reagent and thereafter mixing same for a second predetermined period of time and measuring the emitted light level; and h. calculating the bacterial concentration.
Claim 37
Dependent Claim
17 words
The method of claim 29 wherein said pumping is accomplished by a plurality of peristaltic pump channels.
Claim 38
Dependent Claim
15 words
The method of claim 29 wherein said first predetermined period of time is 2 minutes.
Claim 39
Dependent Claim
15 words
The method of claim 29 wherein said second predetermined period of time is 6 seconds.
Claim 40
Dependent Claim
18 words
The method of claim 29 wherein said water sample is prefiltered prior to said water sample reservoir formation.
Claim 41
Dependent Claim
19 words
The method of claim 29 wherein said water sample undergoes bacterial concentration prior to said water sample reservoir formation.
Claim 42
Dependent Claim
16 words
The method of claim 34 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 43
Dependent Claim
22 words
The method of claim 29 wherein said water sample is prefiltered and undergoes bacterial concentration prior to said water sample reservoir formation.
Claim 44
Dependent Claim
16 words
The method of claim 36 wherein said bacterial concentration is accomplished by a hollow fiber membrane.
Claim 45
Dependent Claim
78 words
A chemiluminescent method for rapidly assaying bacteria in a water sample by detecting iron porphyrins in the water sample comprising treating the sample by: (a) first adding hydrogen peroxide to a portion of said water sample, then adding an aliquot of the sample to luminal reagent, and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the bacteria concentration.
Claim 46
Dependent Claim
78 words
A chemiluminescent method for rapidly assaying bacteria in a water sample by detecting iron porphyrins in the water sample comprising treating the sample by: (a) first adding hydrogen peroxide to a portion of said water sample, then adding an aliquot of the sample to luminal reagent, and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the bacteria concentration.
Claim 47
Dependent Claim
18 words
The method of claim 38 wherein bacteria are concentrated prior to treatment of the sample with hydrogen peroxide.
Claim 48
Dependent Claim
11 words
The method of claim 38 wherein said blank is deionized water.
Claim 49
Dependent Claim
17 words
The method of claim 38 wherein said standard is a known amount of bacteria in deionized water.
Claim 50
Dependent Claim
16 words
The method of claim 38 wherein emitted light measurements are taken after a six second delay.
Claim 51
Dependent Claim
86 words
A chemiluminescent method for rapidly assaying biomass contained in a saline water sample comprising treating the sample by; (a) filtering the biomass in the sample and retaining both the biomass on the filter and the filtrate, adding a sodium hydroxide-ethanol mixture to the filter and collecting the extract, mixing the extract with luminol reagent and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the biomass concentration.
Claim 52
Dependent Claim
86 words
A chemiluminescent method for rapidly assaying biomass contained in a saline water sample comprising treating the sample by; (a) filtering the biomass in the sample and retaining both the biomass on the filter and the filtrate, adding a sodium hydroxide-ethanol mixture to the filter and collecting the extract, mixing the extract with luminol reagent and measuring the emitted light level; (b) developing a blank and measuring the emitted light level; (c) developing a standard and measuring the emitted light level; and (d) calculating the biomass concentration.
Claim 53
Dependent Claim
23 words
The method of claim 43 wherein said blank employs the filtrate of the saline water followed by extraction with a sodium hydroxide-ethanol solution.
Claim 54
Dependent Claim
23 words
The method of claim 43 wherein said standard employs a known amount of catalase which has been added to a sodium hydroxide-ethanol solution.
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