Rapid detection of
A device for differentially enumerating colonies of coliform and Escherichia coli microorganisms is provided. The device comprises a water-impermeable first sheet; a water-impermeable second sheet attached to the first sheet; a dry, rehydratable culture medium comprising a lactose-fermentation indicator system, a β-D-glucuronidase indicator system, and a first cold-water soluble gelling agent adhered to the first sheet, the culture medium disposed in a microbial growth zone; and a second cold-water-soluble gelling agent adhered to the second sheet. The microbial growth zone is disposed between the first sheet and the second sheet. The first sheet and second sheet are configured to retard passage of carbon dioxide therethrough. Methods of using the device are also provided.
1 . A device for differentially enumerating colonies of coliform and Escherichia coli microorganisms, the device comprising:
a water-impermeable first sheet;
a water-impermeable second sheet attached to the first sheet;
a dry, rehydratable culture medium adhered to the first sheet;
wherein the culture medium comprises:
organonitrogenous nutrients that facilitate growth of coliform microorganisms;
a lactose-fermentation indicator system comprising D-lactose, a first inducer compound that enhances production of β-galactosidase, and a pH indicator;
a β-D-glucuronidase indicator system comprising 5-bromo-4-chloro-3-indolyl-β-D-glucuronide and at least one compound that enhance β-glucuronidase activity in E. coli;
a redox indicator;
an effective amount of at least one agent that selectively inhibits growth of non-coliform microorganisms; and
a first cold-water soluble gelling agent;
a microbial growth zone disposed between the first sheet and the second sheet, wherein an area of the culture medium adhered to the first sheet defines the growth zone; and
a second cold-water-soluble gelling agent adhered to the second sheet;
wherein the first sheet is attached to the second sheet so that the culture medium is facing the second cold-water-soluble gelling agent;
wherein the first sheet and second sheet are configured to facilitate the trapping of carbon dioxide gas;
wherein the culture medium adhered to the first sheet has a coating weight of about 130 mg/100 cm 2 to about 195 mg/100 cm 2 ;
wherein lactose is disposed in the dry culture medium adhered to the first sheet, wherein the lactose is present in the dry culture medium; and
wherein the second sheet has a lower carbon dioxide permeability than 40 um-thick biaxially-oriented polypropylene (BOPP).
2 . The device of claim 1 , further comprising D-glucuronic acid disposed in the microbial growth zone.
3 . The device of claim 1 , wherein the first sheet, the second sheet, or both the first and second sheets comprise a polyester film.
4 . The device of claim 3 , wherein the second sheet comprises
polyethylene terephthalate or biaxially-oriented polypropylene;
Wherein the first sheet comprises polypropylene, polystyrene or photoprint paper with a polyethylene.
5 . The device of claim 1 , wherein the growth zone is defined by a spacer adhered to the first sheet.
6 . The device of claim 1 , wherein the first cold-water soluble gelling agent comprises guar gum.
7 . The device of claim 1 , wherein said D-lactose is present in said culture medium in an amount of about 3 g/1000 mL deionized water to about 10 g/1000 mL deionized water.
8 . The device of claim 1 , wherein the culture medium further comprises a reagent for buffering the culture medium, when reconstituted with an aqueous liquid, at a pH between about 6.5 and about 7.5.
9 . The device of claim 1 , wherein the pH indicator comprises a sulfonephthalein pH indicator.
10 . The device of claim 1 , wherein the organonitrogenous nutrients are selected from a group consisting of yeast extract, porcine peptones, an enzymatic digest of gelatin, an enzymatic digest of animal peptone, and a combination of any two or more of the foregoing organonitrogenous nutrients.
11 . The device of claim 1 , wherein the growth zone is configured to receive an aqueous liquid having a predetermined volume of about 1 milliliter to about 5 milliliters and, upon receiving the predetermined volume, forms a hydrated culture medium that facilitates growth and enumeration of coliform colonies.
12 . The device of claim 1 , wherein the agent that selectively inhibits growth of non-coliform microorganisms is selected from a group consisting of bile salts, sodium dodecyl sulfate, and combinations thereof.
13 . The device of claim 1 , wherein the second cold-water-soluble gelling agent is disposed on the second sheet in the form of dry particles or dry agglomerated particles.
14 . The device of claim 1 , wherein the first inducer compound that enhances production of β-galactosidase comprises isopropyl-β-D-thiogalactopyranoside.
15 . The device of claim 1 , wherein the first inducer compound that enhances production of β-glucuronidase enzyme activity comprises methyl-β-D-glucuronide.
16 . The device of claim 1 , wherein the β-D-glucuronidase indicator system comprises a plurality of compounds that enhance β-glucuronidase enzyme activity in E. coli.
17 . The device of claim 16 , wherein either a first compound of the plurality of compounds that enhance β-glucuronidase enzyme activity in E. coli is selected from methyl-β-D-glucuronide and phenyl-β-D-glucuronide, or wherein a second compound of the plurality of compounds that enhance β-glucuronidase enzyme activity in E. coli is D-glucuronic acid, or wherein a first compound of the plurality of compounds that enhance β-glucuronidase enzyme activity in E. coli is selected from methyl-β-D-glucuronide and phenyl-β-D-glucuronide and wherein a second compound of the plurality of compounds that enhance β-glucuronidase enzyme activity in E. coli is D-glucuronic acid.
18 . The device of claim 1 , further comprising dry infant formula disposed in the microbial growth zone.
19 . The device of claim 1 , wherein the redox indicator is tetrazolium chloride.
20 . The device of claim 10 , wherein said yeast extract is present in said culture medium in an amount of about 5 g/1000 mL deionized water to about 10 g/1000 mL deionized water.
21 . The device of claim 10 , wherein said sodium dodecyl sulfate is present in said culture medium in an amount of about 0.05 g/1000 mL deionized water to about 0.15 g/1000 mL deionized water.