Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-05-28
2001-09-04
Ware, Deborah K. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S252100, C435S832000, C435S252500, C435S253600, C435S834000
Reexamination Certificate
active
06284517
ABSTRACT:
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 pyruva
Burmeister Marshall A.
Ware Deborah K.
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