Rapid read-out biological indicator

• US 6,623,955 B2
• Filed: 05/18/1995
• Issued: 09/23/2003
• Est. Priority Date: 11/29/1988
• Status: Expired due to Term

• Current Assignee: 3M Innovative Properties Company (3M Company)
• Original Assignee: 3M Innovative Properties Company (3M Company)
• Inventors: Foltz, William E., Matner, Richard R., Woodson, Lewis P.
• Primary Examiner(s): LEFFERS JR, GERALD G
• Application Number:
  US08/443,820
• Publication Number:
  US 20030157588A1
• Time in Patent Office: 3,050 Days
• Field of Search: 422/55, 422/56, 422/58, 422/60, 422/61, 422/83, 422/86, 422/101, 435/31, 435/38, 435/808, 435/810, 435/288.1, 435/282.4, 435/18, 435/19, 435/21, 435/23, 435/24
• US Class Current: 435/287.4
• CPC Class Codes: C12Q 1/22 Testing for sterility conditions
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A rapid method of determining the efficacy of a sterilization cycle, and an indicator adapted to perform such method, comprising subjecting to the sterilization cycle a source of active enzyme having activity which correlates with the viability of a microorganism commonly used to monitor sterilization, and incubating the enzyme source, following the completion of the sterilization cycle, with an effective amount of a substrate system capable of reacting with any residual active enzyme to produce a detectable enzyme-modified product.

Claims

1. A method of detecting the presence of viable microorganisms after a sterilization cycle comprising the steps of:

(a) exposing a source of active enzyme to a sterilization cycle;

(b) contacting the active enzyme after the sterilization cycle with an aqueous buffer and a substrate which is specific for and reacts with the active enzyme at about 20-70°

C. to form an enzyme modified product;

(c) adding a color developer which is specific for and reacts with the enzyme modified product to generate a color in the presence of viable microorganisms at about 20-70°

C.;

(d) determining the existence of said color by visual means in about ten minutes to about 60 minutes; and

(e) correlating the existence of said color with the presence of viable microorganisms.

2. The method of claim 1 wherein the viable microorganisms are bacterial spores.

3. The method of claim 2 wherein the viable microorganisms are selected from the group consisting of Bacillus stearothermophilus spores and Bacillus subtilis spores.

4. The method of claim 1 wherein the substrate is selected from the group consisting of:

5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside;

4-methylumbelliferyl-a-D-glucoside;

L-tyrosine-7-amido-4-methylcoumarin;

L-leucine-7-amido-4-methylcoumarin;

L-alanine-7-amido-4-methylcoumarin;

L-phenylalanine-7-amido-4-methylcoumarin;

L-proline-7-amido-4-methylcoumarin;

L-leucyl-2-naphthylamide;

L-valyl-2-naphthylamide; and

L-cystyl-2-naphthylamide.

5. The method of claim 1 wherein the sterilization cycle uses a sterilization means selected from the group consisting of saturated steam, dry heat, radiation, and ethylene oxide.

6. The method of claim 1 wherein steps (b) and (c) are performed between about 20°

C. and about 30°

C.

7. The method of claim 1 wherein the aqueous buffer consists of tris (hydroxymethyl)(aminomethane HCl) in water at a pH of 7.5-9.5.

8. The method of claim 1 wherein the source of the viable microorganisms is impregnated onto a filter.

9. The method of claim 1 wherein the source of active enzyme is the viable microorganisms.

10. In a method of detecting the presence of viable microorganisms after the completion of a sterilization cycle, wherein a source of active enzyme is exposed to the sterilization cycle, is contacted with a substrate specific for the active enzyme after the sterilization cycle to form an enzyme modified product, and then is contacted with a means for detecting the enzyme modified product as an indication of the presence of viable microorganisms, the improvement comprising:

(I) using an aqueous buffer and a substrate which forms a complex with said active enzyme at 20-70°

C.;

(II) using a color developer which reacts with the enzyme modified product to generate a color at 20-70°

C. as the means of detecting the product, determining the existence of said color by visual means in about 10 minutes to about 60 minutes; and

(III) correlating the existence of said color with the presence of viable microorganisms.

11. An indicator for detecting the presence of viable microorganisms after a sterilization cycle comprising:

(a) a filter containing a source of active enzyme;

(b) an aqueous buffer;

(c) a substrate specific for the active enzyme, which substrate reacts with the active enzyme at 20-70°

C.; and

(d) a color developer specific for the substrate, which developer reacts with the substrate in the presence of the active enzyme to generate a color at 20-70°

C. which can be detected by visual means.