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

• US 6,686,173 B2
• Filed: 01/14/2002
• Issued: 02/03/2004
• 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:
  US10/047,048
• Publication Number:
  US 20020110848A1
• Time in Patent Office: 750 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 first testing device and a duplicate second testing device, each comprising a plurality of testing wells and at least two test substrates selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements, and wherein each well contains at least one of said test substrates;

b) preparing a first suspension comprising a first cell preparation, in an aqueous solution, and a second suspension comprising a second cell preparation in an aqueous solution;

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

d) detecting the response of said first and second cell preparations to said at least two test substrates, wherein said response is detected using a single chromogenic compound; 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 testing substrates further comprise 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 chromogenic compound is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.

7. The method of claim 6, wherein said oxidation-reduction indicator is tetrazolium.

8. The method of claim 6, wherein said oxidation-reduction indicator is redox purple.

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

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

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

i) a first testing device and a duplicate second testing device, each containing a plurality of wells and at least two test substrates selected from the group consisting of nitrogen sources, phosphorus sources, sulfur sources, and auxotrophic supplements, and wherein each well contains at least one of said test substrates;

ii) a single chromogenic compound for detecting the response of said organisms to said at least two test substrates;

iii) a first aqueous suspension; and

iv) a second aqueous suspension.

12. The kit of claim 11, wherein said testing substrates further comprise 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.

13. The kit of claim 11, wherein said chromogenic compound is selected from the group consisting of chromogenic substrates, oxidation-reduction indicators, and pH indicators.

14. The kit of claim 13, wherein said oxidation-reduction indicator is tetrazolium.

15. The kit of claim 13, wherein said oxidation-reduction indicator is redox purple.

16. The kit of claim 11, wherein said testing device is selected from the group consisting of microtiter plates and miniaturized testing cards.