JP3274716B2 - Salmonella isolation medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium and a method for selectively separating and detecting Salmonella from food, environment, and clinical materials.

[0002]

2. Description of the Related Art Salmonella is widely known as a causative agent of food poisoning and a causative agent of typhoid disease in humans and animals, and its detection is important as a daily test. Especially chicken, pork, beef,
Inspection of whether or not foods such as eggs, side dishes, drinking water and milk are contaminated with Salmonella is also regulated by the Food Sanitation Law,
Mandatory for daily inspection.

[0003] As a method for detecting Salmonella, the property of Salmonella producing hydrogen sulfide in a thiosulfate-containing medium (TSI medium) is used. The most common method is to generate iron sulfide by reacting with ferrous ions and observe the presence or absence of blackening of colonies. The MLCB agar medium is widely used as the Salmonella separation medium.

[0004]

However, the conventional Salmonella test method using the MLCB medium has a problem that it cannot be distinguished from other hydrogen sulfide-producing bacteria represented by cytorobacter. Accordingly, an object of the present invention is to provide a Salmonella separation medium capable of distinguishing between Cytobacter and Salmonella with high sensitivity.

[0005]

The inventors of the present invention have conducted intensive studies to find a medium in which Salmonella can be detected separately from Cytobacter, and as a result, have found that a conventional medium containing thiosulfate and ferric ion can be used. If a medium containing a certain amount of saccharides that are not decomposed by Salmonella but can be decomposed by cytorobacters to produce an acid is used, cytorobactors produce hydrogen sulfide, but at the same time, decompose the saccharides to produce acid. Since the reaction of hydrogen sulfide and ferric ion does not proceed to produce black colonies because the reaction of hydrogen sulfide and ferric ion does not proceed, it has been found that Salmonella can be selectively detected, and the present invention has been completed. Was.

[0006] That is, the present invention relates to a yeast extract of 0.4-0.6% by weight and a heart extract powder of 0.25-0.35 by weight.
%, Peptone 1.0-2.5%, sodium chloride 0.3
5-0.45%, Mannit 0.25-0.3%, L-
Lysine hydrochloride 0.45 to 0.55%, sodium thiosulfate 0.35 to 0.45%, ammonium ferric citrate 0.
08-0.12%, brilliant gulin 0.0001-
0.00125%, crystal violet 0.000
Salmonella containing 9 to 0.001% and 0.1 to 3.0% of a saccharide which is not degraded by Salmonella and can be decomposed by Cytobacter to produce an acid, and contains no sulfur-containing amino acid. It provides a separation medium.

Another object of the present invention is to provide a method for detecting Salmonella, which comprises inoculating a test sample into the culture medium for Salmonella isolation and culturing, and then detecting the presence or absence of black colonies on the culture medium. .

[0008]

[0009] Examples of saccharides which are not decomposed by Salmonella but can be decomposed by cytorobacter to produce an acid include, for example, sorbitol, lactose, maltose, trehalose, raffinose, galactoside derivatives, lactulose and palatinit. Here, examples of the galactoside derivative include synthetic substrates such as 5-bromo-3-indolyl-β-D-galactoside and 5-bromo-4-chloro-3-indolyl-β-D-galactoside. These sugars are at least 2
A combination of more than one species may be used.

The amount of these saccharides to be mixed in the separation medium is as follows:
0.1 to 3.0% by weight (hereinafter simply indicated as%), particularly preferably 0.2 to 1.0%. If it is less than 0.1%, the generation of black colonies by cytorobacter may not be sufficiently suppressed.

The separation medium of the present invention may contain other components in addition to the above-mentioned components as long as the effects of the present invention are not impaired. Further, the separation medium of the present invention comprises agar 1.3 at the time of use.
~ 1.5% and water are preferably used as an agar plate medium.

In order to detect the presence of Salmonella in a subject using the separation medium of the present invention, the subject is added to the culture medium, and cultured at 30 to 44.5 ° C. for several hours to several tens of hours.
The presence or absence of black colonies on the medium may be visually observed.

[0013] Examples of the subject include clinical materials such as urine and plasma, foods, medicines, cosmetics, beverages, and the like. May be used in a culture medium for enrichment.

[0014]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Lactose was added to a powder of MLCB medium (Nissui Pharmaceutical) having the composition shown in Table 1 so as to have the concentration shown in Table 2, mixed well with purified water, and added at 105 ° C. for 30 minutes. The mixture was dissolved by heating to prepare a plate, which was used as a separation medium. The test strains were 10 strains shown in Table 2.
The strain was grown at 37 ° C for 2 hours using tryptoise bouillon (Nissui).
A culture solution cultured for 4 hours was used. 10 μl of this culture
After the streak was smeared with a loop and cultured at 37 ° C. for 24 hours, the presence or absence of black colonies was determined by visual observation. as a result,
As is evident from Table 2, it was found that Salmonella and Cytobacter could be separated and detected using a medium containing 0.2% or more lactose.

[0016]

[Table 1]

[0017]

[Table 2]

Example 2 A saccharide described in Tables 3 to 5 was added to an MLCB medium to prepare a separation medium, and the presence or absence of black colonies was determined in the same manner as in Example 1. As a result, the sugars described in Tables 3 to 5
%, It was found that Salmonella and Cytobacter could be separated and detected by using a medium containing at least%.

[0019]

[Table 3]

[0020]

[Table 4]

[0021]

[Table 5]

Example 3 Each of the Salmonellas shown in Table 6 was cultured in the same manner as in Example 1 using MLCB and MLCB supplemented with lactose as a test medium, and the culture was serially diluted 10-fold with sterile physiological saline. .1
The medium was inoculated into the medium and smeared with a contra stick. 37 ° C,
After culturing for 24 hours, the number of bacteria and the size of the colony were measured with a stereoscopic microscope. As a result, even in the MLCB medium supplemented with 1% lactose, there was no significant difference in the growth of Salmonella compared to the case without lactose.

[0023]

[Table 6]

Example 4 Citrobacter freen separated from food
The dii36 strain was inoculated into a 1% lactose-supplemented MLCB medium and an MLCB medium, and the presence or absence of a black colony was examined in the same manner as in Example 1. As a result, no black colony was observed in all the strains in the 1% lactose-added MLCB medium. Showed black colonies in all strains.

Example 5 Each of Salmonellas shown in Tables 7 to 9 was cultured in the same manner as in Example 1 using tryptosome agar medium (TPS), MLCB medium, and MLCB supplemented with lactose as test medium, and the culture was sterilized. Serially dilute 10-fold with physiological saline to about 10 ⁴ /0.1
ml, inoculate 0.1 ml of the medium,
It was smeared with a con large stick. After culturing at 37 ° C. for 24 hours, the number of bacteria was counted. As a result, no significant difference was observed in the growth of Salmonella in any medium.

[0026]

[Table 7]

[0027]

[Table 8]

[0028]

[Table 9]

[0029]

According to the present invention, a food medium,
Salmonella in a specimen such as clinical material is clearly distinguished from cytorobacter and detected.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-63-112975 (JP, A) JP-A-6-22791 (JP, A) JP-A-3-67599 (JP, A) (58) Investigation Field (Int.Cl. ⁷ , DB name) C12N 1/00-1/38 C12Q 1/00-1/70 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. Yeast extract 0.4-0.6%, heart extract powder 0.25-0.35%, peptone 1.0-
2.5%, sodium chloride 0.35-0.45%, mannitol 0.25-0.3%, L-lysine hydrochloride 0.45
0.55%, sodium thiosulfate 0.35 to 0.45
%, Ammonium ferric citrate 0.08 to 0.12%, brilliant gulin 0.0001 to 0.00125%, crystal violet 0.0009 to 0.001% and acid which is not decomposed by Salmonella but decomposed by cytorobacter A medium for separating Salmonella, comprising 0.1 to 3.0% of a saccharide capable of producing phenol and containing no sulfur-containing amino acid. 2. A saccharide which is not degraded by Salmonella but can be decomposed by Cytobacter to produce an acid, and is one or more selected from sorbitol, lactose, maltose, trehalose, raffinose, galactoside derivatives, lactulose and palatinit. The medium for Salmonella isolation according to claim 1, which is: 3. A method for detecting Salmonella, which comprises inoculating a sample into the separation medium according to claim 1 and culturing the medium, and then detecting the presence or absence of a black colony on the medium.