Comparative phenotype analysis of two or more microorganisms using a plurality of substrates within a microwell device

• US 6,387,651 B1
• Filed: 05/18/2000
• Issued: 05/14/2002
• Est. Priority Date: 04/12/1995
• Status: Expired due to Term

• Current Assignee: Biolog, Inc.
• Original Assignee: Biolog, Inc.
• Inventors: Bochner, Barry, Panomitros, Eugenia
• Primary Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US09/574,087
• Time in Patent Office: 726 Days
• Field of Search: 435/4, 435/29, 435/34, 435/32, 435/810, 435/975, 435/7.31, 435/7.32
• US Class Current: 435/34
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 2304/20 Redox indicators
• CPC Class Codes: C12Q 2304/22 Resazurin; Resorufin
• CPC Class Codes: C12Q 2304/24 Tetrazolium; Formazan
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 2333/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/911 using fungi
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The present invention relates to growing and testing microorganisms in a multitest format which utilizes a gel forming matrix for the rapid screening of clinical and environmental cultures. The present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments (e.g., the present invention is particularly suited for the growth and characterization of the actinomycetes and fungi). The present invention is also particularly suited for comparative analysis of phenotypic differences between cell types, including strains of microorganisms that have been designated as the same genus and species, as well as other cell types (e.g., mammalian, insect, and plant cells).

Claims

1. A method for comparing at least two cell preparations, comprising the steps of:

a) providing a testing device comprising;

i) a plurality of testing wells, wherein said wells contain at least one test substrate selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements, and ii) at least one chromogenic compound;

b) preparing a first suspension comprising a first cell preparation and a second suspension comprising a second cell preparation, wherein said first and said second cell preparations are each in an aqueous solution comprising a gelling agent, under conditions such that said gelling agent remains ungelled;

c) introducing said first and second suspension into said wells of said testing device;

d) detecting the response of said first and second cell preparations to said at least one test substrate; and

e) comparing the response of said first and second cell preparations.

2. The method of claim 1, wherein said first and second cell preparations comprise microorganisms selected from the group consisting of bacteria and fungi.

3. The method of claim 1, wherein said first and second cell preparations contain cells of the same genus and species.

4. The method of claim 1, wherein said first and second cell preparations contain cells that differ in one or more genes.

5. The method of claim 1, wherein said at least one test substrate further comprises substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.

6. The method of claim 1, wherein said gelling agent is selected from the group consisting of gellan gum, carrageenan, and alginic acid.

7. The method of claim 1, wherein said testing device further comprises a gel-initiating agent, wherein said gel-initiating agent comprises cationic salts.

8. The method of claim 1, wherein said chromogenic compound is selected from the group consisting of chromogenic substrates, fluorogenic compounds, luminogenic compounds, oxidation-reduction indicators, and pH indicators.

9. The method of claim 8, wherein said oxidation-reduction indicators comprise tetrazolium.

10. The method of claim 8, wherein said oxidation-reduction indicators comprise redox purple.

11. The method of claim 1, wherein said testing device is at least one microplate.

12. The method of claim 1, wherein said response is a kinetic response.

13. A method for determining the phenotype differences in at least two cell preparations, comprising the steps of:

a) providing a testing device comprising;

i) a plurality of testing wells, wherein said wells contain one or more gel-initiating agents and at least one testing substrate selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements, and ii) at least one chromogenic compound;

b) preparing a first suspension comprising a first cell preparation, in an aqueous solution comprising a gelling agent, and a second suspension comprising a second cell preparation, in an aqueous solution comprising a gelling agent, under conditions such that said first and second suspensions remain ungelled;

c) introducing said first and second suspension into said wells of said testing device under conditions such that said first and second suspensions form a gel matrix within said wells and said first and second cell preparations are within said gel matrix;

d) detecting the response of said first and second cell preparations to said testing substrate; and

e) comparing the response of said first and second cell preparations.

14. The method of claim 13, wherein said first and second cell preparations comprise microorganisms selected from the group consisting of bacteria and fungi.

15. The method of claim 13, wherein said first and second cell preparations contain cells of the same genus and species.

16. The method of claim 13, wherein said first and second cell preparations contain cells that differ in one or more genes.

17. The method of claim 13, wherein said at least one test substrate further comprises substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.

18. The method of claim 13, wherein said gelling agent is selected from the group consisting of gellan gum, carrageenan, and alginic acid.

19. The method of claim 13, wherein said one or more gel-initiating agents comprise cationic salts.

20. The method of claim 13, wherein said chromogenic compound is selected from the group consisting of chromogenic substrates, fluorogenic compounds, luminogenic compounds, oxidation-reduction indicators, and pH indicators.

21. The method of claim 20, wherein said oxidation-reduction indicators comprise tetrazolium.

22. The method of claim 20, wherein said oxidation-reduction indicators comprise redox purple.

23. The method of claim 13, wherein said testing device is at least one microplate.

24. The method of claim 13, wherein said response is a kinetic response.

25. A kit for determining the phenotypes of at least two organisms, comprising:

i) a testing device comprising;

i) a plurality of wells, wherein said wells contain at least one testing substrate selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements, and ii) at least one chromogenic compound;

ii) a first aqueous suspension comprising a gelling agent; and

iii) a second aqueous suspension comprising a gelling agent.

26. The kit of claim 25, wherein said gelling agent is selected from the group consisting of gellan gum, carrageenan, and alginic acid.

27. The kit of claim 25, wherein said wells of said testing device further contain one or more gel-initiating agents.

28. The kit of claim 27, wherein said one or more gel-initiating agents comprise cationic salts.

29. The kit of claim 25, wherein said at least one testing substrate further comprises substrates selected from the group consisting of carbon sources, amino peptidase substrates, carboxy peptidase substrates, oxidizing agents, reducing agents, mutagens, amino acid analogs, sugar analogs, nucleoside analogs, base analogs, dyes, detergents, toxic metals, inorganics, and antimicrobials.

30. The kit of claim 25, wherein said chromogenic compound is selected from the group consisting of chromogenic substrates, fluorogenic compounds, luminogenic compounds, oxidation-reduction indicators, and pH indicators.

31. The kit of claim 30, wherein said oxidation-reduction indicators comprise tetrazolium.

32. The kit of claim 30, wherein said oxidation-reduction indicator comprise redox purple.

33. The kit of claim 25, wherein said testing device is selected from the group consisting of microtiter plates and miniaturized testing cards.