Culture device and methods for enumerating mold colonies

Abstract

A thin film culture device for enumerating mold colonies is provided. The device comprises water-resistant first and second substrates with a growth region disposed therebetween, a dry, cold water-soluble gelling agent disposed in the growth region, and an effective amount of a calcium-chelating compound disposed in the growth region. The effective amount of calcium-chelating compound is capable of reducing a rate of lateral enlargement of the colony-forming unit growing in the culture device relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region, wherein reducing the rate of lateral enlargement of the colony-forming unit does not substantially delay detection of the colony. A corresponding method is also provided.

1 . A thin-film culture device, comprising:
 a water-resistant first substrate;   a water-resistant second substrate;   a growth region disposed between the first and second substrates;   a dry, cold-water-soluble gelling agent disposed in the growth region; and   an effective amount of calcium-chelating compound disposed in the growth region;   wherein, when the growth region is hydrated with a predetermined volume of aqueous liquid and inoculated with a colony-forming unit of a mold species, the effective amount of calcium-chelating compound is capable of reducing a rate of lateral enlargement of the colony-forming unit growing in the culture device relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region;   wherein reducing the rate of lateral enlargement of the colony-forming unit does not substantially delay detection of the colony.   
     
     
         2 . The thin-film culture device of  claim 1 , further comprising a calcium salt disposed in the growth region. 
     
     
         3 . The thin film culture device of  claim 1 , wherein the calcium-chelating compound and/or the calcium salt, if present, is disposed in a first dry coating. 
     
     
         4 . The thin film culture device of  claim 1 , wherein the dry, cold-water-soluble gelling agent is disposed in the first dry coating and/or in a second dry coating. 
     
     
         5 . The thin film culture device of  claim 1 , wherein the effective amount of calcium-chelating compound is sufficient to reduce a first average colony diameter of a mold microorganism by at least 30%, relative to a second average colony diameter of the mold microorganism grown in a substantially identical thin film culture device that lacks the calcium-chelating compound, after 48 hours of incubation at a temperature suitable for growing the mold microorganism. 
     
     
         6 . The thin film culture device of  claim 1 , wherein the calcium-chelating compound comprises ethylenediamine tetraacetic acid, ethyleneglycol tetraacetic acid, or citrate. 
     
     
         7 . The thin film culture device of  claim 6 , wherein the calcium-chelating compound comprises disodium ethylenediamine tetraacetic acid dihydrate, wherein the disodium ethylenediamine tetraacetic acid dihydrate is disposed in a dry coating in the growth region at a coating density of about 0.5 mg to about 12 mg/100 cm 2 . 
     
     
         8 . The thin film culture device of  claim 1 , further comprising a nutrient medium to support growth of a mold microorganism. 
     
     
         9 . The thin film culture device of  claim 1 , further comprising an indicator reagent. 
     
     
         10 . The thin film culture device of  claim 1 , further comprising a predefined volume of an aqueous liquid, wherein the effective amount of the calcium-chelating compound disposed in the growth region is dissolved in the aqueous liquid at a concentration effective to reduce a rate lateral enlargement of a colony of a species of mold growing in the culture device that contains the effective amount disposed in the growth region relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region. 
     
     
         11 . The thin film culture device of  claim 1 , further comprising an air-permeable membrane adhered to the first substrate, wherein the air-permeable membrane is substantially coextensive with the growth region of the culture device. 
     
     
         12 . A method for enumerating microorganisms, comprising:
 forming an inoculated culture medium in a growth region of a thin film culture device comprising an effective amount of calcium-chelating compound disposed in the growth region;   incubating the inoculated culture medium for a predetermined period of time sufficient to form a macroscopically-detectable colony of a mold microorganism; and   counting a number of macroscopically-detectable colonies of mold microorganisms in the growth region;   wherein forming the inoculated culture medium comprises hydrating the growth region with a predetermined volume of aqueous liquid;   wherein, when the growth region is hydrated with the predetermined volume of aqueous liquid and inoculated with a colony-forming unit of a mold species, the calcium-chelating compound reduces a rate of lateral enlargement of the colony-forming unit growing in the culture device relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region;   wherein reducing the rate of lateral enlargement does not substantially delay detection of the colony.   
     
     
         13 . The method of  claim 12 , wherein the effective amount of calcium-chelating compound does not substantially delay detection of a yeast microorganism, if present, that would otherwise be detected within the predetermined period of time. 
     
     
         14 . The method of  claim 13 , further comprising counting a number of macroscopically-detectable yeast colonies in the growth region. 
     
     
         15 . The method of  claim 12 :
 wherein forming an inoculated culture medium comprises depositing an aqueous sample into the thin film culture device;   wherein, prior to depositing the aqueous sample, the thin film culture device comprises a nutrient medium, an indicator reagent, and/or the effective amount of the calcium-chelating compound.   
     
     
         16 . The method of  claim 12 , wherein incubating the inoculated culture medium for a predetermined period of time comprises incubating the inoculated culture medium between about 40 hours and about 96 hours. 
     
     
         17 . The method of  claim 12 , wherein counting a number of macroscopically-detectable colonies comprises obtaining an image of a growth region of the thin film culture device. 
     
     
         18 . A kit, comprising:
 a thin film culture device for growing and enumerating mold microorganisms; and   a container holding a predetermined quantity of a calcium-chelating compound;   wherein a portion of the predetermined quantity, when disposed in a growth region of the culture device after the device is inoculated, is sufficient to reduce a rate of lateral enlargement of a colony-forming unit of a mold species growing in the culture device relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region;   wherein reducing the rate of lateral enlargement of the colony-forming unit does not substantially delay detection of the colony.   
     
     
         19 . The kit of  claim 18 , wherein the thin film culture device comprises:
 a water-resistant first substrate;   a water-resistant second substrate;   a growth region disposed between the first and second substrates;   a dry, cold water-soluble gelling agent disposed in the growth region; and   an effective amount of a calcium-chelating compound disposed in the growth region;   wherein, when the growth region is hydrated with a predetermined volume of aqueous liquid and inoculated with a colony-forming unit of a mold species, the effective amount of calcium-chelating compound is capable of reducing a rate of lateral enlargement of the colony-forming unit growing in the culture device relative to the rate of lateral enlargement of a colony of the same mold species growing in an otherwise identical culture device that does not contain the effective amount disposed in the growth region;   wherein reducing the rate of lateral enlargement of the colony-forming unit does not substantially delay detection of the colony.   
     
     
         20 . The kit of  claim 18 , further comprising a reagent selected from the group consisting of a nutrient, a detection reagent, a selective agent, a buffering agent, a dye, and a mixture comprising any two or more of the foregoing reagents.