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US-12624379-B2 - Rapid detection of E. coli in a thin film culture device

US12624379B2US 12624379 B2US12624379 B2US 12624379B2US-12624379-B2

Abstract

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.

Inventors

  • Sailaja Chandrapati
  • Alec J. TEAGARDEN
  • Haley A. SADDORIS

Assignees

  • NEOGEN FOOD SAFETY US HOLDCO CORPORATION

Dates

Publication Date
20260512
Application Date
20180402

Claims (20)

  1. 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. 2 . The device of claim 1 , further comprising D-glucuronic acid disposed in the microbial growth zone.
  3. 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. 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. 5 . The device of claim 1 , wherein the growth zone is defined by a spacer adhered to the first sheet.
  6. 6 . The device of claim 1 , wherein the first cold-water soluble gelling agent comprises guar gum.
  7. 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. 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. 9 . The device of claim 1 , wherein the pH indicator comprises a sulfonephthalein pH indicator.
  10. 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. 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. 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. 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. 14 . The device of claim 1 , wherein the first inducer compound that enhances production of β-galactosidase comprises isopropyl-β-D-thiogalactopyranoside.
  15. 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. 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. 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. 18 . The device of claim 1 , further comprising dry infant formula disposed in the microbial growth zone.
  19. 19 . The device of claim 1 , wherein the redox indicator is tetrazolium chloride.
  20. 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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a national stage filing under 35 U.S.C. 371 of PCT/US2018/025636, filed Apr. 2, 2018, which claims the benefit of U.S. Provisional Application No. 62/480,630, filed Apr. 3, 2017, the disclosure of which is incorporated by reference in its/their entirety herein. BACKGROUND In food and beverage safety testing, the presence or absence of coliform bacteria is considered to be important evidence of quality. The amount of coliform bacteria permitted in beverages and in certain foods (for example, dairy products) is regulated in many countries around the world. Coliform bacteria include fecal coliforms, such as Escherichia coli. The presence of fecal coliforms in a food or water sample is used as a primary indicator of fecal contamination of the food or water and of the possible presence of other pathogenic microorganisms. Methods for detecting, identifying, and/or enumerating microorganisms in food samples often vary according to the nature of the food and the types of microorganisms that are likely to be found in the samples. Compendia of methods for testing food samples include Standard Methods for the Examination of Dairy Products, 27.sup.th Edition, published by The American Public Health Association, Washington, D.C., and the Bacteriological Analytical Manual (“BAM”), published by the U.S. Food and Drug Administration, Washington, D.C. Solid foods are usually suspended in aqueous media and mixed and/or pulverized to obtain a liquid homogenate of the food material, which can be used in methods of quantitative microbial analysis. The above-referenced methods, however, are generally relatively expensive, involve multiple steps, require about 24- to 48-hour incubation periods before colonies can be counted, and/or require relatively sophisticated instrumentation and/or relatively highly trained personnel. There is a need for faster methods for enumerating viable coliform and E. coli microorganisms in food and beverage samples. SUMMARY In general, the present disclosure relates to detection and enumeration of microorganisms in a sample. In particular, the present disclosure relates to culture devices that can be used to grow colony-forming units (CFUs) of coliform bacteria in a semi-solid culture medium and to differentially enumerate coliform CFUs and E. coli CFUs. The inventive culture device is particularly useful for detecting and differentiating colonies of weakly-aerogenic E. coli, thereby providing more-accurate counts of the microorganisms present in a sample. In addition, the culture medium used in the device provides faster detection and enumeration of coliform bacteria and E. coli than prior thin-film culture devices The present disclosure also provides methods of using the culture devices. The inventive methods disclosed herein provide for growth, detection, and differentiation of coliform and E. coli bacteria. The methods permit faster and more-accurate enumeration of coliform and E. coli colonies than prior methods that used thin-film culture devices for enumerating coliform and E. coli colonies. In one aspect, the present disclosure provides a device for differentially enumerating colonies of coliform and Escherichia coli microorganisms. The device can comprise a water-impermeable first sheet; a water-impermeable second sheet attached to the first sheet; a dry, rehydratable culture medium comprising a first cold-water soluble gelling agent adhered to the first sheet; a microbial growth zone disposed between the first sheet and the second sheet; a lactose-fermentation indicator system disposed in the microbial growth zone; a β-D-glucuronidase indicator system disposed in the growth zone; a redox indicator system and a second cold-water-soluble gelling agent adhered to the second sheet. The culture medium can comprise organonitrogenous nutrients that facilitate growth of coliform microorganisms, a lactose-fermentation indicator system, a β-D-glucuronidase indicator system, a redox indicator and an effective amount of at least one agent that selectively inhibits growth of non-coliform microorganisms. An area of the culture medium adhered to the first sheet defines the growth zone. The lactose-fermentation indicator system comprises D-lactose, a first inducer compound that enhances production of β-galactosidase, and a pH indicator. The β-D-glucuronidase indicator system comprises 5-bromo-4-chloro-3-indolyl-β-D-glucuronide and a at least one compound that enhances production of β-glucuronidase enzyme activity. The redox indicator system preferably comprises Tetrazolium chloride (TTC) The first sheet is attached to the second sheet so that the culture medium is facing the second cold-water-soluble gelling agent. The first sheet and second sheet are configured to retard passage of carbon dioxide therethrough. In any of the above embodiments, the device comprises a plurality of compounds that enhance β-glucuronidase activity in E. coli, wherein th