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

• US 6,046,021 A
• Filed: 06/16/1998
• Issued: 04/04/2000
• Est. Priority Date: 04/12/1995
• Status: Expired due to Term

• Current Assignee: Biolog, Inc.
• Original Assignee: Biolog, Inc.
• Inventors: Bochner, Barry
• Primary Examiner(s): Lankford, Jr., Leon B.
• Assistant Examiner(s): TATE, CHRISTOPHER ROBIN
• Application Number:
  US09/098,066
• Time in Patent Office: 658 Days
• Field of Search: 435/4, 435/29, 435/32, 435/34, 435/7.31, 435/7.32, 435/822, 435/911
• 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 2333/355 from Nocardia (G)
• CPC Class Codes: G01N 2333/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/911 using fungi

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 determining the phenotype differences in at least two cell preparations, comprising the steps of:

a) providing a testing device comprising a plurality of testing wells, wherein said wells contain a testing substrate and one or more gel-initiating agents;

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, such that 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.

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 gelling agent is selected from the group consisting of Gelrite™

, carrageenan, and alginic acid.

6. The method of claim 1, wherein said testing substrates are selected from the group consisting of carbon sources, nitrogen sources, sulfur sources, phosphorus 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.

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

8. The method of claim 1, further comprising a colorimetric indicator.

9. The method of claim 8, wherein said colorimetric indicator is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.

10. The method of claim 9, wherein said oxidation-reduction indicator is tetrazolium.

11. The method of claim 9, wherein said oxidation-reduction indicator is redox purple.

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

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

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

i) a microplate testing device containing a plurality of wells, wherein said wells contain one or more gel-initiating agents and one or more testing substrates;

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

iii) a second aqueous suspension comprising a gelling agent.

15. The kit of claim 14, wherein said testing substrates are selected from the group consisting of carbon sources, nitrogen sources, sulfur sources, phosphorus 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.

16. The kit of claim 14, wherein said gelling agent is selected from the group consisting of Gelrite™

, carrageenan, and alginic acid.

17. The kit of claim 14, wherein said gel initiating agent comprises cationic salts.

18. The kit of claim 14, wherein said testing device further comprises a colorimetric indicator selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.

19. The kit of claim 18, wherein said oxidation-reduction indicator is tetrazolium.

20. The kit of claim 18, wherein said oxidation-reduction indicator is redox purple.