MXPA97001207A - Culture medium for a quick count of bacterias colifor - Google Patents

Abstract

The invention relates to products and processes used to determine the presence of bacteria in a sample and particularly relates to a culture medium that can be used in products and processes to enable easy detection and enumeration of Enterobacteriaceae. The bacterial culture medium that facilitates easy detection and counting of bacteria is a mixture of gelatin peptone and yeast extract, lactose or glucose, sodium chloride, bile salts, guar gum and an excess amount of sulfonphthalein dye sufficient to provide a high concentration of dye in close proximity to growing bacteria to allow detection and counting of growing bacteria

Description

CULTIVATION FOR A QUICK COUNT OF BACTERIA COLI FORMES This invention relates in general to products and methods used to determine the presence of bacteria in a sample and in particular refers to a culture medium that can be used in products and methods to allow a rapid enumeration of Enterobacteriaceae, including coliform bacteria.

BACKGROUND Classical methods to determine the presence and number of bacteria in a sample are time-consuming, tedious and labor-intensive. Typically, a technician has to prepare reagents and nutrients, mix the nutrients with agar, heat the mixture, pour the mixture into a petri dish, let the agar gel, obtain a test sample, dilute the test sample, add a aliquot the diluted sample to the agar, incubate the inoculated plate for 24-48 hours and finally count the number of bacterial colonies growing in the REF: 23939 petri dish. Products and procedures that reduce the preparation time and allow a rapid, early counting of the bacteria would be very well received by those who work in this field. An example of a product that greatly simplifies the aforementioned preparation time is a dry culture device for cultivating microorganisms, which is described in U.S. Patent 4,656,783 to Hansen et al. In A typical device reported by Hansen et al., A dry powder soluble in cold water, containing a gelling agent, microbial growth nutrients, is applied on a waterproof substrate. A transparent cover, which allows reading therethrough, is applied on an interior surface with an acrylate adhesive containing an indicator dye, and powder gelling agent is attached to the coated substrate. When the device is used, an amount The predetermined one of an aqueous sample is typically placed in contact with the applied substrate and the cover is placed on the sample and the substrate. The aqueous sample hydrates the soluble dry powder which then forms a gelled medium capable of maintaining the microbial growth. During the growth period, the indicator dye attached to the cover reacts in the presence of visible microorganisms to give a detectable response that allows visualization of bacterial colonies that are cultured in the culture device. A dry culture device based on the Hansen et al report can be obtained commercially as PETRIFILM coliform counting plates (Catalog No. 6400, 3M, St. Paul, MN). The dry culture devices of Hansen et al. Are much simpler to use than conventional systems of gelled agar medium / petri dish because it is not required to heat and mix the culture medium, agar and other reagents and then add the mixture to the petri dishes to pour plates. In addition, Hansen and collaborators' devices are compact and easy to remove and therefore easier and safer to use. Despite the multiple advantages that Hansen and collaborators' devices have over conventional types of culture systems, inoculated thin film plates have yet to be incubated for 24-48 hours before the number of bacteria can be determined. . The ability to detect the presence or determine the number of bacteria earlier can be very valuable in many circumstances. For example, early detection and rapid counting of bacteria is important in the food industry. Currently, the determination of bacteria only after an incubation time of 24-48 hours requires processors to delay the distribution of food products and may allow the production of large quantities of contaminated products. Early detection of bacteria in food products would allow the processor to release food products for distribution earlier in the year because contamination or lack of contamination could be established earlier. further, a processor could locate and correct a source of bacterial contamination without having to discard large quantities of contaminated products. Thus, the detection of bacterial contamination in less than 24-48 hours would be very beneficial for the producers of food products. Although the food industry would clearly benefit from early determination of bacterial contamination, other industries would also welcome the opportunity to detect bacteria more quickly. There is a need for products and procedures that allow early detection and rapid enumeration of Enterobacteriaceae such as coliform bacteria.

BRIEF DESCRIPTION OF THE INVENTION This invention overcomes the shortcomings of the current products and processes mentioned above, providing products and methods that allow early detection and rapid enumeration of Enterobacteriaceae such as coliform bacteria. One embodiment of the present invention is a bacterial culture medium that facilitates the early detection and rapid counting of coliform bacteria growing in the medium (RCCM, rapid coliform counting medium). The medium is a mixture of tryptose, lactose, sodium chloride, bile salts, guar gum and an excess amount of phenol red sufficient to provide a high concentration of phenol red very close to the growing bacteria, to allow detection and the count of growing bacteria in less than 12 hours. Alternatively, a much more useful medium for Enterobacteriaceae replaces lactose with glucose in the medium (RECM, rapid means of counting Enterobacteriaceae). The preferred glucose content range is 2.5-20 g / 1. A preferred liquid culture medium for the detection of coliforms contains between about 7-14 g / 1 of gelatin peptone and 3-18 g / 1 of yeast extract or 10-20 g / 1 of tryptosa, 2.5-20 g / 1 of lactose, 2.5-7.5 g / 1 of sodium chloride, 1.35-1.65 g / 1 of bile salts, 2.5-7.5 g / 1 of guar gum and 0.16-5.0 g / 1 of phenol red. A particularly preferred liquid culture medium contains about 15 g / 1 of tryptose or 7 g / 1 of gelatin peptone and 9 g / 1 of yeast extract, 10 g / 1 of lactose, 5 g / 1 of sodium chloride, 1.5 g / 1 of bile salts, 5 g / 1 of guar gum and 1.25 g / 1 of phenol red. In a further variation of the medium it has been found that other indicators of the sulfonaphthalein type, for example bromocresol purple and chlorophenol red, are also useful at high concentrations to provide early detection of Enterobacteriaceae such as coliforms. The culture medium of this invention can be used in broths, in agar or in thin film devices such as PETRIFILM plates. When used in PETRIFILM plates the culture medium is applied to a surface of the device and the medium is then preferably dried. When the preferred culture medium is in a dry state on a thin film, the medium contains approximately 4.8 mg / in2 of tryptose, 1.6 mg / in2 of lactose, 1.6 mg / in2 of sodium chloride, 0.5 mg / in2 of salts bile, 1.6 mg / in2 of guar gum and 0.4 mg / in2 of phenol red When the dried medium is rehydrated, the components of the culture medium mentioned above are in the same concentrations as in the liquid culture media The dry medium may be partially or completely rehydrated before inoculation with test bacteria Another embodiment of the present invention is a method for detecting the presence of Enterobacteriaceae such as coliform bacteria in a sample. aliquot of the sample containing bacteria to a culture medium comprising triptose, lactose or glucose, sodium chloride, bile salts and an excess amount of sulfon indicator aftalein such as bromocresol purple, chlorophenol red or preferably phenol red to provide a high indicator concentration very close to the bacteria. The bacteria are then cultured in the presence of the culture medium and the presence of bacteria is determined by detecting the color change of the indicator as the growing bacteria produce metabolites. Using this method the detection and counting of coliform bacteria is possible in less than about 12 hours and preferably in less than about 8 hours. The detection of bacteria such as coliform bacteria in the culture medium can be done visually or using an instrument. A suitable instrument is described in US Patent Application Serial No. 08/168, 681 filed on December 13, 1993. Another embodiment of this invention is a device for detecting bacterial growth in a sample. A preferred device includes a waterproof, self-supporting substrate and a transparent cover. The present culture medium is applied on the water-impermeable, self-supporting substrate, and then dried to provide a high concentration of sulfonaphthalein indicator such as phenol red very close to the growing bacteria to allow the detection and counting of the bacteria growing in less than 12 hours. The detection of the bacteria growing in the device is carried out rapidly (either visually or using an instrument) when the purple or red color of the medium changes to a yellow color in the presence of the acidic bacterial metabolites typical of the Enterobacteriaceae such as coliform bacteria. In another alternative embodiment, the present culture medium also contains a second indicator, triphenyltetrazolium chloride. When used in the culture medium, this second indicator provides confirmation of early detection and rapid counting of bacteria. More specifically, after the presence of coliform bacteria has been detected by the color change of a sulfonaphthalein indicator, the growing bacteria continue to produce acids. When there are sufficient colonies that produce acids, in growth, the medium eventually changes completely from red or purple to yellow. After approximately 24 hours and when the medium has changed from red or purple to yellow, it is possible to detect the color change of the triphenyl tetrazolium chloride in the medium caused by the growth of the bacterial colonies. In the presence of bacteria, triphenyltetrazolium chloride changes to a red color. The color change of the triphenyltetrazolium chloride allows the confirmation of early counts associated with the color change of a sulfonaphthalein indicator such as phenol red. This last confirmation is also supported by the presence of gas bubbles around the bacteria, produced by the bacteria. When triphenyltetrazolium chloride is used in the culture medium, the amounts of this indicator in the culture medium are between about 0.025-0.250 g / 1, a preferred amount is about 0.150 g / 1. When triphenyltetrazolium chloride is used in a thin film device, the dry medium preferably contains about 0.02 mg / in2 (0.0031 mg / in2) of triphenyltetrazolium chloride. The triphenyltetrazolium chloride can be provided as part of the culture medium 14 or it can be a part of a coating on the cover.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 is an illustration of a device containing the culture medium of the present invention.

DETAILED DESCRIPTION This invention provides products and methods that can be used to detect the presence of Enterobacteriaceae, including coliform bacteria, in a sample in less than about 12 hours (coliform bacteria include gram-negative rods, which ferment lactose while other Enterobacteriaceae can use glucose). Although a variety of products and methods have been used to detect Enterobacteriaceae, particularly coliform bacteria, a detection and counting time of less than 12 hours is significantly less than the detection times of conventional products or methods. Early detection and rapid counting of coliform bacteria in most samples have been problematic for several reasons. In most cases, the coliform bacteria present in samples have been subjected to stresses and do not grow at an optimum level. In order to provide optimal growth (and thus provide the earliest detection time), the stressed bacteria must be given a period of time to recover from the induced stress. The present invention provides a medium that is believed to allow rapid recovery of coliform bacteria. This medium includes known reagents and nutrients that can be obtained commercially. These reagents and nutrients include a mixture of peptone and yeast extract, such as tryptose, lactose, sodium chloride, and bile salts which can be obtained from Accumedia, Inc. Baltimore, MD. The medium further contains guar gum which is commercially available from Rhone-Poulenc, Inc., Kreuzlinger, Switzerland, phenol red which is commercially available from Aldrich ical Company, Inc., Milwau ee, Wl, and triphenyltetrazolium chloride. can be obtained commercially from AMRESCO, Solón, OH. The reagents and preferred materials are weighed and mixed using conventional methods. The present culture medium includes a pH indicator of sulfonaphthalein, for example, phenol red, bromocresol purple, chlorophenol red and the like. These indicators provide similar results, with variations due to the pH at which the color changes occur. Bromocresol purple changes from purple to and above pH 6.8 to yellow to and below pH 5.2. The phenol red is red at pH 8.2 and changes to yellow at pH 6.8. The chlorophenol red is red at pH 6.2 and changes to yellow at pH 4.8. These variations allow the adequacy of the indicator system when a medium that changes to a slightly different pH is desired. The phenolsulfonaphthalein core on which these dyes are based is shown below.

Because these indicators have a common core they share similar desirable properties such as adequate water solubility and relatively low toxicity for the Enterobacteriaceae. In particular, the present culture medium includes the preferred pH indicator, phenol red. Phenol red is a known indicator that changes color from red to yellow in the presence of acid. As a bacterial colony grows, the colony produces metabolic acids that react with the indicator and produce colored areas of yellow that surround the colony. This indicator has been used in other culture media but is generally used in very small amounts, typically less than about 10-30 mg / l (Manual of Methods of General Bacteriology, page 440 (1981)). Specifically, phenol red has been reported in various culture media at levels between about 18-24 mg / l (BBL Laboratory Products and Procedures Manual, page 131 (1973)). According to the present invention, however, the amounts of phenol red are substantially greater than the amounts of phenol red or other indicators that are generally used in the reported culture media. For example, the use of an approximately ten times excess of phenol red with respect to amounts used in conventional media, more than 160 mg of phenol red per liter provides the benefits of early detection and rapid counting. In addition, the use of an excess of about one thousand times more than the amounts used in conventional media, more than 1000 mg of phenol red per liter, provides an increased color contrast and is the preferred concentration in the present medium. Surprisingly, coliform bacteria seem to recover and grow very well in medium containing such a large excess of a sulfonephthalein indicator such as phenol red and there is apparently no toxicity to coliform bacteria at those concentrations. As much as about 5000 mg of phenol red per liter, the upper limit of solubility for phenol red in water has been found to be non-toxic to coliform bacteria. It may be that the large excess of phenol red serves to act as a buffer for the medium and thus promotes recovery and / or growth because it is believed that bacterial coliform growth may be pH sensitive. The use of amounts of phenol red that are sufficient to provide a buffer capacity to the medium is not an accepted practice, since the indicators are generally not reactive that are used in large quantities or are selected to provide buffer capacity to a solution . Another benefit of the ability to use excess amounts of sulfonaphthalein indicators such as phenol red in the medium to provide some buffering capacity to the medium is the prevention of the diffusion of the metabolic acids in the medium. The uncontrolled diffusion of acids through the medium may allow the colored areas of yellow surrounding the growing colonies to overlap or merge. When the yellow colored areas overlap or come together, the resulting colony counts are difficult to obtain or are inaccurate. Another unexpected benefit of using a large excess of bromocresol purple, phenol red or chlorophenol red, is that the contrast between the strong color, for example bright red of phenol red or bright red-violet of chlorophenol red or Bromocresol purple purple in neutral or basic solutions and the yellow color of bromocresol purple, phenol red or chlorophenol red in the presence of acids is maximized. The maximum contrast between the bright color of the medium and the yellow color of the areas surrounding the growing coliform bacteria allows the visual detection of coliform bacteria in a much more temperate time compared to the detection time of conventional products or procedures . In this specification, the phase "excess amount of phenol red sufficient to cause a high concentration of phenol red very close to the growing bacteria to allow detection and counting of the growing bacteria in less than 12 hours" means a a concentration of phenol red or an equivalent sulfonaphthalein indicator greater than about 160 mg / l and preferably greater than 1000 mg / l allowing visualization or instrument detection of a color change from purple or red to yellow caused by coliform bacterial metabolites . In another variation the indicator means includes a buffer, preferably a buffer that provides an optimum at about pH 7 such as a phosphate buffer comprising monobasic or dibasic sodium phosphate. This variation is particularly useful when you want to limit the size of the dye areas to smaller areas. Figure 1 illustrates a thin film, dry culture device, suitable for use with the medium of the present invention. Briefly, the device is described in U.S. Patent Nos. 4,565,783 and 5,089,413, which describe methods for preparing and using these types of culture devices. The thin film culture device 10 includes a body having a substrate 12 impermeable to water, self-supporting. The substrate 12 is preferably a relatively rigid material made of a waterproof material that does not absorb water such as polyester, polypropylene or polystyrene. Other suitable water-impermeable materials include substrates such as paper containing a water-impermeable polyethylene coating. The upper surface of the substrate 12 is coated with a layer of culture medium 14 is then dried to provide a dry medium in the substrate 12. Alternatively, an adhesive layer can be applied to the substrate 12, which adhesive serves to maintain a medium of culture that can be applied as a powder. The adhesive should be sufficiently transparent when it is hydrated to allow the growth of bacterial colonies on the surface of the substrate through the coated substrate to be seen. The adhesive should also be applied to the substrate with a thickness that allows the substrate to be uniformly applied with dry culture medium without fully incorporating the medium in the adhesive. If the liquid culture medium of this invention has to be used in a dry form or as a dry powder, the reagents, the nutrients and the indicator such as phenol red are dried. The culture medium of this invention can be rapidly dried by heating the liquid medium in an oven of approximately 104.4 ° C (220 ° F) until essentially all the water in the liquid has evaporated. If the medium is heated after the water has evaporated, however, the medium begins to degrade. A foam spacer 16 having a circular opening in the foam is adhered to the surface of the substrate 12 covered with the medium. The foam spacer covering the periphery of the substrate 12 defines the area to be inoculated with a sample and serves to prevent the sample from leaking from the substrate. In an alternative embodiment, a device may not include a foam layer containing the sample. In this sample device it is contained in the substrate by the medium components only. A cover 20 is attached to an edge of an upper surface of the foam spacer 16. The cover 20 is preferably made of a film or transparent cover material to facilitate counting of the bacterial colonies present in the substrate. In addition, the cover 20 is preferably impermeable to bacteria and water vapor to avoid the risk of contamination and deterioration of the components. A preferred material for use as a cover 20 is biaxially oriented polypropylene.

In use, a predetermined amount of inoculum typically about one milliliter of inoculum is added to the device illustrated in Figure 1 by running back the cover 20 and adding an aqueous test sample or water to the center of the substrate 12. The cover 20 is then replaced on the substrate 12 and the inoculum is uniformly distributed on the substrate. A convenient tool to do this is an already heavy circular template which is also used to confine the inoculum to a specific area of the substrate 12. As the inoculum comes into contact and is distributed over the substrate 12, the culture medium the substrate 12 is hydrated to form a nutrient gel that maintains growth. The inoculated device is then incubated for a predetermined time after which the number of bacterial colonies growing on the substrate can be counted through the transparent cover 20. Although the use of the culture medium of this invention on a film device thinner is described above, those skilled in the art will recognize that the culture medium can be used in other culture devices known in the art. For example, the culture medium can be used as a broth and used to grow bacteria in suspension or the culture medium can be used to grow bacteria on known agar plates. The following examples are intended to provide additional details and modalities related to the practice of the present invention. These examples are provided for illustrative purposes and should not be construed to limit the scope of the present invention as defined in the appended claims.

Example 1.- Growth of coliform bacteria in medium of rapid coliform count This example illustrates that a preferred liquid culture medium of this invention (RCCM, rapid coliform count) can be used to grow coliform bacteria in broth, agar, or thin film plate. The medium used in this example contained 15 g / 1 triptose, 5 g / 1 lactose, 5 g / 1 sodium chloride, 1.5 g / 1 bile salts, 5 g / 1 guar gum, 0.050 g / 1 of triphenyltetrazolium chloride and 1.25 g / 1 of phenol red (all components could be obtained commercially from the sources mentioned above) with the exception that triphenyltetrazolium chloride was not used in the broth medium. Several bacteria indicated in Table 1, below, were initially cultured for 18-24 hours in trypticase soy broth (Difco Laboratories, Inc. Detroit, MI) at 35 ° C. The bacteria mentioned in the list in Table 1 were obtained from Sillicker Laboratories, Chicago, IL (indicated by "s" after the name of the bacterium) or were isolated from quality control used by 3M Microbiology Products Laboratory, St. Paul, MN. Those skilled in the art will recognize that strains or equivalent species of bacteria can be obtained commercially or can be isolated using well-known methods or procedures. After approximately 24 hours of growth in trypticase soy broth, the growing cultures containing approximately 108-109 bacteria / ml were serially diluted approximately 106-107 times in Butterfields Standard Methods Buffer (SMB Fisher Scientific, Minneapolis, MN). An aliquot of the diluted culture (approximately one ml) was used to inoculate a petri dish, a glass tube with a screw cap or a PETRIFILM plate containing RCCM.

For agar culture, the aliquots of culture were added to petri dishes and were then covered with RCCM and agar (approximately 12 ml of medium containing approximately% w / v agar) and then incubated for 24 hours at 35 ° C. . For culture in a broth, the aliquots of culture were added to the tubes with screw cap containing RCCM (approximately 10 ml) and a Durham tube. The tubes were then capped and also incubated for 24 hours at 35 ° C. For the cultivation in a thin film, a RCCM layer was forced through a small hole to cover a 7.5 ml polyester substrate film (Imperial Chemical Industries, Wilmington, DE) at room temperature. The coated polyester film was then dried for about 1-20 minutes at about 93.3-121.1 ° C (200-250 ° F). an 18 ml polystyrene foam spacer layer was cut to cover the polyester film and a circular opening was cut in the polystyrene foam spacer. A surface of the polystyrene foam was coated with an isooctilacrylate / pressure sensitive acrylamine adhesive (96/4 wt.% Acrylate-acrylamide ratio) and the polystyrene foam was adhered to the coated surface of the polyester film. A transparent polypropylene film was cut to cover the laminated polyester / polystyrene foam film. One surface of the polypropylene film (0.04 mm (1.6 mils, 3M, St. Paul, MN), was coated with a pressure sensitive isooctyl acrylamide / acrylamide adhesive (96/4% by weight ratio acrylate and acrylamide) ) and covered with a layer of guar gum (Rhone-Poulenc, Inc. Kreuzlinger, Switzerland) A layer of tape covered with adhesive on both sides (3M, St. Paul, MN) was placed on an exposed edge of the spacer. polystyrene foam and the rubber-containing surface of the polypropylene film was adhered to the polystyrene foam spacer along one edge.The culture aliquots (one ml) were placed in the opening of the polystyrene foam spacer, the Polypropylene film was used to cover the inoculum and the thin film was incubated for 24 hours at 35 ° C. After incubation for 24 hours, the petri dishes, the glass tubes and the thin film plates were evaluated with resp ecto to the presence of acid zones that were identified as yellow areas on the red background of the plate and / or the presence of gas bubbles. The broth cultures were evaluated with respect to the change in color from red to yellow and the presence of gas bubbles in the durham tubes. The data indicated in the list in Table 1 below indicate that the RCCM was selective for the culture of coliform bacteria.

TABLE 1 arteries RCCH inoculum - RCCN broth - RCCH agar - PETRIFILH CC film (Cfu / Tsa) Cren. Acid / Gas Believe, Acid Crecii. Acid / 6th Growing up. Acid / 6as E. coli 565s 30 G ('- +) G (<') - E. coti 471 s 66 6 (+ *) G [go) - S. sap.7B9s 43 NG (j '_) NG (- • ) N6 N6 B. pul 1212s 10 NG (JJ NG (-) N6 NG E. fecaelis EN1094 43 N6 (j J NG (-) N6 NG B. aureus 50s 41 NG (JJ NG (-) NG N6 E. fecaelis SF 39 NB (j J NG i--) NG N6 E. fecaelis 3H 37 NG (JJ NG (-) N6 N6 S. aureus 48s 117 N6 (_ '_) NG (• -) NG NG L. dulbreckii 914s 40 N6 (_) N6 (-) NG (- / -) NG (- / -) . typhiauriut 451s 95 8 (- -) G (• • 1 G < * / -) 6 (- / -) Y. ent. 572s 93 6 (* '*) G (<) 6 6 l. eonocytog 23s 70 N6 (- '-) 6 (--I NG N6 E. fecaelis 732s 20 N6 (-1 -) NG (-) NG NG 3. saintpaul 373s 49 G (*' -) G (-) ND (- / -) ND (- / -) P. vulgaris 760s 60 6 ('' -) G (- -) 6 G E. coli 503s 42 G (+ > *) 6 (< •) ND ND C. freundii 614s 29 G (+ / +) 6 • I ND ND Freundii 433s 27 6 (+ J '*) G (< •) ND ND E. coli 563s 53 6 (* i •) 6 (' - ) ND ND K. pneu 26 49 6 (+ '*) G (< y) ND ND E. aerog 39 95 G (*' "•) G ') ND ND E. aggloaerans 44s 41 6 (+ +) G ['y) ND ND E, coli 555s 94 6 (+ J *) 6 (< •) ND (' / +) ND (-7+) E. doacae CS 98 6 (+ '+) 6 (< HG 6 6. nettport 347s 92 6 t + -) G "'•) ND (* / *) ND (* • / *) E. sakazaki C3 73 G (+' +) 8 (HG (+ / ') 6 ( + / +) K. o.ytoca C4 48 G (+ It) G { < y) G (< • / *) 6 ("• / *) H. alveí C2 80 6 (+ 1 *) G (y) G (+ / +) 6 (+ / +) B. liquefaciens Cl 75 G (+ '+) G (>) G 6 E. coli 5615 76 G (+' *) G (y) ND ND C. freundii 17 96 G (+ '"•) 6 (and ) ND ND i. Oxytoca 33 97 G (+ 1 *) G (y) ND (+ / ') ND («• / *) t.coh 149 80 G (+ t *) N6 (-) G 6 E. fecaelis EN1062 70 NG (- '-) G (NG NG E. coli 6275 86 G (+ 1 *) S (i-) ND ND E. aggl 611s 136 G (+' -) G (H ND ND E. coli 633s 71 6 (+ "+) NG (-) ND ND White 0 NG (- I-) NG (-) NG NG B - growing N6 - no growth HD - not determined 6as - (-) no gas bubbles, ( +) fteido gas bubbles - (-) without acid zone, (+) acid zone EXAMPLE 2.- Effect of the indicator dye concentration.

This example indicates that excess amounts of the indicator dye such as phenol red, i.e., amounts of phenol red greater than about 160 mg / l, provide detection and early counting of the coliform bacteria. In this example several bacteria were isolated from quality control used by 3M Microbiology Products Laboratory, St. Paul, MN. These bacteria included Serratia liquefaciens (3M strain Cl which was used at three different dilutions, approximately 25 bacteria / ml, 50 bacteria / ml and 75 bacteria / ml), Hafnia alvei (3M strain C2), Enterobacter sakazaki (3M strain C3), Klebsiella oxytoca (3M strain C4), Enterobacter cloacae (3M strain C5), and Escherichia coli (E. coli 149, ATCC Accession No. 55535 which was used at three different dilutions approximately 25 bacteria / ml, 50 bacteria / ml and 75 bacteria / ml). Strains or species of equivalent bacteria would be readily recognized by those skilled in the art. The bacteria were cultured and diluted as described in Example 1 and aliquots of culture were added to thin film plates as described in Example 1 with the exception that the concentrations of phenol red in the medium applied on the film of polyester ranged from 0.04-2.5 g / 1. The data in Table 2, below, indicate in a list the percentage of colonies that were counted at 12 hours compared to the number of colonies that were counted at 24 hours. The count at 24 hours was done by identifying colonies that produced gas and that were detected by the color change of triphenyltetrazolium chloride. The data indicate that quantities of phenol red in excess of 160 mg / l allowed consistent early detection and rapid counting as well as providing a more rapid quantification of the bacteria that produced acid.

TABLE 2 Effect of the concentration of phenol red or Example 3- Comparative example In one experiment, thin-film plates containing the culture medium of this invention (RCCM) were compared with commercially available PETRIFILM coliform counting plates (3M, St. Paul, MN) and with conventional pour plates containing agar and violet red bile (Difco Laboratories (Inc.). Thin film plates containing RCCM were prepared as described in Example 1. Aliquots used to inoculate the thin film plates were taken from milk samples obtainable at the request of the Dairy Quality Control Institute, Minneapolis, MN, which were diluted as described in Example 1. For each different sample aliquots of one ml were added to three plates each having a different type of medium and then the plates inoculated were incubated at 35 ° C. Each of the inoculated plates were visually evaluated every hour, in addition, the plates with RCCM and those with thin film PETRIFILM were evaluated every thirty minutes by taking images of the plates with a camera at two different wavelengths. The images at both wavelengths were then digitized and stored electronically. The stored images were then processed by dividing the images of the two wavelengths and then subtracting the divided image of the divided image calculated from the images made thirty minutes earlier. This method of image analysis is described in U.S. Patent 5,403,722 to Floeder et al. The detection and counting of colony forming units of the three media were determined manually. The data provided by the comparison described above are indicated in the form of a list in the Table below. The data states that the means of this invention allow for earlier detection and counting when compared to any other means.

TABLE 3 MEDIA TRAINING UNITS COLONY TIME / ML (HOURS) (duplicate plates) Red bile agar 41/31 24 violet 15/15 counting plates 24 coliforms PETRIFILM RCCM (visual) 31/39 10 RCCM (instrument) 36/35 8 In another experiment, the film plates containing the culture medium of this invention (RCCM) were compared to PETRIFIL coliform count plates (3M, St. Paul, MN) with a PETRIFILM coliform count plate having red phenol or neutral red (another commonly used indicator obtainable from Sigma-Aldrich Corp., Milwaukee, Wl) and with a thin film plate coated with a medium that was identical to RCCM except that the phenol red was replaced by red neutral, a different indicator commonly used. The thin film plates containing the different media and indicators were prepared as described in Example 1 and were inoculated with aliquots of diluted sample containing the bacteria indicated in the list in Table 4, below. The bacteria used in this example were used in Example 2, above. The data indicate the time required to count one hundred percent of the colonies that were observed after 24 hours as detected by the color change of the triphenyl tetrazolium chloride and the formation of gas bubbles.

TABLE 4 Time to reach a count equivalent to the 24-hour count bacteria Cl C2 C3 C4 C5 149 half-indicator RCCM 9 hr 11 hr 12 hr 9 hr 12 hr 10 hr phenol red RCCM CNR CNR CNR CNR CNR CNR neutral red PCC * 10 hr 10 hr 12 hr 8 hr 12 hr 9 hr red of phenol PCC * CNR CNR 13 hr CNR CNR CNR neutral red CNR - counts that can not be read 24 hr count determined by gas bubble formation and color change of triphenyltetrazolium chloride * PCC - PETRIFILM coliform count plates Example 4- Comparative example In this experiment the thin film plates containing variations of the culture medium of this invention (RCCM and RECM) were compared with the commercially available coliform count plates (PCC-NR, 3M, St. Paul, MN) They used neutral red as the indicator dye. Film plates containing RCCM and RECM were prepared as described in Example 1. However, the amounts of components differed somewhat and the dibasic monobasic potassium phosphate was added according to the list in Table 5 below.

TABLE 5 Media formulations * Ingredient Coliform Enterobacteriaceae (RCCM) (RECM) Peptone G1 14 g / 1 14 g / i Yeast extract1 18 g / 1 6 g / i Sodium chloride2 10 g / 1 10 g / i Bile salts1 3 g / 1 3 g / i Potassium phosphate, 2 g / 1 2 g / i monobasic3 Potassium phosphate, 6 g / 1 6 g / i dibasic3 Lactose1 20 g / 1 - Glucose1 - 20 g / i Rubber guar4 10 g / 1 11 g / i Deionized water, 1 liter 1 1 itro pH = 7.0 ± 0.1 Sources of ingredients 1 Accumedia 2 Sigma Chemical Company 3 Mallinckrodt Rhone-Poulenc * The coating solutions are twice the weight of the final coating.

To the formulations of the media one of the dyes, bromocresol purple, chlorophenol red or phenol red (all can be obtained from Accumedia), was added at coating concentrations (twice the final concentration (dried)) as shown in Table 5 and the formulations were knife-coated on the base and dried at 104 ° C (220 ° F) for 5 to 10 minutes. The bacterium E. coli 149 (ATCC No. 55535) was grown overnight at 35 ° C in tryptic soy broth (Dimed Corporation, St. Paul MN). The culture was diluted approximately 10-7 fold in Butterfields buffer (Fischer Scientific, Chicago, IL) to approximately 20-50 colony forming units / milliliter. Duplicate plates of each type of dye and concentration were inoculated with one ml of the bacterial suspension and incubated at 35 ° C for the remainder of the experiment. The plates were observed with respect to the presence of yellow zones, indicating the presence of acid-forming bacteria. The results are presented as a percentage of the final number of colonies at 24 hours on the plates in Table 6 and Table 7.

These results show that all sulfonaphthalein dyes provide a faster detection of Enterobacteriaceae when the dyes are used in high concentrations.

TABLE 6 E. coli counts in the middle of Enterobacteriaceae (RECM) EB / NR = Medium / neutral red indicator of Enterobacteriaceae BCP = bromocresol purple CPR = chlorophenol red PR = phenol red TABLE 7 It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (9)

1. A bacterial culture medium, characterized in that it facilitates the early detection and counting of the Enterobacteriaceae cultured in the medium comprises gelatin peptone and yeast extract, lactose or glucose, sodium chloride, bile salts, guar gum and an excess amount of a sulfonaphthalene dye sufficient to provide a high concentration of dye very close to the growing bacteria to allow the detection and counting of the growing bacteria.

2. The culture medium according to claim 1, characterized in that the concentration of the dye is greater than about 160 mg / l.

3. The culture medium according to claim 1, characterized in that the concentration of the dye is greater than about 1000 mg / l.

4. The culture medium according to claim 1, characterized in that the dye is selected from bromocresol purple, phenol red and chlorophenol red.

5. The culture medium according to claim 1, characterized in that the medium further comprises triphenyltetrazolium chloride.

6. The culture medium according to claim 4, characterized in that it contains approximately 10-20 g / 1 with tryptosa or 7-14 g / 1 of gelatin peptone, 3-18 g / 1 of yeast extract, 2.5-20 g / 1 of lactose or 2.5-20 g / 1 of glucose, 2.5-7.5 g / 1 of sodium chloride, 1.35-1.65 g / 1 of bile salts, 2.5-7.5 g / 1 of guar gum, 0.025-0.250 g / 1 of triphenyltetrazolium chloride and 0.16-5.0 g / 1 of dye.

7. The method for detecting the presence of Enterobacteriaceae in a sample, characterized in that it comprises the steps of adding an aliquot of the sample containing bacteria to a culture medium comprising a mixture of gelatin peptone and yeast extract, lactose or glucose, chloride of sodium, bile salts, guar gum and an excess amount of dye sufficient to provide a high concentration of dye very close to the bacteria; you cultivate the bacteria in the presence of the culture medium; and detect the color change of the dye as growing bacteria metabolize.

8. The culture medium according to claim 7, characterized in that the dye is selected from bromocresol purple, phenol red and chlorophenol red.

9. A device for detecting coliform bacterial growth in a sample, characterized in that it consists essentially of a waterproof, self-supporting substrate, a foam spacer and a transparent cover, characterized in that a culture medium comprising a mixture of gelatin peptone and extract of yeast, lactose or glucose, sodium chloride, bile salts, guar gum and an excess amount of a sulfonaphthalein dye is applied to the water-impermeable substrate, self-supporting enough to provide a high concentration of dye very close to bacteria growing to allow detection and counting of growing bacteria.