Abstract:
An isolation plating medium and mixture for simultaneously identifying Bacillus species are disclosed. The specific bacilli identified are  Bacillus thuringiensis  and  Bacillus cereus.  The medium and mixture contain nutrients, inhibitory ingredients to inhibit the growth of other bacteria yeast and molds and a chromogenic substrate. The substrate changes color in response to the production of phosphatidylinositol-specific phospholipase C for the identification of the bacteria.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to the presumptive identification of bacteria, and in particular to the presumptive identification the microorganisms  Bacillus cereus  and  Bacillus thuringiensis.    
     Chapter 35 of the  Compendium of Methods for the Microbiological Examination of Foods,  American Public Health Association, 1992, questions whether  Bacillus thuringiensis  is a separate species or a variety of  Bacillus cereus  because of the cultural similarity of the microorganisms. Here they are considered separate species.  Bacillus cereus  and  Bacillus thuringiensis  are found on a variety of foods, and have been implicated in food poisoning of humans. For this reason and the fact that they are similar in characteristics, it is desirable to consider both in the process of making a presumptive identification from a mixed sample. 
     The  Compendium of Methods for the Microbiological Examination of Foods,  supra, describes the Kim-Goepfert agar and the mannitol yolk polymyxin agar for presumptive identification of  Bacillus cereus  from a mixed sample, and points out that these plating media are not 100% selective and may be difficult to interpret. 
     The paper entitled Phosphatidylinositol-Specific Phospholipases C from  Bacillus cereus  and  Bacillus thuringiensis  by O. H. Griffith, J. J. Volwerk and A. Kuppe, Methods in Enzymology, Vol. 197 Academic Press, Inc. 1991, investigates the production of the enzyme Phosphatidylinositol-Specific Phospholipases C produced by both  Bacillus cereus  and  Bacillus thuringiensis.  M. Ryan, J. Huang, O. H. Griffith, J. F. W. Keana, and J. J. Volwerk describe detection of Phosphatidylinositol-Specific Phospholipase C produced by  Bacillus cereus  and  Bacillus thuringiensis  with a chemiluminescent substrate in the paper entitled A Chemiluminescent Substrate for the Detection of Phosphatidylinositol-Specific Phospholipase C, Analytical Biochemistry, Vol. 214, pages 548-556 (1993). In a paper entitled Isolation and Detection of Listeria monocytogenes Using Fluorogenic and Chromogenic Substrates for Phosphatidylinositol-Specific Phospholipase C, by L. Restaino (the present inventor), E. W. Frampton, R. M. Irbe, Günter Schabert, and Hans Spitz, Journal of Food Protection, Vol 62, No. 3, 1999, Pages 244-251, a chromogenic substrate is disclosed for Listeria responsive to the production of phosphatidylinositol-specific phospholipase C. 
     SUMMARY OF THE INVENTION 
     It is a principle object of the present invention to provide a plating medium with a chromogenic substrate for the presumptive identification of  Bacillus cereus  and  Bacillus thuringiensis  from a mixed sample, and to identify both species of Bacillus symultaneosly. 
     The inventor has recognized that when disposed in a growth medium both  Bacillus cereus  and  Bacillus thuringiensis  secrete the enzyme phosphatidylinositol-specific phospholipase C into the growth medium. It is an object of the present invention to provide a plating medium for the presumptive identification of  Bacillus cereus  and  Bacillus thuringiensis  that has a phosphatidylinositol-specific phospholipase C responsive chromogenic substrate, thus providing a medium that simultaneously responds to both  Bacillus cereus  and  Bacillus thuringiensis  bacteria. The inventor recognized that the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate is responsive to Phosphatidylinositol-Specific Phospholipase C secreted by  Bacillus cereus  and  Bacillus thuringiensis  from his work on Listeria reported in the paper entitled Isolation and Detection of Listeria monocytogenes Using Fluorogenic and Chromogenic Substrates for Phosphatidylinositol-Specific Phospholipase C, supra, and it is an object of the present invention to provide a plating medium utilizing this chromogen for the presumptive identification of  Bacillus cereus  and  Bacillus thuringiensis.    
     The plating medium, according to the present invention, comprises (1) a nutrient media that promotes the growth of  Bacillus cereus  and  Bacillus thuringiensis  under conditions promoting incubation, (2) at least one ingriedient that resusitates damaged Bacillus cells under incubation, (3) at least one ingredient that promotes generation of  Bacillus cereus  spores under incubation, (4) at least one ingredient that under incubation inhibits the growth of Bacillus bacteria other than  Bacillus cereus  and  Bacillus thuringiensis  and other related bacteria, (5) at least one ingredient that inhibits the growth of yeast and molds under incubation, (6) the substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate, (7) at least one ingredient that promotes the expression of the enzyme to react with the substrate, and (8) at least one ingredient that solidifies the mixture. 
     DETAILED DESCRIPTION OF THE INVENTION 
     It is necessary that the Bacillus bacteria consume nutrients and grow in order for the bacteria to secrete enzymes. Hence the plating medium must have a rich nutrient base. In order to promote the growth of the various strains of Bacillus bacteria, the plating media of the present invention include one or more of the ingredients proteose peptone, LAB LEMCO (meat extract) powder, and yeast extract. In the preferred medium described throughout this specification, all three of these ingredients are in the plating medium and form the nutrient base. 
     The preferred plating medium includes sodium pyruvate to facilitate the resuscitation of damaged Bacillus cells, and magnesium sulfate to promote germination of  Bacillus cereus  spores. 
     In any plating medium, the growth of cells of bacteria other than the bacteria of interest complicates or completely frustrates reading of the plate, and hence it is desirable or necessary to inhibit the growth of species other than the one or ones of interest. The media of the present invention must suppress all strains of Bacillus other than  Bacillus cereus  and  Bacillus thuringiensis  and related bacteria. For this purpose, the media of the present invention contain one or more of the ingredients lithium chloride, ceftazidime, polymixin B sulfate, the third and fourth generation of cephalosporins, and moxactan. The preferred plating medium contains lithium chloride, ceftazidime, and polymixin B sulfate. 
     The preferred medium contains cycloheximide to inhibit the growth of yeast and molds. The chromogenic substrate that changes color responsive to the presence of phosphatidylinositol-specific phospholipase C in the preferred medium is 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. 
     Ingredients that permit the activation of the enzyme phosphatidylinositol-specific phospholipase C in the plating media are bovine serum and powdered silicates. In the preferred embodiment, this ingredient is bovine serum. 
     The plating media also contains at least one ingredient to maintain the pH of the medium in a suitable range, namely, potassium phosphate (monobasic) and/or sodium phosphate (dibasic). In the preferred embodiment, both potassium phosphate and sodium phosphate are used in the media. 
     An ingredient must be added to the mixture to solidify the mixture. In the preferred composition, this ingredient is agar. The formula for the preferred embodiment of the plating media is set forth in Table 1. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 CHEMICAL 
                 SUPPLIER 
                 GRAMS/LITER 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Proteose peptone 
                 Difco 
                 10.00 
               
               
                 LAB LEMCO powder 
                 Oxoid 
                 5.00 
               
               
                 Yeast extract 
                 Difco 
                 6.00 
               
               
                 Sodium pyruvate 
                 Biosynth 
                 10.00 
               
               
                 Potassium phosphate 
                   
                 0.24 
               
               
                 (monobasic} 
               
               
                 Sodium phosphate 
                   
                 2.50 
               
               
                 (dibasic) 
               
               
                 Magnesium sulfate 
                   
                 0.06 
               
               
                 Anhydrous 
               
               
                 Cycloheximide 
                   
                 0.20 
               
               
                 Lithium chloride 
                 Sigma 
                 2.00 
               
               
                 Agar 
                 Difco 
                 15.00 
               
               
                 Bovine Serum 
                 Bayer 
                 4.20 
               
               
                 82-067 
               
               
                 Ceftazidime 
                 Glaxo Wellcome 
                 Sufficient to suppress 
               
               
                   
                   
                 Bacillus other than 
               
               
                   
                   
                   B. cereus  and 
               
               
                   
                   
                 
                   B. thuringiensis 
                 
               
               
                 5-bromo-4-chloro-3- 
                 Biosynth 
                 0.35 
               
               
                 indoxyl-myo-inositol- 
               
               
                 1-phosphate. 
               
               
                 Polymixin B sulfate 
                 Sigma 
                 0.013 
               
               
                   
               
             
          
         
       
     
     Prior to the preparation of the plating medium, the ingredients are admixed into four components. The first component includes proteose peptone, potassium phosphate (monobasic), LAB LEMCO powder, and cycloheximide. The second component contains yeast extract, sodium pyruvate, and magnesium sulfate. The third component contains sodium phosphate (dibasic), lithium chloride, and agar. The fourth component is the remaining ingredients, each of which is maintained separately under its prescribed stage conditions until the plating medium is to be produced. 
     The composition is prepared by admixing the three components set forth above, under sterile conditions, and each of the first three components is admixed. Thereafter, one at a time, the remaining four components, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate, bovine serum, ceftazidine, and polymixin B sulfate, are added to the mixture. The composition is then placed in petri dishes and stored under proper conditions overnight. 
    
    
     EXAMPLE I 
     The bacterial strains indicated in Table 2 were applied to the petri dishes referred to above, and incubated at 35 degrees Celsius for a period of 24 hours. Thereafter, the surfaces of the platting media in the petri dishes were observed, and produced the following results. 
     
       
         
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Number of 
                   
               
               
                 Bacteria 
                 Strains 
                 Colonial Morphology 
               
               
                   
               
             
             
               
                 
                   Bacillus cereus 
                 
                 8 
                 Turquoise flat dull colonies; 
               
               
                   
                   
                 2-7 mm with and without 
               
               
                   
                   
                 turquoise halos 
               
               
                 
                   Bacillus thuringiensis 
                 
                 3 
                 Turquoise flat dull colonies; 
               
               
                   
                   
                 2-8 mm with and without 
               
               
                   
                   
                 turquoise halos 
               
               
                 
                   Bacillus circulans 
                 
                 1 
                 White domed dull colonies; 
               
               
                   
                   
                 1-2 mm 
               
               
                 
                   Bacillus megaterium 
                 
                 2 
                 No growth 
               
               
                 
                   Bacillus licheniformis 
                 
                 3 
                 No growth 
               
               
                   Bacillus subtilis ,  Bacillus   
                 1 strain each 
                 No growth 
               
               
                   brevis ,  Bacillus lentus , 
               
               
                   Bacillus pumilus ,  Bacillus   
               
               
                   spaericus , 
               
               
                   PaeniBacillus macerans , 
               
               
                   PaeniBacillus polymyxa , 
               
               
                   Bacillus mycoides , and 
               
               
                 
                   Bacillus insolitus 
                 
               
               
                 
                   Listeria monocytogenes 
                 
                 3 
                 No growth to turquoise domed 
               
               
                   
                   
                 colonies; pinpoint to &lt;1 mm 
               
               
                 
                   Listeria ivanovii 
                 
                 1 
                 Turquoise domed colonies; 
               
               
                   
                   
                 pinpoint to &lt;1 mm 
               
               
                   Listeria innocua ,  Listeria   
                 1 strain each 
                 White domed colonies; 
               
               
                   seeligeri , and  Listeria   
                   
                 pinpoint to &lt;1 mm 
               
               
                 
                   welshimeri 
                 
               
               
                 
                   Enterococcus faecium 
                 
                 4 
                 No growth to white domed 
               
               
                   
                   
                 colonies; pinpoint 
               
               
                 
                   Enterococcus faecalis 
                 
                 2 
                 No growth to white domed 
               
               
                   
                   
                 colonies; pinpoint 
               
               
                 
                   Enterococcus avium 
                 
                 3 
                 No growth 
               
               
                 
                   Staphylococcus aureus 
                 
                 5 
                 No growth 
               
               
                 Micrococcus sp., 
                 1 strain each 
                 No growth 
               
               
                   Pediococcus cerevisiae , 
               
               
                 
                   Staphylococcus 
                 
               
               
                   epidermidis , 
               
               
                 and  Staphylococcus   
               
               
                 
                   saprophyticus 
                 
               
               
                 Gram negative species* 
                 7 
                 No growth 
               
               
                   
               
               
                 *One strain each of  Pseudomonas aeruginosa ,  Escherichia coli ,  Enterobacter agglomerans ,  Salmonella derby ,  Salmonella typhimurium ,  Klebsiella pneumoniae , and  Escherichia coli  0157:H7.  
               
             
          
         
       
     
     From Table 2, it is clear that  Bacillus cereus  and  Bacillus thuringiensis  produce significantly large colonies on the plating medium and are further readily distinguishable by their turquoise color. 
     It should also be noted that of the Bacillus genus, only  Bacillus cereus  and  Bacillus thuringiensis  produce significant colonies on the preferred medium. Table 3 compares the phosphatidylinositol-specific phospholipase C production of Bacillus species. 
     
       
         
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                 NUMBER OF 
                   
               
               
                   
                 ATCC 
                 STRAINS 
                 ENZYME 
               
               
                 SPECIES 
                 NUMBERS 
                 TESTED 
                 PRODUCTION 
               
               
                   
               
             
             
               
                 
                   Bacillus cereus 
                 
                 11778, 
                 8 
                 + 
               
               
                   
                 13061, 
               
               
                   
                 14549 and 
               
               
                   
                 21281 
               
               
                 
                   Bacillus thuringiensis 
                 
                 33680 and 
                 3 
                 + 
               
               
                   
                 39152 
               
               
                 
                   Bacillus circulans 
                 
                 4513 
                 1 
                 − 
               
               
                 
                   Bacillus megaterium 
                 
                 14581 
                 2 
                 − 
               
               
                 
                   Bacillus licheniformis 
                 
                 10716 and 
                 3 
                 − 
               
               
                   
                 11946 
               
               
                 
                   Bacillus subtilis 
                 
                 37015 
                 1 
                 − 
               
               
                 
                   Bacillus brevis 
                 
                 8246 
                 1 
                 − 
               
               
                 
                   Bacillus pumilus 
                 
                 7061 
                 1 
                 − 
               
               
                 
                   Bacillus sphaericus 
                 
                 14577 
                 1 
                 − 
               
               
                 
                   Bacillus macerans 
                 
                 8244 
                 1 
                 − 
               
               
                 
                   Bacillus polymyxa 
                 
                 842 
                 1 
                 − 
               
               
                 
                   Bacillus mycoides 
                 
                 — 
                 1 
                 − 
               
               
                 
                   Bacillus insolitus 
                 
                 23299 
                 1 
                 − 
               
               
                   
               
             
          
         
       
     
     Table 3 shows that the generally known species of Bacillus, except for  Bacillus cereus  and  Bacillus thuringiensis,  are not producers of phosphatidylinositol-specific phospholipase C, and accordingly these species will not produce colonies on the preferred plating medium. 
     Although variations in the plating medium of the preferred embodiment are set forth above, other variations will become apparent to those skilled in the art. It is therefore intended that this invention be not limited to the foregoing specification, but rather only to the appended claims