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Last Updated: March 29, 2024

Claims for Patent: 5,610,029


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Summary for Patent: 5,610,029
Title: Medium for detecting target microbes in a sample
Abstract:The invention features a medium for the detection of target microbes in a liquified environmental or biological sample in less than 18 hours.
Inventor(s): Ehrenfeld; Elizabeth (Falmouth, ME), Fricker; Colin (Reading, GB3), Townsend; David E. (Scarborough, ME)
Assignee: Idexx Laboratories, Inc. (Westbrook, ME)
Application Number:08/423,134
Patent Claims:1. A target microbe-specific medium which allows the detection of the presence or absence of a target microbe in a liquified environmental or biological sample comprising:

a) an effective amount of vitamin, amino acid, element and salt ingredients operable to allow viability and reproduction of said target microbe at a rate such that any detectable change in said sample observable within 18 hours for a sample with less than 10 target microbes per 100 ml; and

b) an effective amount of one or more nutrient-indicators provided in an amount sufficient to produce a detectable characteristic signal in the medium during growth of the target microbe; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to confirm the presence or absence of the target microbe in the sample.

2. The target microbe-specific medium of claim 1 wherein said nutrient indicator alters the color of said target microbe-specific medium.

3. The target microbe-specific medium of claim 2 wherein said nutrient indicator alters the color of said target microbe-specific medium in the visible wavelength range.

4. The target microbe-specific medium of claim 2 wherein said nutrient indicator comprises a moiety capable of fluorescing and detectable in a wavelength range visible after exposure to an excitation light source.

5. The target microbe-specific medium of claim 1 wherein said target microbe is a coliform.

6. The target microbe-specific medium of claim 5 wherein said coliform is E. coli.

7. The target microbe-specific medium of claim 1 wherein said nutrient indicator is a .beta.-D-glucuronidase substrate.

8. The target microbe-specific medium of claim 7 wherein said .beta.-D-glucuronidase substrate is selected from the group consisting of orthonitrophenyl-.beta.-D-glucuronide, .beta.-naphthalamide-.beta.-D-glucuronide, .alpha.-naphthol-.beta.-D-glucuronide, and 4-methylumbelliferyl-.beta.-D-glucuronide.

9. The target microbe-specific medium of claim 1 wherein said nutrient indicator is a .beta.-D-galactosidase substrate.

10. The target microbe-specific medium of claim 9 wherein said .beta.-D-galactosidase substrate is selected from the group consisting of orthonitrophenyl-.beta.-D-galactopyranoside and 4-methylumbelliferyl-.beta.-D-galactopyranoside.

11. The target microbe-specific medium of claim 1 wherein said nutrient indicator is a .beta.-glucosidase substrate.

12. The target microbe-specific medium of claim 1 wherein said nutrient indicator is a L-pyronidonyl aminopeptidase substrate.

13. The target microbe-specific medium of claim 1 wherein said nutrient indicator is a L-alanine aminopeptidase substrate.

14. The target microbe-specific medium of claim 1 further comprising an antibiotic which prevents non-target microbes from metabolizing said nutrient indicator.

15. The target microbe-specific medium of claim 14 wherein said antibiotic operates against gram positive microbes or non-target gram negative microbes.

16. The target microbe-specific medium of claim 1 wherein said medium is a non-sterile, water soluble powder.

17. The target microbe-specific medium of claim 1 further comprising an amount of sodium pyruvate sufficient to assist the recovery of metabolically injured cells.

18. The target microbe-specific medium of claim 1 comprising effective amounts of some or all of the following ingredients to allow detection of coliforms in a liquid sample 18 hours:

a) Nitrogen;

b) Amino acids necessary for or conducive to microbe growth selected from the group consisting of Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine;

c) Elements selected from the group consisting of Calcium, Chloride, Cobalt, Copper, Iron, Lead, Magnesium, Manganese, Phosphate, Potassium, Sodium, Sulfate, Sulfur, Tin, Zinc, Biotin, Choline, Folic Acid, Inositol, Nicotinic Acid, PABA, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, and Thymidine;

d) Vitamins selected from the group consisting of Biotin, Choline, Cyanocobalamin, Folic Acid, Inositol, Nicotinic Acid, PABA, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, Niacin and Thymidine;

e) one or more inducers of enzyme activity;

f) one or more nutrient indicators; and

g) one or more precursors of amino acids or DNA.

19. A medium comprising 4.5 to 5.5 g/liter ammonium sulfate or an equivalent amount of ammonium ions, one or more buffers in an amount equivalent to 6.0 to 7.5 g/liter HEPES free acid, 4.7 to 5.8 g/liter HEPES sodium salt, 0.12 to 0.17 g/liter D-gluconic acid, 0.036 to 0.044 g/liter sodium sulfite or an eouivalent source of sulfite ions, 0.001 to 0.003 g/liter amphotericin B or an equivalently effective amount of an antifungal agent, 0.09 to 0.11 g/liter magnesium sulfate or an equivalent source of magnesium ions, 0.450 to 0.550 g/liter ONPG or an equivalent amount of a nutrient indicator, 0.05 to 0.10 g/liter MUG or an equivalent amount of a nutrient indicator, 0.0004 to 0.0006 g/liter zinc sulfate (heptahydrate) or an equivalent source of zinc ions, and 0.0004 to 0.0006 g/liter manganese sulfate or an equivalent source of manganese ions; Alanine at least 0.025 g/liter, Arginine at least 0.030 g/liter, Aspartic Acid at least 0.056 g/liter, Glutamic Acid at least 0.1021 g/liter, Glycine at least 0.015 g/liter, Cystine at least 0.002 g/liter, Histidine at least 0.0058 g/liter, Isoleucine at least 0.0145 g/liter, Leucine at least 0.0301 g/liter, Lysine at least 0.0342 g/liter, Methionine at least 0.0119 g/liter, Phenylalanine at least 0.0181 g/liter, Serine at least 0.0287 g/liter, Threonine at least 0.0181 g/liter, Tryptophan at least 0.0036 g/liter, Tyrosine at least 0.0064 g/liter, and Valine at least 0.0235 g/liter; and Iron (at least 0.00165 mg/l) Calcium, Phosphate, Potassium, Sodium, Chloride, Cobalt, Copper, Lead and Manganese; and Biotin at least 0.0005 mg/l, Choline at least 0.05 mg/l, Folic Acid at least 0.00075 mg/l, Inositol at least 0.06 mg/l, Niacin at least 0.00385 mg/l, PABA at least 0.020 mg/l, Pantothenic Acid at least 0.01095 mg/l, Pyridoxine at least 0.0015 mg/l, Riboflavin at least 0.0028 mg/l, Thiamine at least 0.0037 mg/l, Thymidine at least 0.010 mg/l, and Cyanocobalamin in trace amounts.

20. A target microbe specific medium comprising:

a) 4.5 to 5.5 g/liter ammonium sulfate or an equivalent amount of ammonium ions, one or more buffers in amounts equivalent to 6.0 to 7.5 g/liter HEPES free acid and 4.7 to 5.8 g/liter HEPES sodium salt, 0.13 to 0.16 g/liter D-gluconic acid, 0.036 to 0.044 g/liter sodium sulfite or an equivalent amount of sulfite ions, 0.0009 to 0.0011 g/liter amphotericin B or an equivalently effective amount of antifungal agent, 0.09 to 0.11 g/liter magnesium sulfate or an equivalent amount of magnesium ions, one or more nutrient indicators in amounts equivalent to 0.450 to 0.550 g/liter ortho-nitrophenyl-.beta.-D-galactopyranoside (ONPG) or 0.05 to 0.10 g/liter 4-methylumbelliferyl-.beta.-D-glucuronide (MUG)), 0.0004 to 0.0006 g/liter zinc sulfate or an equivalent amount of zinc ions, and 0.0004 to 0.0006 g/liter manganese sulfate or an equivalent amount of manganese ions, a nitrogen content of at least 1.1 to 1.7 g/liter; and

b) amino acids required for growth of the target microbe, in effective amounts to promote growth, selected from the group consisting of: Alanine, Arginine, Aspartic Acid, Glutamic Acid, Glycine, Proline, Cystine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Serine, Threonine, Tryptophan, Tyrosine, and Valine; and

c) one or more agents in amounts sufficient to aid growth of microbes selected from the group consisting of: Iron (at least 0.00165 mg/l), Calcium, Phosphate, Potassium, Sodium, Chloride, Cobalt, Copper, Lead and Manganese; and

d) one or more vitamins in amounts sufficient to aid growth of microbes selected from the group consisting of: Biotin, Choline, Cyanocobalamin, Folic Acid, Inositol, Nicotinic Acid, Niacin, PABA, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, and Thymidine; and

e) one or more agents active to inhibit growth of non-coliform microbes.

21. A method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample comprising the step of thermally equilibrating said liquid sample with a target microbe-specific detection medium having a nutrient indicator operable to alter a detectable characteristic of the sample if said target microbe is present in said sample to incubation temperature in a water bath prior to placing said liquid sample in a dry incubator and monitoring the sample to determine whether said detectable characteristic is altered.

22. The method of claim 21 wherein said incubation temperature is 30.degree. to 41.degree. C. and is maintained for more than 10 minutes.

23. A method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample within 18 hours comprising the steps of:

a) mixing said liquid sample with a medium which includes an effective amount of via, amino acid, element and salt ingredients operable to allow viability and reproduction of said target microbe in the presence of a nutrient-indicator; and an effective amount of a nutrient-indicator which is provided in an amount sufficient to support growth of said target organism until a detectable characteristic signal is produced in the medium during growth; and said nutrient-indicator being operable to alter a detectable characteristic of the sample if metabolized by the target microbe so as to cinform the presence or absence of the target microbe in the sample;

b) warming said liquid sample and medium mixture to an incubation temperature of 30.degree. to 41.degree. C. and said liquid sample is maintained at said incubation temperature for 10 minutes or more; and

c) monitoring the sample to determine whether said detectable characteristic has been altered.

24. The method of claim 23 wherein said medium further comprises an effective amount of at least one antibiotic which inhibits growth of one or more potential non-target microbes.

25. The method of claim 23 wherein said medium comprises:

a) Nitrogen;

b) Amino acids necessary for or conducive to microbe growth selected from the group consisting of Alanine, Arginine, Aspartic Acid, Cystine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine;

c) Elements selected from the group consisting of Calcium, Chloride, Cobalt, Copper, Iron, Lead, Magnesium, Manganese, Phosphate, Potassium, Sodium, Sulfate, Sulfur, Tin, Zinc, Biotin, Choline, Folic Acid, Inositol, Nicotinic Acid, PABA, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, and Thymidine;

d) Vitamins selected from the group consisting of Biotin, Folic Acid, Pyridoxine, Riboflavin, Thiamine, Vitamin B12, Niacin and Pantothenic Acid;

e) one or more inducers of enzyme activity;

f) one or more nutrient indicators; and

g) one or more precursors of amino acids or DNA.

26. The method of claim 23 wherein said medium comprises:

a) 4.5 to 5.5 g/liter ammonium sulfate, one or more buffers in amounts equivalent to 6.0 to 7.5 g/liter HEPES free acid and 4.7 to 5.8 g/liter HEPES sodium salt, 0.13 to 0.16 g/liter D-gluconic acid, 0.036 to 0.044 g/liter sodium sulfite, 0.0009 to 0.0011 g/liter amphotericin B, 0.09 to 0.11 g/liter magnesium sulfate, one or more nutrient indicators in amounts equivalent to 0.450 to 0.550 g/liter ortho-nitrophenyl-.beta.-D-galactopyranoside (ONPG) or 0.05 to 0.10 g/liter 4-methylumbelliferyl-.beta.-D-glucuronide (MUG)), 0.0004 to 0.0006 g/liter zinc sulfate, and 0.0004 to 0.0006 g/liter manganese sulfate, a nitrogen content of at least 1.1 to 1.7 g/liter including an amino nitrogen content of at least 0.06 g/liter; and

b) amino acids required for growth of the target microbe, in effective amounts to promote growth, selected from the group consisting of: Alanine, Arginine, Aspartic Acid, Glutamic Acid, Glycine, Proline, Cystine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Serine, Threonine, Tryptophan, Tyrosine, and Valine; and

c) one or more agents in amounts sufficient to aid growth of microbes selected from the group consisting of: Iron (at least 0.00165 mg/l), Calcium (at least 0.0003 g/liter), Phosphate (at least 0.0005 g/liter), Potassium (at least 0.004 g/liter), Sodium (at least 0.03 g/liter), Chloride, Cobalt, Copper, Lead and Manganese; and

d) one or more vitamins in amounts sufficient to aid growth of microbes selected from the group consisting of: Biotin, Choline, Cyanocobalamin, Folic Acid, Inositol, Nicotinic Acid, Niacin, PABA, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, and Thymidine; and

e) one or more agents active to inhibit growth of non-coliform microbes.

27. A method for detecting the presence or absence of a target microbe in an environmental or biological liquid sample comprising the steps of mixing a target microbe specific medium of any of claims 1-20 with said liquid sample to form a liquid mixture, and incubating the mixture at a temperature sufficient to allow detection of said target microbe in 18 hours when said sample has less than 10 target microbes per 100 ml.

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