US5079144A
Expired / Lapsed
US
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Microorganism testing with a hydrolyzable fluorogenic substrate
Assignee: Siemens Healthcare Diagnostics Inc
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Filing: 1990-03-21
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Granted: 1992-01-07
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Expires: 2010-03-21
Abstract
Abstract A test for microbial growth is carried out by culturing a microorgnism to be tested in a lliquid culture medium containing an enzymatically hydrolyzable fluorogenic substrate for a period of less than about 7 hours, measuring fluorescence of the culture medium resulting from hydrolysis of the fluorogenic substrate by growth of the microorganism, continuing culturing of the microorganism in the culture medium for an additional period, and measuring microorganism growth by a non-fluorescent method as a confirmatory test of growth determined from fluorescence. Preferably, the additional culture period is overnight and the non-fluorescent method is by visual inspection. The culture medium may be in the wall of a microtitre plate, and the culture medium can contain an antibiotic to test for sensitivity of a microorganism to the antibiotic or to test for minimum inhibitory concentration of the antibiotic.
Inventors
- 👤 Anthony H. Carr
- 👤 Robert A. Badley
- 👤 Ian Jobling
- 👤 Thomas J. Sands
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 18:07
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12Q1/18; C12Q2334/00; C12Q2334/20; C12Q2334/22; C12Q2337/00; C12Q2337/20; C12Q2337/22; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S435/81
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
144 words
A method for carrying out a microbial culture test comprising innoculating a microorganism under test into a liquid culture medium including at least one enzymatically hydrolysable fluorogenic substrate contained in a well of a microtitre plate cultivating the innoculated microorganism in the liquid culture medium by incubation for a period of less than about 7 hours, thereafter measuring fluorescence yield of the incubated liquid culture, medium resulting from enzymatic hydrolysis of the fluorogenic substrate by microbial growth to detect growth of the microorganism continuing the incubation in the microtitre plate well of the same portion of incubated liquid culture medium on which the fluorescent yield was measured for a further, overnight, culture period to permit further growth of the microorganism under test and effecting a confirmatory test of culture growth of the microorganism by visual inspection for a button of growth in the well.
Claim 2
Independent Claim
144 words
A method for carrying out a microbial culture test comprising innoculating a microorganism under test into a liquid culture medium including at least one enzymatically hydrolysable fluorogenic substrate contained in a well of a microtitre plate cultivating the innoculated microorganism in the liquid culture medium by incubation for a period of less than about 7 hours, thereafter measuring fluorescence yield of the incubated liquid culture, medium resulting from enzymatic hydrolysis of the fluorogenic substrate by microbial growth to detect growth of the microorganism continuing the incubation in the microtitre plate well of the same portion of incubated liquid culture medium on which the fluorescent yield was measured for a further, overnight, culture period to permit further growth of the microorganism under test and effecting a confirmatory test of culture growth of the microorganism by visual inspection for a button of growth in the well.
Claim 3
Dependent Claim
28 words
A method according to claim 1, in which the incubation for less than about 7 hours is carried out for a period in the range of 3.5-5 hours.
Claim 4
Dependent Claim
17 words
A method according to claim 1, in which the fluorogenic substrate is a hydrolysable derivative of 4-methyl-coumarin.
Claim 5
Dependent Claim
24 words
A method according to claim 3, in which the concentration of said fluorogenic substrate is in the range of 30 micromolar to 300 micromolar.
Claim 6
Dependent Claim
29 words
A method according to claim 1, in which said fluorescence yield of said culture is measured by a fluorimeter having a xenon lamp light source and a photomultiplier detector.
Claim 7
Dependent Claim
46 words
A method according to claim 1, in which said fluorescence yield of said culture with a culture volume in the range of 50-100 microliters is measured by a fluorimeter having a range of sensitivity capable of detecting 0-14 micromolar concentration of fluorophor in said culture volume.
Claim 8
Dependent Claim
36 words
A method according to claim 1, for carrying out a test for sensitivity of the microorganism under test to an antibiotic agent, in which the liquid culture medium contains the antibiotic agent throughout the culture period.
Claim 9
Dependent Claim
57 words
A method according to claim 7, for carrying out a bacteriological M.I.C. (minimum inhibitory concentration) test wherein a series of microtitre plate wells is used to form a series of liquid culture mediums containing a dilution series of an antibiotic, and from growth or lack of growth in each well assessing a M.I.C level for the antibiotic.
Claim 10
Dependent Claim
59 words
A method according to claim 1, in which the liquid culture medium contains at least one fluorogenic substrate so as to yield fluorescent production upon growth of each one of the following microorganisms: Bacillus subtilis Staphylococcus aureus Streptococcus pyogenes Streptococcus faecalis Klebsiella edwardsii Klebsiella pneumoniae Proteus vulgaris Proteus mirabilis Escherichia coli Pseudomonas aeruginosa Citrobacter freundii Serratia marcescens Salmonella anatum.
Claim 11
Dependent Claim
37 words
A method according to claim 1, in which at least one fluorogenic substrate selected from the group consisting of 4-methyl-umbelliferyl phosphate, 4-methyl-umbelliferyl C 6-16 fatty acid ester, and 7-(N)-L-(aminoacyl or peptidyl)-7-amido-4-methyl-coumarin, is included in the culture medium.
Claim 12
Dependent Claim
27 words
A method according to claim 8, in which the incubation for less than about 7 hours carried out for a period in the range of 3.5-5 hours.
Claim 13
Dependent Claim
17 words
A method according to claim 8, in which the fluorogenic substrate is a hydrolysable derivative of 4-methyl-coumarin.
Claim 14
Dependent Claim
24 words
A method according to claim 8, in which the concentration of said fluorogenic substrate is in the range of 30 micromolar to 300 micromolar.
Claim 15
Dependent Claim
59 words
A method according to claim 8, in which the liquid culture medium contains at least one fluorogenic substrate so as to yield fluorescent production upon growth of each one of the following microorganisms: Bacillus subtilis Staphylococcus aureus Streptococcus pyogenes Streptococcus faecalis Klebsiella edwardsii Klebsiella pneumoniae Proteus vulgaris Proteus mirabilis Escherichia coli Pseudomonas aeruginosa Citrobacter freundii Serratia marcenscens Salmonella anatum.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-01-06 | 1995-07-08 | 1996-01-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-01-06 | 1999-07-08 | 2000-01-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-01-06 | 2003-07-08 | 2004-01-07 | $7700.00 | Window Closed / Lapsed |