Compositions for the isolation and quantitation of bacillus cereus in thin film dehydrated culture medium

Abstract

This present disclosure is directed to testing food, beverages, and environmental matrices for the presence of microbes.

Description

COMPOSITIONS FOR THE ISOLATION AND QUANTITATION OF BACILLUSCEREUS IN THIN FILM DEHYDRATED CULTURE MEDIUMCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 654,850, filed on May 31, 2024, the contents of which are hereby incorporated by reference in its entirety.

[0002] FIELD OF THE INVENTION

[0003] The field of the invention relates generally to testing food, beverages, and environmental matrices for the presence of microbes.

[0004] BACKGROUND

[0005] Biochemical characteristics of bacteria serve as a fingerprint for identification and are often used as markers to identify the presence or absence of target organisms in samples. Enzyme markers are of particular interest since they can be queried using many tools in the present day. This is especially true in the food industry where the detection of certain classes of organisms serves as an indicator of food quality and safety. The specific enzymes and subsequent biochemical characteristics produced by organism classes of interest can be detected via multiple modalities often involving the use of chromogenic or fluorogenic indicators. When incorporated into growth media, these indicators, especially the precipitating kind are particularly useful since they illuminate the colonies via color or light that enables their easy visualization and localization. This feature enables the end user to quantify the number of colonies on the growth medium thereby enabling the determination of levels of the target organisms in the test sample.

[0006] Organisms belonging to the Bacillus cereus group are Gram-positive bacteria that are ubiquitous in the environment and are commonly isolated from the soil, water, air and foodproducts especially dried and processed food stuffs such as grains, cereals, pastas, flours, spices and powdered milks, amongst others. These organisms can form spores which enable them to be resistant to a wide variety of food processing steps including thermal treatments, drying etc. Foods contaminated with spores of . cereus, especially at levels known to cause food poisoning, are problematic and therefore the easy detection of B. cereus in these food stuffs is of utmost importance.

[0007] Agar based media containing egg yolk such as the MYP or PEMBA are recommended by standard methods such as ISO or FDA-BAM for the isolation of B. cereus group of organisms from foodstuffs. These agar media use the egg yolk reaction as a substrate for the enzyme lecithinase which produces a white precipitate around the colony of the B. cereus group of organisms on the surface of the agar medium. Several constraints exist with the preparation of these agar media. For instance, the egg yolk is heat labile and cannot be added to the liquid agar medium prior to sterilization. As such, the agar media must be sterilized and subsequently cooled and tempered to 50 °C at which point the egg yolk is added to the bottle per manufacturer’ s instructions. This post sterilization manipulation of the agar medium introduces the possibility of contamination which can compromise the end use results. Additionally, the egg yolk itself is highly microbially susceptible and requires storage under refrigeration conditions adding additional impositions to the end user. These manipulation steps readily convey the complicated nature of the procedure involved to prepare the B. cereus agar medium for use.

[0008] U.S. Pat. No. 6,284,517, EP-B-1219628 and U.S. Pat. No. 6,558,917 describe the use of chromogenic or fluorescent media based on the detection of Phosphatidylinositol-specific phospholipase C (PI-PLC; enzyme classification: EC 4.6.1.13). Flicker et al., 2008 describe some challenges with these media, for instance of generating false-negative results especially with organisms belonging to the B. cereus group which do not exhibit PI-PLC activity (suchas B. mycoides), or a low PI-PLC activity (B. anthracis). Furthermore, the authors describe false positive results encountered with Gram-positive bacteria expressing such an enzyme activity despite the presence of selective agents. While US7,309,580 attempts to overcome some of the limitations described above with the use of a combination of chromogenic substrates specific to phosphatidylcholine-specific phospholipase C (PC-PLC, enzyme classification: EC 3.1.4.3) and a chromogenic substrate of PI-PLC that result in two different colored colonies or a combined third color, Fricker et al, 2008 demonstrate incorrect identification of atypical strains by this medium.

[0009] Similarly, US 10, 351,896 describes the use of chromogenic and / or fluorogenic carboxylesterase and / or triacylglycerol-lipase substrates to detect the presence of B. cereus group of organisms. While the use of these very specific substrates may circumvent the challenges associated with the use of egg yolk above, they can result in false negative (several strains of B. cereus not giving the signature response within 24 hour thereby needed an extended incubation time which can be a disadvantage for the test) and / or false positive (B. pumilus, B. sublilis). both phenomena of which results as exemplified in Tables 3 and 4 (US10,351,896). The observance of the false negative and false positive responses is further described in the same tables as sensitivity and specificity rates.

[0010] Additionally, US20230143236 builds upon the use of the PIPLC enzyme system above in conjunction with a substrate for the enzyme alpha glucosidase to overcome some of the aforementioned challenges with false positives along with increasing the stringency of selectivity. Unfortunately, these approaches do not resolve the problems of false positivity and false negativity encountered. For instance, it is well known that Geobacillus stearothermophilus constitutively expresses the enzyme alpha glucosidase and can often be present in thermally processed food stuffs potentially generating a false positive result.

[0011] The examples above reiterate the technical challenges that need to be overcome in order to have a reliable and easy to use detection system for Bacillus cereus group of organisms. There is a need to develop a growth-based detection system that can help the food industry isolate and enumerate organisms belonging to B. cereus group of bacteria from food and environmental samples to ensure the safety of these systems.

[0012] This background information is provided for the purpose of making information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the information disclosed herein constitutes prior art against the present invention.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] Figure 1. Schematic of prototype plates. The broth coated hydrophobic bottom film was first adhered onto 20 mil polystyrene foam substrate (American FujiSeal (Shaumburg, IL) with a 2 3 / 8thinch circle that serves as a zone for sample inoculation followed by adherence to a powder coated top film comprising Tetrazolium chloride (TTC) indicator described above, with the aid of a double sided adhesive tape (3M Company, St. Paul, MN). The cover sheet and substrate sheet of each device were oriented so that the coated surfaces were facing each other.

[0001] Figure 2. Exemplary culture device. In particular, a top perspective view, partially in section, of a microbiological growing device of the present disclosure.

[0016] Figure 3. Cross sectional view of device of FIG. 1.

[0017] Figure 4. Top view of a grid pattern that can be printed on the first sheet or the second sheet of the device of FIG. 1.

[0018] Figures 2 and 3 illustrate certain aspects of a device according to the present disclosure. The microbiological growth device (10) includes body member comprising a self- supporting waterproof first sheet 12 having an upper and lower surface. 12 can be made of a variety of materials as described in US Pat. No., 4,565,783 (incorporated by reference). To facilitate counting of colonies, the first sheet 12 may have a pattern (e.g., sq. grid pattern) printed as shown in FIG. 4. The first sheet 12 is optionally coated with an adhesive 14 which serves to hold the culture medium 16 that comprises one or more cold water-soluble gelling agents and nutrients (which may be delivered in a dry powder form or a coated, dried liquid formulation) to facilitate and select for growth of target organisms in a uniform monolayer that facilitates easy hydration.

[0019] Adhered to first sheet 12 (directly or indirectly) is a second sheet 22- preferably transparent to facilitate colony viewing and or counting and substantially impermeable to water vapor and bacteria. Generally, second sheet 22 may have properties similar to first sheet 12 with suitable materials being preferably PP (e.g., 40 pm thick biaxially oriented polypropylene (BOPP) or less preferably polyester (e.g., polyethylene terephthalate PET) that is about 70 pm thick with a density of 1.38 g / mm3).

[0020] Adhesive 14, or alternate adhesive 24 can also be coated on second sheet 22 which serves to hold a dry rehydratable culture medium comprising one or more gelling agents incorporated as powder 26. Powder 26 is coated onto the adhesive 24, preferably as a uniform monolayer, as described in U.S. Pat. No., 4,565,783 (incorporated by reference). Adhesives 14 and 24 may be solvent based or water soluble. In some embodiments, the adhesive has a thickness range of about 5 mm to about 13 mm. In further embodiments, the adhesive as disclosed herein allows for microbial colony visibility through the film coated with adhesive.Suitable adhesives include pressure-sensitive adhesives as described in US Pat. No., 4,565,783 (incorporated by reference). Heat activated adhesives; water activated adhesives may also be used. Components to differentiate various types of bacteria such as selective agents (e.g., antibiotics), dyes, enzyme substrates, etc. can be introduced to the growth device 10 in multiple locations, for e.g., Adhesive 14, Adhesive 24, and growth medium 16 which is substantially water free.

[0002] Powder 26 should have sufficient surface area exposed in a microbial growth zone to act in concert with the dry culture medium 16 to absorb a predefined volume of aqueous liquid sample deposited in growth zone of the device. In any embodiment, the microbial growth zone is defined by a spacer element 18 applied to the surface of first sheet 12 coated with the culture medium 16. The spacer element 18 has an aperture 20 (circular, square, rectangular) cut through the center to expose the dry culture medium such that it can form a microbial growth zone when hydrated and in concert with powder 26 located on second sheet 22. Closed cell polystyrene foam is one example of a suitable material for spacer 18, but any material which is hydrophobic (non-wetting), inert to microorganisms, and capable of withstanding sterilization maybe used. The aperture 20 forms a perimeter of the microbial zone in the culture medium, wherein upon hydration with a desired, predetermined volume, the device forms a hydrated culture medium that facilitates growth and enumeration of target organisms.

[0003] The dry rehydratable culture medium 16 comprises nutrients that facilitate the growth of gram-positive Bacillus organisms while providing components that will help differentiate between Bacillus cereus group of organisms and non-Bacillus cereus bacteria, while inhibiting the growth of gram-negative bacteria and non-Bacillus gram positive bacteria, via the use of selective agents such as antibiotics. The effective amount is selected so that , when a predetermined volume of aqueous liquid is deposited in the growth zone , the rehydrated culture medium has a concentration of the at least one agent that is sufficiently high toselectively inhibits growth of gram negative bacteria and some non-target gram positive microorganisms (such as Staphylococcus aureus) but not so high that it substantially inhibits the growth of the target B. cereus group of organisms.

[0004] The culture medium 16 of devices of the present disclosure is cold-water soluble. The “cold-water solubility” of the culture medium employed in the devices of the present invention includes one or more cold-water soluble gelling agent in the culture medium. Suitable cold- water soluble gelling agents for inclusion in culture medium 16 include both natural and synthetic gelling agents which form solutions in water at room temperature. Gelling agents including a non-exhaustive list of xanthan gum, guar gum, locust bean gum, for example form solutions in water at room temperature and are suitable cold-water soluble gelling agents for providing a dry rehydratable culture medium that is "cold-water soluble” according to the present disclosure. Preferred cold-water soluble gelling agents for culture medium 16 include for example, guar gum and xanthan gum, these gelling agents being useful individually or in combination with one another and in combination with other cold-water soluble gelling agents.

[0005] Bacillus cereus serves as a useful indicator of food safety and quality. The rapid detection and enumeration of these organisms is highly advantageous to the food and beverage industry. In addition to producing the enzyme lecithinase, which is a feature exhibited by >95% of organisms belonging to the B. cereus sensu lato group, B. cereus can be classified as a gram positive aerobic or facultative anaerobic spore former, exhibiting pathogenic properties via formation of an enterotoxin. The ability to detect the lecithinase reaction by the end user, without the need to handle the highly microbially susceptible egg emulsions is a significant advantage afforded by a thin film growth device described.

[0021] DESCRIPTION

[0022] Definitions

[0023] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated invention, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).

[0026] The use of “or” means “and / or” unless stated otherwise.

[0027] The use of “a” or “an” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate.

[0028] The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and / or “consisting of.”

[0029] As used herein, the term “about” refers to a ±10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0030] As used herein, the term “about” refers to a ±10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0031] Any ranges given either in absolute terms or in approximate terms are intended to encompass both, and any definitions used herein are intended to be clarifying and not limiting. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges (including all fractional and whole values) subsumed therein.

[0032] The term as used herein “sample” refers to an amount isolated from an entity (such as food, air, biological fluid or tissue, etc.) for analysis. The sample may be of industrial or clinical origin, that is to say, according to a non-exhaustive list, an air specimen, a water specimen, a specimen taken from a surface, a part or a manufactured product, or a product of food origin. Among the samples of food origin, mention may, in a non-exhaustive manner, be made of a sample of milk products (yogurts, cheeses, etc.), of meat, of fish, of eggs, of fruit, of vegetables, of water or of a beverage (milk, fruit juice, soda, etc.). These samples of food origin may also come from prepared dishes or sauces. Finally, a food sample may be derived from an animal feed, such as in particular animal meals. The sample may be of biological origin, i.e., animal, vegetable or human origin. It may then correspond to a specimen taken from a biological fluid (whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretion), a tissue specimen or isolated cells. This specimen can be used as it is or, prior to analysis, undergo a preparationof enrichment, extraction, concentration or purification type, according to methods known to those skilled in the art.

[0033] “Culture device”, as used herein, refers to an article adapted to house a film comprising a liquid formulation as disclosed herein.

[0034] “Target microorganism”, as used herein, refers to a particular microorganism (i.e., a species of microorganism, or a group of species (e.g., sensu lato group) or a particular group of microorganisms (e.g., a particular genus of microorganisms, Bacillus bacteria, antibioticresistant bacteria) to be detected.

[0035] The term “food” as used herein is generally used to refer to a solid, liquid (e.g., including, but not limited to, solutions, dispersions, emulsions, suspensions, etc., and combinations thereof) and / or semi-solid comestible composition. Examples of foods include, but are not limited to, meats, poultry, eggs, fish, seafood, vegetables, fruits, prepared foods (e.g., soups, sauces, pastes), grain products (e.g., flour, cereals, breads), canned foods, milk, other dairy products (e.g., cheese, yogurt, sour cream), fats, oils, desserts, condiments, spices, pastas, beverages, water, beer, animal feed, other suitable comestible materials, and combinations thereof.

[0036] As used herein, the term “gram-positive bacteria” collectively refers to a group of bacteria that give a positive result in a Gram stain test. These bacteria have a cell wall composed of a thick layer of a substance called peptidologlycan. The term “gram-negative bacteria” as used herein collectively refers to a group of bacteria that give a negative result in a Gram stain test.

[0037] The terms “culture” and “growth” of microorganisms as used herein refers to a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under conditions conducive for their growth. More particularly it is the method of providing a suitable culture medium and conditions to facilitate at least one cell division of amicroorganism. Culture media include solid, semisolid, or liquid media containing nutrients and necessary physical growth parameters necessary for microbial growth.

[0038] As used herein, the term “enrichment” refers to a culture method of selectively enriching the growth of a specific microorganism by providing medium and conditions with specific and known attributes that favors the growth of that particular microorganism. The enrichment culture's environment positively influences the growth of a selected microorganism (“the target”) and / or negatively influence the growth of other microorganisms resulting in selective growth of the target.

[0039] As used herein, the term “hydrophobic” refers to a material that exhibits a water contact angle of 90° or larger on its surface.

[0040] The term “powder”, as used herein, refers to particulate material of one or more gelling agents or nutrients having an average diameter suitable for use in the thin film culture device(s) of the present invention, preferably a diameter of about 10-400 microns more preferably a diameter of about 30-90 microns.

[0041] “TTC” as used herein is an abbreviation for the dye triphenyl tetrazolium chloride.

[0042] “B. cereus ” refers to the Bacillus cereus group of organisms (for example the B. cereus sensu lato group.

[0043] “MYP" refers to mannitol yolk polymyxin.

[0044] “PEMBA” refers to polymyxin egg yolk mannitol bromothymol blue agar.

[0045] “FDA-BAM” refers to the Bacteriological Analytical Manual of the Food and Drug Administration.

[0046] The term “culture medium” and “culture media” are used interchangeably herein with the terms “nutrient medium” and “nutrient medium formulation”.

[0047] The term “broth formulation” is used interchangeably herein with the term “liquid formulation” as disclosed herein.

[0048] As used herein, the term “selective agent” refers to a chemical compound that is added to a nutrient medium to isolate a particular bacterial species or genus. This refers to any element, compound, or composition that functions to inhibit the growth of one type of microorganism (e.g., gram-negative bacterium) relative to another type of microorganism (e.g., gram-positive microorganism) and thereby facilitate the growth and / or identification of microorganisms grown on the thin film culture device(s) according to the present disclosure.

[0049] The term “gelling agents” is used interchangeably herein with to refer to “gums”, which is known in the art. Non-limiting examples of such gelling agents include: guar gum, xanthan gum, hydroxy ethyl cellulose, carboxymethyl cellulose, polyacrylamide, locust bean gum, algin, and combinations of two or more of the foregoing.

[0050] Substantially water free- designates coating which has a water content no greater than about the water content of the dehydrated coating once it has equilibrated with the ambient environment. In particular, the coating is a dehydrated form of the broth that was initially hydrated and mixed in water. During preparation, the broth goes through coating using methods disclosed herein and is heated to remove water. The coating needs to be dry. However, the “substantially water free” dehydrated coating disclosed herein may have moisture based on humidity of the surrounding environment.

[0051] “Indicator system”, as used herein, refers to one or more of any of the following and any combination of one or more of the following: a chromogenic enzyme substrate, a fluorogenic enzyme substrate, a redox indicator (e.g., triphenyltetrazolium chloride, methylene blue), a metabolizable nutrient, pH indicator. “Metabolizable nutrient” refers to any molecule that can be used by a predetermined indicator organism and / or a predetermined target microorganism to produce biomass and / or energy. The use of the metabolizable nutrient by the microorganisms directly or indirectly results in a pH or other detectable ionicchange in an aqueous medium that is in fluid contact with the microorganism. A“differentiating” indicator system is an indicator system that can be used to distinguish two nonidentical microorganisms based on their respective reactivities with a component(s) of the indicator system(s).

[0052] The term “adhesive” as used herein refers to an adhesive that does not inhibit microbial growth.

[0053] The use of term “vitamin mix” as used herein refers to a peptone enhanced with vitamins. In some embodiments the vitamin mix is a casein peptone enhanced with vitamins. The Casein digest peptone is a general-purpose growth peptone produced by the enzymatic digestion of casein that is used for the cultivation of most bacteria, fungi, and some protozoa. Casein is the main protein of milk, and a rich source of amino nitrogen.

[0054] Compositions disclosed herein and culture devices including the same can be used to differentially detect and quantify the presence of B. cereus.

[0055] One aspect of the invention pertains to a nutrient medium formulation comprising a protein, yeast extract, sodium pyruvate, and a selective agent for inhibition of gram negative and non-Bacillus organisms. In some embodiments, the nutrient medium formulation disclosed herein is for selective growth of Bacillus cereus group of organisms. In further embodiments, said selective agent is for inhibition of Bacillus cereus group of organisms.

[0056] Another aspect of the invention pertains to a liquid formulation comprising a nutrient medium disclosed herein and one or more gelling agent. In some embodiments, the nutrient medium disclosed herein includes a cold-water soluble gelling agent such as guar gum, xanthan gum and locust bean.

[0057] A further aspect of the invention pertains to a film comprising a liquid formulation disclosed herein disposed on the surface of water-proof substrate (first sheet 12).

[0058] A yet further aspect of the invention pertains to a culture device for growing microorganisms, comprising: a body member comprising a self-supporting, water-proof substrate (12) having upper and lower surface; a coating of a cell culture medium according to any of the preceding embodiments adhered to the upper surface of the substrate, a cover sheet (22) having an inner-facing surface and an outer-facing surface, the cover sheet adhered to at least a portion of the body member; and wherein said coversheet comprises an adhesive (14 or 24) and a dry composition (powder 26) comprising one or more cold water-soluble gelling agents (such as a mixture of xanthan and guar gum) adhered to the inner-facing surface of the cover sheet; wherein the device optionally includes an indicator (e.g., a redox indicator such as tetrazolium indicator) either disposed in an adhesive in the cover sheet, substrate, or a combination thereof; wherein the device includes a microbial growth zone disposed between the substrate and the cover sheet; wherein the cover sheet is adhered to the body member so that the upper surface of the substrate faces the inner-facing surface of the cover sheet.

[0059] Another aspect of the invention pertains to a method of detecting the presence or absence of a microorganism in a sample, comprising: providing a liquid sample suspected of containing a microorganism; contacting a predetermined amount of the sample with the microbial growth zone disposed between the substrate and the cover sheet of a culture device according to any of the preceding embodiments; incubating said device; andobserving an indication of the presence or absence of microbial growth.

[0060] The pH indicator as used in the nutrient medium formulation disclosed herein may be selected according to criteria known in the art such as, for example, pH range, compatibility with the indicator and / or target microorganisms, and solubility. In some embodiments, a salt form of the pH indicator may be used, for example, to increase the solubility of the pH indicator in an aqueous mixture. Nonlimiting examples of suitable pH indicator dyes include, for example, thymol blue, tropaeolin 00, methyl yellow, methyl orange, bromophenol blue, bromocresol green, methyl red, bromothymol blue, phenol red, neutral red, phenolphthalein, thymolphthalein, alizarin yellow, tropaeolin O, nitramine, trinitro benzoic acid, thymol blue, bromophenol blue, tetra bromophenol blue, bromocresol green, bromocresol purple, methyl red, bromothymol blue, phenol red, Congo red, and cresol red.

[0061] In some embodiments, the culture device disclosed herein may include a buffering agent. The buffering agent can be provided in the first and / or second cold water-soluble gelling agent composition or the culture medium. Nonlimiting examples of suitable buffering agents include phosphate compounds (e.g., sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate), sodium carbonate, MOPS (2-[N-Morpholino] ethane sulfonic acid) free acid, and MOPS sodium salt. Preferred gelling agents include guar gum, locust bean gum and xanthan gum, these gelling agents being useful individually or, in any embodiment, in combination with one another.

[0062] A plurality of indicator agents may be used for detecting a presence of a bacterium. Suitable indicator agents include, but are not limited to, chromogenic enzymatic substrates (e.g., indoxyls), and redox indicators (e.g., tetrazolium indicators).

[0063] “Liquid medium formulation” can also be referred to as “culture medium formulation” as well as “broth medium formulation” and “broth formulation”. The liquid medium formulation may include one or more gelling agents, suitable nutrients, salts, ions, proteins, carbon sources, and selective agents. The liquid medium of the present disclosure can include at least one selective agent that selects for growth of certain gram-positive bacteria.

[0064] It is to be understood that both the foregoing descriptions are exemplary, and thus do not restrict the scope of the invention.

[0065] LIST OF EMBODIMENTS

[0066] The following is a non-limiting list of embodiments:1. A nutrient medium formulation comprising a protein, yeast extract, sodium pyruvate, and one or more selective agents for inhibition of gram-negative organisms and non- Bacillus organisms.2. The nutrient medium formulation of embodiment 1, wherein said protein is skim milk and / or peptic digest of animal tissue.3. The nutrient medium formulation of embodiment 1, wherein said selective agent is ceftazidime and / or Polymyxin B.4. The nutrient medium formulation of embodiment 1, wherein said sodium pyruvate has a concentration in the range of about 5.0 g / L to about 40 g / L.5. The nutrient medium formulation of embodiment 4, wherein said sodium pyruvate has a concentration of 15 g / L or 30 g / L or about 15 g / L to about 30 g / L.6. The nutrient medium formulation of embodiment 2, wherein said skim milk has a concentration in the range of about 5.0 g / L to about 60 g / L.7. The nutrient medium formulation of embodiment 6, wherein said skim milk has a concentration in the range of about 10 g / L to about 45 g / L.8. The nutrient medium formulation of embodiment 7, wherein said skim milk has a concentration of 30 g / L, 40 g / L, or about 30 g / L to about 40 g / L.9. The nutrient medium formulation of embodiment 1, wherein said yeast extract has a concentration in the range of about 0.25 g / L to about 5.0 g / L.10. The nutrient medium formulation of embodiment 9, wherein said yeast extract has a concentration in the range of about 0.5 g / L to about 2.0 g / L, 1-5 g / L, or about 0.55 g / L.11. The nutrient medium formulation of embodiment 3, wherein polymyxin B has a concentration in the range of about 7,000 IU to about 50,000 IU or about 7,000 IU to about 15,000 IU.12. The nutrient medium formulation of embodiment 11, wherein polymyxin B has a concentration in the range of about 7,000 IU to about 20,000 IU.13. The nutrient medium formulation of embodiment 3, wherein ceftazidime has a concentration of 10 mg / L or 12.5 mg / L or 10 mg / L to about 15 mg / L.14. The nutrient medium formulation of embodiment 1, wherein said formulation further comprises one or more salts, one or more buffering agents, one or more amino acids, one or more nucleosides, one or more chromogenic indicators, one or more lecithin components, and a pH indicator.15. The nutrient medium formulation of embodiment 14, wherein said pH indicator is Bromocresol purple (BCP).16. The nutrient medium formulation of embodiment 14 wherein said salt is sodium chloride.17. The nutrient medium formulation of embodiment 14, wherein said buffering agent is chosen from dibasic K phosphate, monobasic K phosphate, or a combination thereof.18. The nutrient medium formulation of embodiment 14, wherein said amino acid is alanine.19. The nutrient medium formulation of embodiment 14, wherein said nucleoside is inosine.20. The nutrient medium formulation of embodiment 14, wherein said chromogenic indicator is 5-bromo-4-chloro-3-inoxyl beta glucoside.21. The nutrient medium formulation of embodiment 14, wherein said lecithin component is egg emulsion.22. The nutrient medium formulation of embodiment 21, wherein said egg emulsion is present in a concentration in the range of about 30 mL / L to about 120 mL / L.23. The nutrient medium formulation of embodiment 21, wherein said egg emulsion is present in a concentration of 100 mL / L or 50 mL / L or about 40 mL / L to about 100 mL / L.24. The nutrient medium formulation of any of the preceding embodiments, wherein said formulation further comprises water.25. The nutrient medium formulation of embodiment 24, wherein said water is deionized water.26. The nutrient medium formulation of embodiment 1, wherein said medium formulation further comprises a vitamin mix.27. The nutrient medium formulation of embodiment 26, wherein said vitamin mix is present in a concentration of about 7.5 g / L.28. A liquid formulation comprising a nutrient medium according to any of the preceding embodiments and one or more gelling agents.29. The liquid formulation of embodiment 28, where said one or more gelling agents is guar gum, xanthan gum, locust bean, or any combination thereof.30. The liquid formulation of embodiment 28, wherein the gelling agent is guar gum, xanthan gum, or any combination thereof.31. A film comprising a liquid formulation according to any of the preceding embodiments disposed on the surface of water-proof surface.32. The film of embodiment 31, wherein the water-proof surface comprising paper or plastic (such as PET).33. The film of embodiment 31, wherein the water-proof surface maybe opaque, transparent, white or colored.34. A culture device for growing microorganisms, comprising: a body member comprising a self-supporting, water-proof substrate having upper and lower surface; a coating of a liquid formulation according to any of the preceding embodiments adhered to the upper surface of the substrate, a cover sheet having an inner-facing surface and an outer-facing surface, the cover sheet adhered to at least a portion of the body member; and wherein said coversheet comprises a dry composition comprising one or more cold water-soluble gelling agents (such as a mixture of xanthan and guar gum) adhered to the inner-facing surface of the cover sheet; wherein the device includes a microbial growth zone disposed between the substrate and the cover sheet; wherein the cover sheet is adhered to the body member so that the upper surface of the substrate faces the inner-facing surface of the cover sheet.35. A method of detecting the presence or absence of a microorganism in a sample, comprising: providing a liquid sample suspected of containing a microorganism;contacting a predetermined amount of the sample with the microbial growth zone disposed between the substrate and the cover sheet of a culture device according to any of the preceding embodiments incubating said device; and observing an indication of the presence or absence of microbial growth.36. The method of embodiment 35, wherein said microorganism is bacteria.37. The method of embodiment 36, wherein said bacteria belongs to the genus Bacillus.38. The method of embodiment 37, wherein said bacteria is Bacillus cereus group of organisms.39. The method of embodiment 35, wherein said sample is food (e.g., dried and processed foods such as cereals, grain, beverages, raw milks, slurries, sauces, dips, ice creams, etc.).40. The method of embodiment 35, wherein said sample can be diluted in routinely used buffers.41. The method of embodiment 35, wherein said sample can be diluted in Butterfield’s phosphate buffer, buffered peptone water, distilled water, or letheen buffer.42. The method of embodiment 35, wherein the device includes an indicator (e.g., tetrazolium indicator, redox indicator, etc.) either disposed in an adhesive in the cover sheet, on the substrate or a combination thereof. For example, the indicator may be disposed in the adhesive on the cover sheet. In some embodiments, the indicator is disposed in adhesive 14 on first sheet 12, or in adhesive 24 on cover sheet 22 (see Figures 1 and 2).43. A culture device for growing microorganisms comprising a film according to any of the preceding embodiments.

[0067] EXAMPLES

[0068] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, described herein.

[0069] Example 1. Preparation of Nutrient Medium Formulation

[0070] The following components were weighed in 2 L stainless steel containers according to the amounts listed in Table 1.

[0071] Table 1. Exemplary Nutrient Medium Formulations

[0074] General Method for Preparing Broth Culture Medium Formulation: A broth formulation involving components in Table 1 along with a first and second gelling agents was prepared as follows:

[0075] A dried, powdered gelling agent comprising Guar gum and a secondary gelling agent Keltrol® Xanthan gum (CP Kelco) each was weighed out separately and combined as a gum mixture. One liter of de-ionized water was added to a stainless-steel beaker and mixed with a Silverson High Shear Mixer with a homogenizer blade. The nutrient mixes consisting of nutrients listed as comparative examples above were added individually along with the gelling agents to the beaker with continued mixing. The mixture was then subj ected to heat and brought up to 80 °C with continuous mixing. Once the desired temperature was reached, the beaker was removed from the heat and allowed to cool to ambient temperature (22 °C - 25 °C). At this point, the antimicrobial agents and egg emulsion were added to the broth and mixed well. Once cooled sufficiently, the contents were transferred to a plastic jar and stored in the refrigerator until further use.

[0076] The chilled broth culture medium was then knife coated (gap setting about 0.3 mm) onto the upper surface of the first sheet comprising a hydrophobic paper (a bleached kraft paper coated with a water-resistance polymeric layer) containing a layer of iso-octyl Acrylate Acrylamide Copolymer (96:4) adhesive (RD1273, 3M Company, St. Paul, MN) as outlined in US 11414641. The resulting coated film was dried in an oven at 93 °C- 104 °C. for 1-30 minutes. The coated film was cut into 76 mm wide by 102 mm long sections that formed the substrate sheet of the device. A foam spacer (polystyrene foam; 76 mm wide by 102 mm long by 0.57 mm thick) with a circular opening (60 mm in diameter) was adhesively laminated to the coated side of the substrate sheet. The circular opening was positioned near the center of the foam layer and defined the growth zone of the device.

[0077] Example 2. Adhesive Coated Biaxial Polypropylene

[0078] Bi-axially oriented polypropylene (BOPP) coated with the same iso-octyl Acrylate Acrylamide Copolymer (96:4) adhesive (RD 1273, 3M Company, St. Paul, MN) containing triphenyl tetrazolium chloride (TTC, Sigma #17779), per procedures outlined in US 4,476,226 and US 5,089,413 was subsequently powder coated to capacity with dry powdered Guar along with a secondary gelling agent Keltrol® Xanthan gum, per example 11 of U.S. Pat. No. 4,565,783 and served as a cover sheet for the B. cereus prototypes.

[0079] Example 3. Preparation of Prototype Petrifilm Plates

[0080] Prototype plates were prepared according to the procedures outlined in US Pat. Nos., 4,476,226 and US 5,089,413 (incorporated by reference). Broth coated hydrophobic bottom substrate (12 with 14) was first adhered onto 20 mil polystyrene foam (20) spacer (Amerian Fuji Seal, Inc.) with a 2 3 / 8thinch circle (18) that serves as a zone for the sample inoculation followed by adherence to a powder coated top film (22) comprising an adhesive (24) containing Tetrazolium chloride (TTC) indicator, with the aid of a double sided adhesive tape (3M Company, St. Paul, MN). The cover sheet (22) and substrate sheet (12) of each device were oriented so that the coated surfaces were facing each other. See Figs. 2 and 3.

[0081] Example 4. Inoculation and Incubation of Plates

[0082] A panel of test organisms (Table 2) was used for the evaluation of the prototypes. Briefly, the panel comprised of multiple Bacillus cereus strains that appear as TTC reducing red colored colonies with associated lecithinase activity. Additionally, the panel included beta glucosidase producing differential strains of non-Bacillus cereus-Bacillus organisms that did not produce lecithinase to enable easy visual differentiation from the signature response of the Bacillus cereus strains. Finally strains of E. coli and Staphylococcus aureus served as exclusivity strains whose growth is significantly diminished or eliminated from the plate since they do not belong to the Bacillus cereus group. The panel of strains were tested on the comparative formulations outlined in Table 1 with a goal of modulating the formulationtowards a well-defined and visible lecithinase zone around the Bacillus cereus colony, a differential response with the non-Bacillus cereus strains and minimal or diminished growth of non-Bacillus bacteria.

[0083] TABLE 2. The following table depicts the bacteria colony morphology on the Bacillus cereus plates.

[0084] Example 5. Testing Petrifilm Plates

[0085] The test organisms were obtained from Microbiologies (St. Cloud, MN) as a Kwik Stik and propagated on Tryptic Soy Agar medium per manufacturer’s instructions. The working stock cultures of the organisms were maintained on sterile TSA agar, followed by a sub culturing in Tryptic Soy broth for a period of 18-24 hours at 35 °C prior to use. Dilution of organisms: An overnight culture of the test organism was serially diluted in Butterfield’s buffer as needed to provide countable colonies on experimental plates. 1 ml of the diluted sample was plated onto the example prototype Bacillus cereus plates and incubated at 30 °C for 18-24 hours. Following incubation, plates were observed for red colored colonies with prominent white lecithinase zone production in case of inclusive strains, blue colored colonies for differential strains and minimized or diminished growth for organisms whose growth is selected against on the prototype plates. Images of plates were obtained at 18-24 hours for the prototype plates inoculated with the panel outlined in Table 2 using the Petrifilm RAC algorithm in Petrifilm Plate Reader Advanced (Formerly 3M Company, St. Paul, MN). The number of colonies in the case of the inclusive strains was enumerated via manual counting.

[0086] Example 6. Signature Morphology and Appearance of Microorganisms on Petrifilm Plates

[0087] The target Bacillus cereus plates demonstrated the signature morphology of a red colony with a prominent white zone across all strains tested and all the comparative examples (Table 2). Polymyxin B did not have the predicted dose response behavior of increasing lethality typically observed with increasing dose of the antibiotic, as seen in Example A vs the others. In fact, it was observed that the levels needed to prevent the growth of E. coli was significantly less than that typically recommended for this application.

[0088] As described earlier, an oft-encountered challenge is the occurrence of False positives and false negatives. The typical strategies employed by researchers is the ramping up ofselective agents as described in the cited literature above. While this increased selectivity via the use of multiple antibiotics and antifungals is effective at reducing non target organisms, as cited previously, it also significantly increases the odds of false negatives which is non desirable for the end user and for accurate detection of the presence of this pathogen in foods.

[0089] Staphylococcus aureus is one such organism that can appear as a false positive on most media, especially that containing egg yolk by producing a white zone around the colony, which in the presence of a reducing agent such as TTC appears red. The modulation of certain components such as yeast extract in the medium directly impacts the appearance and growth of this target medium and enables a selective response without largely increasing amounts or types of antibiotics.

[0090] Additionally, it was observed that all the non-target Bacillus strains (that is non- . cereus- Bacillus strains such as B. licheniformis, B. subtilis, B. pumilus) preferentially utilized the beta glucoside substrate over the reduction of the redox indicator, TTC that typically occurs via the electron transport chain in the bacterial cell wall. Since these organisms have both fermentative and oxidative metabolic pathways it was intriguing that they hydrolyzed the beta glucoside substrate to form a blue colony instead of reducing the TTC to formazan resulting in a red colony. In contrast the B. cereus preferentially reduces the TTC to form a red colony. This strategy for obtaining differential response provides a benefit over traditional strategies using mannitol fermentation. Mannitol fermentation is indicated in traditional strategies as a yellow diffuse zone around the colony due to a pH change effected by a pH indicator. The challenge with this approach is that the acid responsible for the yellowing of the medium can diffuse far from the colony often overlapping a target B. cereus colony which is non mannitol fermenting, causing confusion in enumeration and difficulties in interpretation for the end user. By leveraging the beta glucoside hydrolysis via utilization of a chromogenic indicator, theabove problem is solved wherein the non-target Bacillus colonies appear blue and are easy to identify and interpret.

[0091] Example 7. Count Plate Performance Summary

[0092] The Petrifilm Bacillus cereus Count (BC) Plate is a selective and differential sample- ready-culture-medium system which contains proprietary nutrients, a cold-water-soluble gelling agent, chromogenic indicators and a lecithinase substrate that facilitates colony enumeration. This medium is used for the enumeration of the Bacillus cereus group, also known as the B. cereus sensu Jato group, which are aerobic, spore forming, Gram positive rods.

[0093] The purpose of inclusivity and exclusivity testing is to ensure that the Petrifilm Bacillus cereus Count Plate can detect Bacillus cereus while discriminating from non-Bacillus cereus bacteria. The term “inclusivity” as used herein is defined as the ability of a method to detect the target analyte from a wide range of strains. The term “exclusivity” as used herein is defined as the lack of interference from a relevant range of non-target strains.

[0094] The purpose of the food study is to show performance in matrices of interest on Petrifilm Bacillus cereus Count Plate as compared to the ISO 7932 Method. The selected matrices may have been chosen because they present potential common reservoirs for Bacillus organisms and / or are regulated categories of interest. These matrices have a variety of background flora levels and are typically artificially inoculated for testing to assess recovery against a standard reference method.

[0095] Example 8. Method- Inclusivity and Exclusivity Testing

[0096] Pure bacteria cultures were derived from purchased culture collection preparations or from frozen stock cultures. Inclusivity and exclusivity cultures were cultured in a non-selective growth medium and plated at a concentration within the countable range of the Petrifilm Bacillus cereus Count Plate. Organisms were tested according to the Petrifilm Bacillus cereus Count Plate product instructions and incubated at both 30°C and 35°C for 20-24 hours.

[0097] Of the 38 isolates tested, 38 were detected by Petrifilm Bacillus cereus Count Plate at both 30°C and 35°C.

[0098] Of the 32 isolates tested, 32 of the isolates were inhibited, produced atypical colonies (blue colonies or pinpoint red colonies with no cream / white precipitate around the colony) or were not detected on the Petrifilm Bacillus cereus Count Plate at 30°C and 35°C. Those that produced atypical colonies include Bacillus species, Enterococcus faecalis, Listeria sp. and Staphylococcus aureus.

[0099] Table 3. Inclusivity List (n=38)

[0100] Table 4. Exclusivity List (n=38)

[0101] Example 9. Food Study

[0102] Samples were prepared at a 1 : 10 dilution in Butterfield's Phosphate Buffer and tested according to the Petrifilm Bacillus cereus Count Plate product instructions and incubated at both 30° C and 35°C for 20-24 hours. Additionally, samples were spread across MVP agar and incubated at 30°C for 18-48 hours following the ISO 7932 standard. Results from the Petrifilm Bacillus cereus Count Plate method were not statistically different from the reference method.

[0103] Table 5. Food Study Performance (n=120 foods tested)

[0104] Table 6. List of Foods Tested

Claims

CLAIMSWe claim:

1. A nutrient medium formulation comprising a protein, yeast extract, sodium pyruvate, and one or more selective agents for inhibition of gram-negative organisms and non- Bacillus organisms.

2. The nutrient medium formulation of claim 1, wherein said protein is skim milk and / or peptic digest of animal tissue.

3. The nutrient medium formulation of claim 1 , wherein said selective agent is ceftazidime and / or Polymyxin B.

4. The nutrient medium formulation of claim 1, wherein said sodium pyruvate has a concentration in the range of about 5.0 g / L to about 40 g / L.

5. The nutrient medium formulation of claim 4, wherein said sodium pyruvate has a concentration of 15 g / L or 30 g / L or about 15 g / L to about 30 g / L.

6. The nutrient medium formulation of claim 2, wherein said skim milk has a concentration in the range of about 5.0 g / L to about 60 g / L.

7. The nutrient medium formulation of claim 6, wherein said skim milk has a concentration in the range of about 10 g / L to about 45 g / L.

8. The nutrient medium formulation of claim 7, wherein said skim milk has a concentration of 30 g / L, 40 g / L, or about 30 g / L to about 40 g / L.

9. The nutrient medium formulation of claim 1, wherein said yeast extract has a concentration in the range of about 0.25 g / L to about 5.0 g / L.

10. The nutrient medium formulation of claim 9, wherein said yeast extract has a concentration in the range of about 0.5 g / L to about 2.0 g / L, 1-5 g / L, or about 0.55 g / L.

11. The nutrient medium formulation of claim 3, wherein polymyxin B has a concentration in the range of about 7,000 IU to about 50,000 IU or about 7,000 IU to about 15,000 IU.

12. The nutrient medium formulation of embodiment 11, wherein polymyxin B has a concentration in the range of about 7,000 IU to about 20,000 IU.

13. The nutrient medium formulation of claim 3, wherein ceftazidime has a concentration of 10 mg / L or 12.5 mg / L or 10 mg / L to about 15 mg / L.

14. The nutrient medium formulation of claim 1, wherein said formulation further comprises one or more salts, one or more buffering agents, one or more amino acids, one or more nucleosides, one or more chromogenic indicators, one or more lecithin components, and a pH indicator.

15. The nutrient medium formulation of claim 14, wherein said pH indicator is Bromocresol purple (BCP).

16. The nutrient medium formulation of claim 14 wherein said salt is sodium chloride.

17. The nutrient medium formulation of claim 14, wherein said buffering agent is chosen from dibasic K phosphate, monobasic K phosphate, or a combination thereof.

18. The nutrient medium formulation of claim 14, wherein said amino acid is alanine.

19. The nutrient medium formulation of claim 14, wherein said nucleoside is inosine.

20. The nutrient medium formulation of claim 14, wherein said chromogenic indicator is 5-bromo-4-chloro-3-inoxyl beta glucoside.

21. The nutrient medium formulation of claim 14, wherein said lecithin component is egg emulsion.

22. The nutrient medium formulation of claim 21, wherein said egg emulsion is present in a concentration in the range of about 30 mL / L to about 120 mL / L.

23. The nutrient medium formulation of claim 21, wherein said egg emulsion is present in a concentration of 100 mL / L or 50 mL / L or about 40 mL / L to about 100 mL / L.

24. The nutrient medium formulation of any of the preceding embodiments, wherein said formulation further comprises water.

25. The nutrient medium formulation of claim 24, wherein said water is deionized water.

26. The nutrient medium formulation of claim 1, wherein said medium formulation further comprises a vitamin mix.

27. The nutrient medium formulation of claim 26, wherein said vitamin mix is present in a concentration of about 7.5 g / L.

28. A liquid formulation comprising a nutrient medium according to any of the preceding embodiments and one or more gelling agents.

29. The liquid formulation of claim 28, where said one or more gelling agents is guar gum, xanthan gum, locust bean, or any combination thereof.

30. The liquid formulation of claim 28, wherein the gelling agent is guar gum, xanthan gum, or any combination thereof.

31. A film comprising a liquid formulation according to any of the preceding embodiments disposed on the surface of water-proof surface.

32. The film of claim 31, wherein the water-proof surface comprising paper or plastic33. The film of claim 32, wherein said water-proof surface comprising paper or plastic is PET.

34. The film of claim 31 , wherein the water-proof surface maybe opaque, transparent, white or colored.

35. A culture device for growing microorganisms, comprising: a body member comprising a self-supporting, water-proof substrate having upper and lower surface; a coating of a liquid formulation according to any of the preceding embodiments adhered to the upper surface of the substrate,a cover sheet having an inner-facing surface and an outer-facing surface, the cover sheet adhered to at least a portion of the body member; and wherein said coversheet comprises a dry composition comprising one or more cold water- soluble gelling agents (such as a mixture of xanthan and guar gum) adhered to the inner-facing surface of the cover sheet; wherein the device includes a microbial growth zone disposed between the substrate and the cover sheet; wherein the cover sheet is adhered to the body member so that the upper surface of the substrate faces the inner-facing surface of the cover sheet.

36. A method of detecting the presence or absence of a microorganism in a sample, comprising: providing a liquid sample suspected of containing a microorganism; contacting a predetermined amount of the sample with the microbial growth zone disposed between the substrate and the cover sheet of a culture device according to any of the preceding embodiments incubating said device; and observing an indication of the presence or absence of microbial growth.

37. The method of claim 36, wherein said microorganism is bacteria.

38. The method of claim 37, wherein said bacteria belongs to the genus Bacillus.

39. The method of claim 38, wherein said bacteria is Bacillus cereus group of organisms.

40. The method of claim 36, wherein said sample is food.

41. The method of claim 40, wherein said food is dried and processed foods such as cereals, grain, beverages, raw milks, slurries, sauces, dips, ice creams, poultry, meat, seafood, pet food, dairy, and baby food.

42. The method of claim 36, wherein said sample can be diluted in routinely used buffers.

43. The method of claim 36, wherein said sample can be diluted in Butterfield’s phosphate buffer, buffered peptone water, distilled water, or letheen buffer.

44. The method of claim 36, wherein the device includes an indicator either disposed in an adhesive in the cover sheet, on the substrate or a combination thereof. For example, the indicator may be disposed in the adhesive on the cover sheet. In some embodiments, the indicator is disposed in adhesive 14 on first sheet 12, or in adhesive 24 on cover sheet 22.

45. The method of claim 44, wherein said indicator is tetrazolium indicator, or redox indicator.

46. A culture device for growing microorganisms comprising a film according to any of the preceding embodiments.

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