Claims for Patent: 4,925,789

Summary for Patent: 4,925,789

Title:	Method and medium for use in detecting target microbes in situ in a specimen sample of a possibly contaminated material
Abstract:	The presence or absence of a predetermined target microbe in a sample is determined by adding a testing medium to the sample, or vice versa. The testing medium provides a selective growth medium for the target microbe and includes a specific nutrient which only the target microbe can metabolize. This specific nutrient is modified by attaching a sample-altering moiety thereto, thereby converting the nutrient to a nutrient-indicator. The sample-altering moiety is activated to alter the sample only if the specific nutrient is metabolized by the target microbe. The sample-altering moiety can be a material which changes the color of the sample (visible or non-visible) or changes an electrical characteristic of the sample, or alters some other detectable characteristic of the sample. The testing media does not have to be kept sterile, and the testing procedure does not have to be performed in a sterile environment.
Inventor(s):	Edberg; Stephen C. (Orange, CT)
Assignee:
Application Number:	06/880,305
Patent Claims:	1. A specific medium for combination with a specimen sample of a material suspected to be contaminated to determine the presence of absence of a target microbe in the specimen sample, and which can detect the presence of said target microbe without the need of performing a preliminary target microbe growth step, said medium comprising operative amounts of essential vitamins and elements needed to support growth of said target microbe and a nutrient-indicator which is the primary nutrient in the medium and which is substantially the only nutrient in said medium which can be metabolized by said target microbe to the extent needed to support continued reproductive growth thereof, and which cannot be metabolized by other viable microbes in the specimen, to that extent, said nutrient-indicator including a metabolizable moiety and a sample-altering moiety, the latter of which is released only when said nutrient-indicator is metabolized by said target microbe, whereupon a sensible characteristic of the sample is altered. 2. The medium of claim 1 further comprising an effective amount of antibiotic material to render any competing non-target microbes which may be present in the sample unable to metabolize said nutrient-indicator, said antibiotic material being ineffective against said target microbe. 3. The medium of claim 1, wherein said sample-altering moiety is a chromogen which, when released by metabolization of the nutrient-indicator, will alter the color of the specimen sample. 4. A specific medium for addition to a specimen sample of a material suspected to be contaminated with E. coli to determine the presence or absence of E. coli in the specimen sample, and which can detect the presence of E. coli without the need of performing a preliminary E. coli growth step, said medium comprising operative amounts of essential vitamins and elements needed to support growth of E. coli and a chromogenic substrate for .beta.-glucuronidase enzyme which is substantially the sole carbon source nutrient in the medium and which is metabolizable by E. coli to the extend needed to support continued reproductive growth thereof, and which, when metabolized, releases a chromogen which alters the color of the specimen there being no other nutrients in the medium which are able to support substantial reproductive growth of E. coli in the sample. 5. The medium of claim 4 wherein said chromogenic substrate of B-glucuronidase is a glucuronide selected from the group consisting of orthonitrophenyl-B-D-glucuronide, B-naphthalamide-B-D-glucuronide, alpha-napthol-B-D-glucuronide, methylumbilliferyl-B-D-glucuronide, bromo-chloro-indole-B-D-glucuronide, and mixtures thereof. 6. The medium of claim 4 further comprising an effective amount of antibiotic material operable to render any competing microbes incapable of metabolizing said chromogenic substrate. 7. The medium of claim 6 wherein said antibiotic material is selectively operable against gram positive bacteria. 8. A specific medium for addition to a specimen sample of a material suspected to be contaminated with gram negative bacteria to determine the presence or absence of gram negative bacteria in the specimen sample, and which can detect the presence of gram negative bacteria without the need of performing a preliminary gram negative bacteria growth step, said medium comprising operative amounts of essential vitamins and elements to support growth of gram negative bacteria, effective amounts of antibiotic material operable to render any yeast and gram positive bacteria in the specimen sample incapable of metabolizing nutrients, and a nutrient chromogenic substrate for L-alanine aminopeptidase which is substantially the sole carbon source nutrient in the medium and which is metabolizable by gram negative bacteria to the extend needed to support continued reproductive growth thereof, and which, when metabolized, releases a chromogen which alters the color of the specimen there being no other nutrients in the medium which are able to support substantial reproductive growth of gram negative bacteria in the sample. 9. The medium of claim 8 wherein said chromogenic substrate of L-alanine aminopeptidase is an alanine selected from the group consisting of L-alanine-B-orthonitrophenyl, B-napthalamide-B-L-alanine, alpha-napthol-B-L-alanine, methylumbilliferyl-B-L-alanine and mixtures thereof. 10. A specific medium for addition to a specimen sample of a material suspected to be contaminated to determine the presence or absence of a target microbe in the specimen sample, and which can detect the presence of said target microbe without the need of performing a preliminary target microbe growth step, said medium comprising operative amounts of essential vitamins and elements needed to support growth of said target microbe and a nutrient-indicator which is the primary nutrient in the medium and which is substantially the only nutrient in said medium which can be metabolized by said target microbe to the extent needed to support continued growth thereof, and, which cannot be metabolized by other viable microbes in the specimen to that extend, said nutrient-indicator including a metabolizable moiety and a sample-altering moiety the latter of which is released only when said nutrient-indicator is metabolized by said target microbe, whereupon a sensible characteristic of the sample is altered, said medium being in the form of a non-sterile, water-soluble powder. 11. A method of testing a specimen suspected to be contaminated to determine the presence or absence of a target microbe in the specimen, said method comprising the steps of: (a) obtaining at least one known volume sample of the specimen; (b) forming a specimen sample and medium mixture by adding to the specimen sample a predetermined amount of a medium which is soluble in the specimen sample and which contains effective amounts of vitamins and elements essential to growth of the target microbe, and a nutrient-indicator which is the primary nutrient in the medium and which is the only nutrient in the medium which the target microbe can metabolize to an extent needed to support continued reproductive growth thereof, and which cannot be metabolized by other viable microbes in the specimen sample to that extend, said nutrient-indicator including a metabolizable moiety and a sample altering moiety, said sample altering moiety being operable to alter a sensible, characteristic of the specimen sample when said nutrient-indicator is metabolized by the target microbe; and (c) monitoring the specimen sample and medium mixture for at least about twenty hours or until said characteristic has been altered to determine the presence or absence of the target microbe. 12. The method of claim 11, wherein said medium is added to a 1.0 ml sample of the specimen, to a 0.1 ml sample of the specimen, and a 0.01 ml sample of the specimen and each specimen sample and medium mixture is monitored for characteristic alteration to verify the concentration of said target microbe in the specimen. 13. The method of claim 12 further comprising the step of incubating the specimen sample-medium mixtures at a temperature in the range of about 20.degree. C. to about 44.5.degree. C. during monitoring thereof. 14. The method of claim 11 further comprising the step of adding to the specimen sample an effective amount of antibiotic material for rendering any microbes, other than the target microbe, incapable of metabolizing said nutrient-indicator. 15. A specific medium for the detection of a target micro-organism in a sample, and which can detect the presence of said target micro-organism without the need of performing a preliminary target micro-organism growth step, said medium comprising a two component mixture, a first component of which includes a combination of elements, vitamins and other essential nutritive materials required to support growth of the target micro-organism but said first component being unable by itself to allow substantial reproductive growth of the target micro-organism or any other viable organism in the sample; and a second component which comprises a primary nutrient ingredient, which is present in operative amounts to allow substantial growth of only the target micro-organism, said ingredient being in the form of a coupled nutrient-indicator such that when the nutrient is metabolized by the target micro-organism, a sensible characteristic of the sample is altered there being no other nutrients in the medium which are able to support substantial reproductive growth of the target micro-organisms in the sample. 16. A specific medium for combination with a specimen sample of a material to simultaneously detect total coliforms and E. coli in the sample, said medium comprising: operative amounts of vitamins and elements needed to support growth of total coliforms and E. coli; a first nutrient-indicator which is the primary nutrient in the medium for total coliforms and which is a nutrient which cannot be metabolized by other viable microbes in the sample to the extend needed to support continued reproductive growth thereof; and a second nutrient-indicator which is the primary nutrient in the medium for E. coli and which is a nutrient which cannot be metabolized by other viable microbes in the sample to the extent needed to support continued reproductive growth thereof; one of said nutrient indicators, when metabolized, producing a visible color change in the specimen, and the other of said nutrient-indicators, when metabolized, producing a normally non-visible color change in the specimen which is rendered visible upon exposure of the sample to an excitation light source there being no other nutrients in the medium which are able to support substantial reproductive growth of total coliforms and E. coli in the sample. 17. The specific medium of claim 16 wherein said first nutrient-indicator is a substrate for .beta.-galactosidase; and said second nutrient-indicator is a substrate for .beta.-glucuronidase. 18. A specific medium for combination with a specimen sample of a material to detect total coliforms in the sample, said medium comprising: operative amounts of vitamins and elements needed to support growth of total coliforms; and a nutrient-indicator which is the primary source of metabolizable carbon in the medium for total coliforms and which is a nutrient which cannot be metabolized by other viable microbes in the sample to the extend needed to support continued reproductive growth of said other viable microbes, said nutrient indicator, when metabolized, producing a visible color change in the specimen, and said nutrient indicator being a substrate for .beta.-galactosidase there being no other nutrients in the medium which are able to support substantial reproductive growth of total coliforms in the sample. 19. A non-sterile, water soluble powdery specific medium for determining the presence or absence of a target microbe in a specimen sample of water suspected to be contaminated, and which can detect the presence of said without the need of performing a preliminary target microbe growth step, said medium comprising about 10% to about 50% by weight of a nitrogen-containing compound, about 0.06% to about 1.0% by weight of amino acids essential to growth of said target microbe, about 0.09% to about 0.60% by weight of vitamins essential to growth of said target microbe, about 0.05% to about 0.70% by weight of elements essential to growth of said target microbe, about 6.4% to about 85% by weight of salts essential to growth of said target microbe, and about 1.20% to about 2.0% of a nutrient-indicator which is substantially the sole carbon source nutrient in the medium and which is the only nutrient in said medium which can be metabolized by said target microbe to the extent needed to support continued reproductive growth thereof and which is a nutrient which cannot be metabolized by other viable microbes in the sample to that extent, said nutrient-indicator, when metabolized by said target microbe, releasing a moiety which will alter a sensible characteristic of the sample.

Details for Patent 4,925,789

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2039-09-23