US5464755A
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US
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Microbiological medium and method of assay
Assignee: BOCHNER BARRY PHD
•
Filing: 1994-04-29
•
Granted: 1995-11-07
•
Expires: 2014-04-29
Abstract
Abstract The present invention is directed to methods and media for the isolation and presumptive identification of various bacteria. In particular, the organisms commonly associated with urinary tract infections are distinguished based on their colonial morphology and color.
Inventors
- 👤 Barry Bochner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 18:09
CPC Classifications
C; C12; C12N; C12N1/00; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12N1/20
Claims Analysis
Total Claims: 55
Claim 1
Independent Claim
188 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate capable of forming a second color upon reacting with arylsulfatase; iii) a milk-derived, opaque reagent; and iv) a tryptophan-containing compound capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said bacterial organisms which generate one or more of said first, second and third colors; and c) examining said test medium for i) the presence of bacterial colonies having said first color, ii) the presence of bacterial colonies having said second color; iii) the presence of bacterial colonies having said third color; iv) the presence of bacterial colonies without said first, second or third colors; and v) the presence of a zone of clearing surrounding said bacterial colonies.
Claim 2
Independent Claim
188 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate capable of forming a second color upon reacting with arylsulfatase; iii) a milk-derived, opaque reagent; and iv) a tryptophan-containing compound capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said bacterial organisms which generate one or more of said first, second and third colors; and c) examining said test medium for i) the presence of bacterial colonies having said first color, ii) the presence of bacterial colonies having said second color; iii) the presence of bacterial colonies having said third color; iv) the presence of bacterial colonies without said first, second or third colors; and v) the presence of a zone of clearing surrounding said bacterial colonies.
Claim 3
Dependent Claim
26 words
The method of claim 1, wherein said medium further comprises at least one coordinating compound selected from the group consisting of manganese, copper and iron ions.
Claim 4
Dependent Claim
20 words
The method of claim 1, further comprising the step of assaying for bacterial organisms having the ability to hydrolyze urea.
Claim 5
Dependent Claim
43 words
The method of claim 3, wherein said assaying for bacterial organisms having the ability to hydrolyze urea comprises the steps of i) placing said bacterial organisms in a solution comprising urea and a pH indicator; and ii) examining for the production of color.
Claim 6
Dependent Claim
42 words
The method of claim 4, wherein the concentration of urea in said solution is approximately five percent and the concentration of said pH indicator is approximately 0.05 percent, and the pH of said solution is adjusted to approximately pH 4 to 7.
Claim 7
Dependent Claim
30 words
The method of claim 5, wherein said pH indicator is selected from the group comprising m-cresol purple, phenol red, thymol blue, bromthymol blue, bromcresol purple, xylenol blue, and cresol red.
Claim 8
Dependent Claim
20 words
The method of claim 1, further comprising the step of assaying for bacterial organisms having the ability to reduce tellurite.
Claim 9
Dependent Claim
51 words
The method of claim 7, wherein said assaying for bacterial organisms having the ability to reduce tellurite comprises the steps of i) placing drops of a tellurite salt solution on colonies suspected of being Enterococcus faecalis; ii) incubating said bacterial organisms; and iii) examining for the production of a black color.
Claim 10
Dependent Claim
16 words
The method of claim 8, wherein said tellurite salt solution comprises approximately one percent potassium tellurite.
Claim 11
Dependent Claim
36 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Escherichia coli, visible to the eye as red in color, with a zone of clearing in the medium surrounding the colonies.
Claim 12
Dependent Claim
38 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Klebsiella pneumoniae, visible to the eye as indigo in color and mucoid, with a zone of clearing in the medium surrounding the colonies.
Claim 13
Dependent Claim
35 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Salmonella choleraesuis, visible to the eye as brown in color, with brown pigment diffusing from the colonies into the medium.
Claim 14
Dependent Claim
39 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Proteus mirabilis, visible to the eye as orange in color, with irregular spreading edges and orange pigment diffusing from the colonies into the medium.
Claim 15
Dependent Claim
46 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Klebsiella oxytoca, visible to the eye as yellow in color, with yellow pigment diffusing from the colonies into the medium, and a zone of clearing in the medium surrounding the colonies.
Claim 16
Dependent Claim
46 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Pseudomonas aeruginosa, visible to the eye as green in color, with green pigment diffusing from the colonies into the medium, and a zone of clearing in the medium surrounding the colonies.
Claim 17
Dependent Claim
36 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Staphylococcus aureus, visible to the eye as yellow in color, with a zone of clearing in the medium surrounding the colonies.
Claim 18
Dependent Claim
38 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Enterococcus faecalis, visible to the eye as small and white in color, with a zone of clearing in the medium surrounding the colonies.
Claim 19
Dependent Claim
35 words
The method of claim 1, wherein said bacterial colonies growing on said test medium are Salmonella choleraesuis, visible to the eye as brown in color, with brown pigment diffusing from the colonies into the medium.
Claim 20
Independent Claim
135 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate capable of forming a second color upon reacting with arylsulfatase; and iii) tryptophan capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said organisms which generate one or more of said first, second and third colors; c) examining said test medium for the presence of bacterial colonies having said first, second, or third color.
Claim 21
Independent Claim
135 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate capable of forming a second color upon reacting with arylsulfatase; and iii) tryptophan capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said organisms which generate one or more of said first, second and third colors; c) examining said test medium for the presence of bacterial colonies having said first, second, or third color.
Claim 22
Dependent Claim
26 words
The method of claim 19, wherein said medium further comprises at least one coordinating compound selected from the group consisting of manganese, copper and iron ions.
Claim 23
Dependent Claim
20 words
The method of claim 19, further comprising the step of assaying for bacterial organisms having the ability to hydrolyze urea.
Claim 24
Dependent Claim
43 words
The method of claim 21, wherein said assaying for bacterial organisms having the ability to hydrolyze urea comprises the steps of i) placing said bacterial organisms in a solution comprising urea and a pH indicator; and ii) examining for the production of color.
Claim 25
Dependent Claim
40 words
The method of claim 22, wherein the concentration of urea in said solution is approximately five percent and the concentration of said pH indicator is approximately 0.05 percent, and the pH of said solution is approximately pH 4 to 7.
Claim 26
Dependent Claim
30 words
The method of claim 23, wherein said pH indicator is selected from the group comprising m-cresol purple, phenol red, thymol blue, bromthymol blue, bromcresol purple, xylenol blue, and cresol red.
Claim 27
Dependent Claim
20 words
The method of claim 19, further comprising the step of assaying for bacterial organisms having the ability to reduce tellurite.
Claim 28
Dependent Claim
51 words
The method of claim 25, wherein said assaying for bacterial organisms having the ability to reduce tellurite comprises the steps of i) placing drops of a tellurite salt solution on colonies suspected of being Enterococcus faecalis; ii) incubating said bacterial organisms; and iii) examining for the production of a black color.
Claim 29
Dependent Claim
16 words
The method of claim 26, wherein said tellurite salt solution comprises approximately one percent potassium tellurite.
Claim 30
Dependent Claim
25 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Escherichia coli, visible to the eye as red in color.
Claim 31
Dependent Claim
27 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Klebsiella pneumoniae, visible to the eye as indigo in color and mucoid.
Claim 32
Dependent Claim
35 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Salmonella choleraesuis, visible to the eye as brown in color, with brown pigment diffusing from the colonies into the medium.
Claim 33
Dependent Claim
39 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Proteus mirabilis, visible to the eye as orange in color, with irregular spreading edges and orange pigment diffusing from the colonies into the medium.
Claim 34
Dependent Claim
32 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Klebsiella oxytoca, visible to the eye as yellow in color, with yellow pigment diffusing from the colonies.
Claim 35
Dependent Claim
32 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Pseudomonas aeruginosa, visible to the eye as green in color, with green pigment diffusing from the colonies.
Claim 36
Dependent Claim
25 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Staphylococcus aureus, visible to the eye as yellow in color.
Claim 37
Dependent Claim
27 words
The method of claim 19, wherein said bacterial colonies growing on said test medium are Enterococcus faecalis, visible to the eye as small and white in color.
Claim 38
Independent Claim
195 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate selected from the group comprising 6-chloro-3-indolyl-β-D-glucuronide, and 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate, indoxyl-3-sulfate, capable of forming a second color upon reacting with arylsulfatase; iii) an opaque reagent containing casein; iv) a tryptophan-containing compound capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said organisms which generate one or more of said first, second and third colors; c) examining said test medium for i) the presence of bacterial colonies having said first color, ii) the presence of bacterial colonies having said second color; iii) the presence of bacterial colonies having said third color; iv) the presence of bacterial colonies without said first, second or third colors; and v) the presence of a zone of clearing surrounding said bacterial colonies.
Claim 39
Independent Claim
195 words
A method for detecting the presence of bacterial organisms in a test sample suspected of containing bacterial organisms comprising the steps of: a) inoculating a solid test medium with said test sample, wherein said medium comprises: i) a chromogenic β-glucuronidase substrate selected from the group comprising 6-chloro-3-indolyl-β-D-glucuronide, and 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, capable of forming a first color upon reacting with β-glucuronidase; ii) a chromogenic arylsulfatase substrate, indoxyl-3-sulfate, capable of forming a second color upon reacting with arylsulfatase; iii) an opaque reagent containing casein; iv) a tryptophan-containing compound capable of forming a third color upon reacting with at least one bacterial enzyme, wherein said first, second and third colors are visually distinguishable; b) incubating said test medium to produce bacterial colonies of said organisms which generate one or more of said first, second and third colors; c) examining said test medium for i) the presence of bacterial colonies having said first color, ii) the presence of bacterial colonies having said second color; iii) the presence of bacterial colonies having said third color; iv) the presence of bacterial colonies without said first, second or third colors; and v) the presence of a zone of clearing surrounding said bacterial colonies.
Claim 40
Dependent Claim
26 words
The method of claim 36, wherein said medium further comprises at least one coordinating compound selected from the group consisting of manganese, copper and iron ions.
Claim 41
Dependent Claim
20 words
The method of claim 36, further comprising the step of assaying for bacterial organisms having the ability to hydrolyze urea.
Claim 42
Dependent Claim
43 words
The method of claim 38, wherein said assaying for bacterial organisms having the ability to hydrolyze urea comprises the steps of i) placing said bacterial organisms in a solution comprising urea and a pH indicator; and ii) examining for the production of color.
Claim 43
Dependent Claim
40 words
The method of claim 39, wherein the concentration of urea in said solution is approximately five percent and the concentration of said pH indicator is approximately 0.05 percent, and the pH of said solution is approximately pH 4 to 7.
Claim 44
Dependent Claim
30 words
The method of claim 40, wherein said pH indicator is selected from the group comprising m-cresol purple, phenol red, thymol blue, bromthymol blue, bromcresol purple, xylenol blue, and cresol red.
Claim 45
Dependent Claim
20 words
The method of claim 36, further comprising the step of assaying for bacterial organisms having the ability to reduce tellurite.
Claim 46
Dependent Claim
51 words
The method of claim 42, wherein said assaying for bacterial organisms having the ability to reduce tellurite comprises the steps of i) placing drops of a tellurite salt solution on colonies suspected of being Enterococcus faecalis; ii) incubating said bacterial organisms; and iii) examining for the production of a black color.
Claim 47
Dependent Claim
16 words
The method of claim 43, wherein said tellurite salt solution comprises approximately one percent potassium tellurite.
Claim 48
Dependent Claim
36 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Escherichia coli, visible to the eye as red in color, with a zone of clearing in the medium surrounding the colonies.
Claim 49
Dependent Claim
38 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Klebsiella pneumoniae, visible to the eye as indigo in color and mucoid, with a zone of clearing in the medium surrounding the colonies.
Claim 50
Dependent Claim
35 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Salmonella choleraesuis, visible to the eye as brown in color, with brown pigment diffusing from the colonies into the medium.
Claim 51
Dependent Claim
39 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Proteus mirabilis, visible to the eye as orange in color, with irregular spreading edges and orange pigment diffusing from the colonies into the medium.
Claim 52
Dependent Claim
43 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Klebsiella oxytoca, visible to the eye as yellow in color, with yellow pigment diffusing from the colonies, and a zone of clearing in the medium surrounding the colonies.
Claim 53
Dependent Claim
43 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Pseudomonas aeruginosa, visible to the eye as green in color, with green pigment diffusing from the colonies, and a zone of clearing in the medium surrounding the colonies.
Claim 54
Dependent Claim
36 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Staphylococcus aureus, visible to the eye as yellow in color, with a zone of clearing in the medium surrounding the colonies.
Claim 55
Dependent Claim
38 words
The method of claim 36, wherein said bacterial colonies growing on said test medium are Enterococcus faecalis, visible to the eye as small and white in color, with a zone of clearing in the medium surrounding the colonies.
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