Medium composition, method and device for selectively enhancing the isolation of anaerobic microorganisms contained in a mixed sample with facultative microorganisms

• US 7,374,905 B2
• Filed: 11/08/2001
• Issued: 05/20/2008
• Est. Priority Date: 11/08/2000
• Status: Expired due to Term

• Current Assignee: Oxyrase, Inc.
• Original Assignee: Oxyrase, Inc.
• Inventors: Myers, Kathy J., Copeland, James C.
• Primary Examiner(s): Navarro; Mark
• Assistant Examiner(s): PORTNER, VIRGINIA ALLEN
• Application Number:
  US10/007,739
• Publication Number:
  US 20030138867A1
• Time in Patent Office: 2,385 Days
• Field of Search: 435/4, 435/15, 435/18, 435/25, 435/26, 435/139, 435/800, 435/262, 435/21, 435/31, 435/32, 435/34, 435/7.32, 435/283.1, 435/287.1, 435/288.3, 435/288.4, 435/289.1, 435/305.1, 435/305.2, 435/305.3, 435/39, 435/254.2, 435/168, 435/173.1, 435/173.9, 435/244, 435/257.1, 435/257.6, 195/103.5, 210/632, 210/717, 210/721, 210/759, 210/909, 438/253, 530/300
• US Class Current: 435/34
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12Q 1/045 Culture media therefor

Abstract

The present invention is directed to a medium, broth or agar, and a method of utilizing the same, in order to isolate and/or identify anaerobes from a mixed sample that contains facultative microorganisms. The medium contains an inhibitor of the electron transport system, such as a salt of azide (N₃⁻), cyanide (CN⁻) or related compounds. These inhibitors are present in an amount sufficient to limit the growth of facultative microorganisms under anaerobic conditions while not inhibiting the growth of the anaerobe microorganisms. Preferably, the inhibitor is present in the amount of from about 0.1 mg/ml to about 1.0 mg/ml in broth medium, and from about 0.01 mg/ml to 1.0 mg/ml in agar medium.

Claims

1. An anaerobic medium composition for the selective growth of anaerobes from a sample that contains at least facultative microorganisms and anaerobes, wherein said medium composition comprises a nutrient medium, a salt of an azide present in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the medium, and oxygen scavenging membrane fragments to create an anaerobic environment, wherein the membrane fragments are derived from a respiratory system of an organism sensitive to azide.

2. The medium composition of claim 1, wherein the medium comprises Brain Heart Infusion, Brucella, CDC Anaerobe, Nutrient, Schaedler, Thioglycollate, or Trypticase Soy.

3. The medium composition of claim 1, wherein the sample is obtained from

a. patients;

b. economically important animals;

or

c. pharmaceutical, or environmental sources.

5. A device for the transport of a sample that contains anaerobes and facultative microbes to enable the recovery of the anaerobes, wherein the device comprises the self-generating anaerobic medium composition of claim 1.

4. A method for the rapid recognition, isolation, or identification of anaerobes from a sample that contains at least facultative microorganisms and anaerobes comprising the following steps:

a. providing a liquid medium composition comprising a nutrient medium and a salt of an azide, wherein the azide is present in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the medium, and oxygen scavenging membrane fragments to create an anaerobic environment, wherein the membrane fragments are derived from the respiratory system of an organism sensitive to azide;

b. inoculating the sample into the liquid medium composition;

c. incubating the inoculated liquid medium composition;

d. determining the presence of growth in the inoculated liquid medium composition, with partial growth being indicative that an anaerobe is present; and

,

e. sampling the inoculated liquid medium composition for further characterization and isolation of the anaerobe organism.

6. A medium composition which allows for the selective growth of an anaerobe contained in a mixed sample also containing at least a facultative microbe comprising:

a microbiological nutrient medium containing a hydrogen donating substance, a plurality of oxygen scavenging membrane fragments which contain an electron transport system which reduces oxygen to water in the presence of a hydrogen donor, and an inhibitor of the electron transport system required for cellular respiration, wherein the inhibitor is present in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the medium, and wherein the oxygen scavenging membrane fragments are derived from respiring bacteria.

7. The medium composition of claim 6, wherein the hydrogen donating substance comprises an organic substrate.

8. The medium composition of claim 6, wherein the hydrogen donating substance comprises lactic acid, succinic acid, alpha-glycerol phosphate, formic acid or malic acid or any of their corresponding salts.

9. The medium composition of claim 6, wherein the oxygen scavenging membrane fragments are derived from the cytoplasmic membranes of Escherichia coli.

10. The medium composition of claim 6, wherein the inhibitor of the electron transport system comprises an azide or cyanide.

11. The medium composition of claim 6, wherein the inhibitor of the electron transport system comprises a salt of an azide or a cyanide.

12. The medium composition of claim 6, wherein the inhibitor of the electron transport system is sodium azide.

13. The medium composition of claim 6, wherein the microbiological nutrient medium comprises Brain Heart Infusion, Brucella, CDC Anaerobe, Nutrient, Schaedler, Thioglycollate or Trypticase Soy medium in broth or agar form.

14. A medium composition which restricts the growth of facultative microbes but not anaerobic microbes comprising a nutrient medium comprising a hydrogen donating organic substrate, one or more oxygen scavenging membrane fragments derived from the cytoplasmic membranes of bacteria, and an inhibitor of the electron transport system required for aerobic respiration in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the medium.

15. The medium composition of claim 14, wherein the oxygen scavenging membrane fragments are derived from the cytoplasmic membranes of Escherichia coli.

16. The medium composition of claim 14, wherein the inhibitor of the electron transport system comprises a salt of azide or cyanide.

17. The medium composition of claim 14, wherein the inhibitor is sodium azide.

18. A medium composition which restricts the exponential growth of facultative microbes but not anaerobic microbes comprising a base medium containing a hydrogen donating substrate, oxygen scavenging membrane fragments derived from the cytoplasmic membranes of Escherichia coli, and a salt of an azide in an amount of about 0.1 mg/ml to 1.0 mg/ml in the medium.

19. A method for the selective growth of an anaerobe from a sample containing a facultative microbe, said method comprising the steps of:

a. providing a medium composition comprising a nutrient medium containing a hydrogen donating substance, a salt of an azide in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the nutrient medium, and oxygen scavenging membrane fragments which contain an electron transport system which reduces oxygen to water in the presence of a hydrogen donor, wherein the membrane fragments are derived from the respiratory system of an organism normally sensitive to azide;

b. inoculating the medium composition with the sample; and

,

c. incubating the medium composition containing the sample.

20. A method for the selective growth of an anaerobe from a sample containing a facultative microbe, said method comprising the steps of:

a. providing an agar plate comprising a nutrient medium, a salt of an azide in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the agar plate nutrient medium, and oxygen scavenging membrane fragments which reduce oxygen to water wherein the membrane fragments are derived from a respiratory system of an organism normally sensitive to azide;

b. providing a liquid broth comprising a nutrient medium and a salt of an azide in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the liquid broth nutrient medium;

c. inoculating the liquid broth with the sample and thereafter incubating the inoculated broth;

d. inoculating the plated agar medium with the liquid broth containing the sample; and

,

e. incubating the plated agar medium inoculated with the liquid broth under anaerobic conditions thereby producing isolated colonies of the anaerobe free of facultative microbe.

21. The method of claim 20, further comprising the step of:

f. selecting isolated colonies of the anaerobes for characterization and identification.

22. A method for the selective enhancement of an anaerobe from a mixed sample also containing a facultative microorganism, said method comprising the steps of:

a. providing a liquid nutrient medium composition containing a biocatalytic oxygen reducing agent and a salt of an azide in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the liquid nutrient medium composition;

b. providing an agar plate comprising a nutrient medium, a salt of an azide in an amount of from about 0.1 mg/ml to 1.0 mg/ml in the agar plate nutrient medium, a biocatalytic oxygen reducing agent, and a hydrogen donating substance;

c. inoculating the liquid medium composition with the mixed sample and thereafter incubating the inoculated broth;

d. inoculating the agar plate with the liquid medium composition containing the mixed sample; and

e. incubating the agar plate containing the mixed sample under anaerobic conditions.

23. The method of claim 22, wherein the biocatalytic oxygen reducing agent comprises oxygen scavenging membrane fragments of bacteria normally sensitive to azide.

24. The method of claim 22, wherein the biocatalytic oxygen reducing agent comprises oxygen scavenging membrane fragments of mitochondrial organelles.

25. The method of claim 23, wherein the bacteria is Escherichia coli.

26. The method of claim 22, wherein the salt of an azide is sodium azide.