Article and method for detecting aerobic bacteria

Abstract

A thin film culture device for detecting aerobic bacteria in a sample is provided. The culture device comprises a self-supporting substrate sheet having a first major surface and a second major surface; a cover sheet attached to the substrate sheet, a sample-receiving zone disposed between the substrate sheet and the cover sheet, a first layer comprising a substantially dry, cold-water-soluble first hydrogel-forming composition adhered to a portion of the sample-receiving zone; and a plurality of indicator agents disposed in at least one layer adhered to the substrate sheet or the cover sheet. The plurality of indicator agents comprises three indicator agents for detecting distinct glycosidase enzyme activities, an indicator agent for detecting an alkyl esterase enzyme activity, an indicator agent for detecting a phosphatase enzyme activity, and a redox indicator. A method of using the culture device is also provided.

1 . A device for culturing and detecting microorganisms, the device comprising:
 a self-supporting substrate sheet having a first major surface and a second major surface;   a cover sheet attached to the substrate sheet;   a sample-receiving zone disposed between the substrate sheet and the cover sheet;   a first layer comprising a substantially dry, cold-water-soluble first hydrogel-forming composition adhered to the first major surface of the substrate sheet; and   a plurality of indicator agents, the plurality of indicator agents comprising:
 three enzyme activity indicator reagents for detecting distinct glycosidase enzyme activities; 
 an enzyme activity indicator reagent for detecting an alkyl esterase enzyme activity; 
 an enzyme activity indicator reagent for detecting a phosphatase enzyme activity; 
 a redox indicator comprising a tetrazolium dye; 
   wherein each of the plurality of enzyme activity indicator reagents comprises a detectable reporter group;   wherein each of the plurality of indicator agents is disposed in at least one layer adhered to the substrate sheet or the cover sheet, wherein the at least one layer is in fluid communication with the sample-receiving zone when a predetermined volume of aqueous liquid is deposited in the sample-receiving zone.   
     
     
         2 . The device of  claim 1 , further comprising a second layer comprising a first adhesive composition disposed between the substrate sheet and the first layer. 
     
     
         3 . The device of  claim 2 , further comprising an air-permeable membrane adhered to the substrate sheet. 
     
     
         4 . The device of  claim 1 , wherein the first hydrogel-forming composition further comprises a nutrient to facilitate growth of an aerobic bacterium, wherein the first hydrogel-forming composition is adhered to at least a portion of the substrate sheet or the cover sheet, wherein the portion is in fluid communication with the sample-receiving zone. 
     
     
         5 . The device of  claim 1 , wherein the cover sheet comprises a first major surface, wherein the first major surface of the cover sheet faces the first major surface of the substrate sheet, wherein the culture device further comprises:
 a third layer comprising a second adhesive composition, wherein the third layer is adhered to a portion of the cover sheet; and   a fourth layer comprising a substantially dry, cold-water-soluble second hydrogel-forming composition, wherein the fourth layer is adhered to the third layer.   
     
     
         6 . The device of  claim 1 , wherein at least one of the plurality of indicator agents is disposed in the first adhesive composition, the second adhesive composition, the first hydrogel-forming composition, and/or the second hydrogel-forming composition. 
     
     
         7 . The device of  claim 6 , wherein at least three of the plurality of indicator agents are disposed in the first adhesive composition and/or the second adhesive composition. 
     
     
         8 . The device of  claim 1 , wherein the first hydrogel-forming composition, and/or the second hydrogel-forming composition comprises a mixture of gelling agents. 
     
     
         9 . The device of  claim 1 , wherein the at least three enzyme activity indicator reagents for detecting distinct glycosidase enzyme activities include a compound to detect alpha-glucosidase enzyme activity, a compound to detect beta-glucosidase enzyme activity, and a compound to detect beta-galactosidase enzyme activity. 
     
     
         10 . The device of  claim 9 , wherein the at least three enzyme activity indicator reagents for detecting distinct glycosidase enzyme activities comprise 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside or a salt thereof, 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside or a salt thereof, and 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside or a salt thereof. 
     
     
         11 . The device of  claim 1 , wherein the enzyme activity indicator reagent for detecting an alkyl esterase enzyme activity comprises 3-indolyl-acetate or a salt thereof. 
     
     
         12 . The device of  claim 1 , wherein the enzyme activity indicator reagent for detecting a phosphatase enzyme activity comprises 5-bromo-4-chloro-3-indolyl-phosphate or a salt thereof. 
     
     
         13 . The device of  claim 1 , further comprising a predefined volume of aqueous liquid disposed between the substrate sheet and the cover sheet in the sample receiving zone. 
     
     
         14 . The device of  claim 1 , wherein the first hydrogel-forming composition or second hydrogel-forming composition further comprises an effective amount of at least one nutrient for growing an aerobic bacterium. 
     
     
         15 . The device of  claim 1 , further comprising a nutrient selected from the group consisting of L-arginine, skim milk, D-trehalose, and a combination of any two or more of the foregoing nutrients. 
     
     
         16 . The device of  claim 1 , wherein the first hydrogel-forming composition or the second hydrogel-forming composition comprises substantially dry agglomerated powders. 
     
     
         17 . A method of detecting an aerobic bacterium in a sample, the method comprising:
 contacting a sample material and an aqueous liquid in the sample-receiving zone of the device of  claim 1  to form an inoculated culture device;   incubating the inoculated culture device for a period of time; and   detecting a bacterial colony in the inoculated culture device.   
     
     
         18 . The method of  claim 17 , wherein detecting a bacterial colony in the inoculated culture device comprises detecting in the inoculated culture device a presence of a formazan dye or the detectable reporter group of at least one of the indicator agents, wherein detecting the presence of the formazan dye or the detectable reporter group is indicative of a presence of a colony of bacteria. 
     
     
         19 . The method of  claim 17 , wherein contacting a sample material with the first hydrogel-forming composition or second hydrogel-forming composition of the device comprises placing the sample in fluid communication with a nutrient to facilitate growth of an aerobic bacterium. 
     
     
         20 . The method of  claim 17 , wherein incubating the inoculated culture device for a period of time comprises incubating the inoculated culture device for about 22 hours to about 26 hours, inclusive.