JPH0265798A - Method for detecting salmonellal bacteria, culture medium and detecting paper for salmonellal bacteria used therefor - Google Patents

Abstract

PURPOSE:To readily detect salmonellal bacteria which are causative bacteria of food intoxication by adding a specimen containing the salmonellal bacteria to a liquid culture medium for pretreatment, culturing the bacteria, applying the resultant culture to a detecting paper impregnated with a culture medium for detection, culturing the bacteria and observing spots appearing on the detecting paper. CONSTITUTION:A specimen containing salmonellal bacteria is added to a liquid culture medium at pH 7.2 containing peptone, mannitol, Na2HPO4, KH2PO4, sodium selenite and Briliant Green dissolved in water for pretreatment and cultured at 33-39 deg.C for 16-26hr. The resultant culture solution is then applied to a detecting paper impregnated with a liquid culture medium for detection at pH 6.8 containing polypeptone, proteose peptone, yeast extract, heart extract, mannitol, NaCl, sodium thiosulfate, ferric ammonium citrate, L-lysine hydrochloride, bile acid salt, lead acetate and copper sulfate dissolved in water and cultured at 33-39 deg.C for 18-26hr. The salmonellal bacteria are then detected from spots appearing on the detecting paper.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a method for detecting Salmonella that can easily detect bacteria of the genus Salmonella that causes food poisoning present in meat, etc., and a method for detecting Salmonella used in the method. related to a medium for use and a detection paper.

[Conventional technology]

Looking at the causative agents that account for the highest number of bacterial food poisoning cases in Japan, the first is Vibrio parahaemolyticus, and the second is Vibrio parahaemolyticus.
Staphylococcus aureus ranks third, followed by Salmonella, and Salmonella is an important food-poisoning bacterium from the perspective of pathogenicity.

Acute gastroenteritis caused by Salmonella occurs when large amounts of Salmonella that have grown in food are ingested orally and are sterilized to some extent as they pass through the stomach, but the surviving bacteria enter the small intestine and multiply again. Most of the symptoms are thought to be caused by ingestion of 7η-dyed foods. In particular, salmonella contamination of meat is a problem that countries around the world are struggling to prevent, and even in Japan, salmonella has been detected at a high rate in meat produced at slaughterhouses and poultry processing plants. Among the foods that cause salmonella food poisoning, these meats and their processed products are said to cause the highest number of outbreaks.

Therefore, to prevent food poisoning caused by Salmonella,
Firstly, food contaminated with Salmonella should be quickly detected through inspection and eliminated.Secondly, foods should be stored at low temperatures to inhibit the growth of contaminated Salmonella, or food should be heated to sterilize the contaminated bacteria. It is important to take countermeasures depending on the test results, and for this purpose, it is necessary to incorporate Salmonella tests into daily work and to perform sufficient quality control.

In this case, as a method for detecting Salmonella, the method described below has conventionally been generally employed.

That is, first, the following ingredients were added to purified water 12: peptone io, o g mannitol 5.0 g sodium monohydrogen phosphate 6.45 g sodium dihydrogen phosphate 2.0 g purified raw bile powder
20.0 g brilliant green
0.0135 g dissolved at pH 7.2 ± 0.1 2
Add the sample to 2M broth and incubate at 35°C for 15-18
Pre-incubate for an hour. This 22M broth inhibits the growth of Durham-positive bacteria, but among intestinal bacteria, the growth of Proteus, which does not ferment mannitol, is only slightly delayed.
Therefore, this medium is used only for culture for Salmonella testing. Next, in purified water 12, the following ingredients peptone were added.
5.0g Lactose 4.0g Sodium phosphate 10.0g Sodium selenite 4.0g dissolved in selenite medium at pH 7.0±0.1.
Add a predetermined amount of 2M broth and culture for enrichment at 43°C for 18 to 24 hours. This medium is an enrichment medium for Salmonella with moderate selectivity, and can be used to enrich Salmonella typhi and Salmonella baratifi A in stool, but Salmonella cholerethuis will not grow on this medium. After that, add the following ingredients to the purified water IP: 5.0g peptone, 7.5g bile salts.
9.0g milk Kikara
1 0.
Og Sodium Citrate 8.5
g Sodium 1,000 sulfate 5.5 g Ferric citrate 1.0 g Neutral Red 0.025 g Brilliant Green 0.33 mg Agar 13.
A predetermined amount of the above selenite medium is transferred to a pH 7.0±0.1 SS agar medium in which 5 g of the selenite medium has been dissolved and solidified, and direct separation culture is performed at 35°C for 18 to 2.1 hours. Thereafter, a colony suspected of containing Salmonella is taken and transplanted onto a confirmation medium such as TSI agar or LIM medium, followed by separate culture, followed by identification of Salmonella.

[Problem to be solved by the invention]

As mentioned above, the detection of Salmonella requires pre-culture with EEM broth, enrichment culture with selenite medium, SS
It is necessary to perform four cultures: direct isolation culture using agar and differential culture using confirmation medium. This is because it is extremely difficult to selectively increase Salmonella alone. Therefore, routinely testing for Salmonella contamination requires enormous amounts of time, equipment, and expense, and the reality is that such testing is difficult to carry out.

The present invention was made in view of the above circumstances, and aims to provide a detection method that allows Salmonella detection to be carried out easily and inexpensively in a short time, as well as a culture medium and detection paper used in the method.

[Means and effects for solving the problems] In order to achieve the above-mentioned object, the present invention contains 9 to 11 g of the following component peptone in 1000 m6 of water.
4.5-5.5g Sodium monohydrogen phosphate 6-7g Potassium dihydrogen nonate 1.5
~2.5g sodium selenite 9~ftg sulfate 0.5~1.5mg brilliant green 0.1~0.3mg dissolved pH
A sample containing Salmonella was added to a pretreatment liquid medium of 7.2 ± 0.2, and after culturing at 33 to 39°C for 16 to 26 hours, the following ingredients polypeptone protease, peptone yeast extract, and heart extract were added to 1000 ml of water. Mannitol sodium chloride J, sodium 1,000 sulfate citrate 1 loss/monium 1,-phosphorus; hydrochloride bile salt lead acetate copper sulfate 9-11 g 4.5-5.5 g 4.5-5.5 g 1 .5-2.5g 2.5-3.5g 3.5-4.5g 3.5-4.5g 0,5-1,5g 4.5-5.5g 2.5-3.5g O, The pretreatment liquid medium after culturing was attached to a detection paper impregnated with a detection liquid medium of pH 6.8 ± 0.2 in which 1 to 0.3 g of 1.5 to 2.5 mg was dissolved. A method for detecting Salmonella is provided, in which Salmonella is detected from spots appearing on the detection paper after culturing at 39°C for 18 to 26 hours.

In addition, the present invention uses water 1000+nj! Contains the following ingredients: peptone 9-11g mannitol
4.5-5.5g Sodium monohydrogen phosphate 6-7g Potassium dihydrogen phosphate 1.5-2
．． 5g Sodium selenite 9-11g Sulfur fJ
Copper 0.5-1.5 mg brilliant green 0.1-0.3 mg are dissolved, pt
Provided is a Salmonella medium in which + is 7.2±0.2.

Furthermore, the present invention contains the following components polypeptone 9 to 11 g proteose peptone 4.5 to 5.5 g yeast extract 4 in 100100 O of water.
, 5-5.5g heart extract 1.5-2
．． 5g Mannit 2.5-3.5g
Sodium chloride 3.5-4.58 Sodium thiosulfate 3.5-4.5g Iron ammonium citrate 0.5-1.5g L-lysine hydrochloride
4.5-5.5g bile salts 2.
5-3.5g Lead acetate 0.1-0.
3g copper sulfate], 5-2.5 m
Provide a Salmonella medium in which g is dissolved and the pH is 6.8±0.2.

Additionally, the present invention provides a detection paper for Salmonella impregnated with the above-mentioned liquid medium for detection.

In the method of the present invention, first, Salmonella is selectively enriched and cultured in a pretreatment liquid medium, and then this Salmonella is transplanted onto a detection paper, and further cultured in a detection liquid medium to produce hydrogen sulfide produced by Salmonella. This is a qualitative test for Salmonella from the spots that appear on the detection paper due to the reaction between the lead acetate poison in the detection medium, and this makes it possible to detect even minute amounts of Salmonella in the sample. That is, in the method of the present invention, by carrying out the first culture using a pretreatment liquid medium, Salmonella is enriched, and at the same time, the proliferation of hydrogen sulfide-producing bacteria other than Salmonella, such as Cytorrhacter and Proteus, is suppressed. Then, after the first selection is completed, Salmonella is selectively grown on a detection paper containing a liquid detection medium to form subonol-1 on the detection paper. Second, if a copper-colored spot appears due to the reaction between hydrogen sulfide and lead acetate, the sample is qualitatively determined to be Salmonella positive.

In this case, in the above pretreatment medium, peptone and mannitol are used as nutrient, sodium monohydrogen phosphate and potassium dihydrogen phosphate are used as p++ stabilizers, and sodium selenite and copper sulfate are used to prevent the growth of bacteria other than Salmonella. Brillian) and Gulino act as pn stabilizers and inhibitors, but in this culture medium, sodium selenite and copper sulfate, which have not been used in this type of culture medium, are used as inhibitors. By using these in combination, Salmonella can be selectively enriched and proliferation of other bacteria can be reliably suppressed. In addition, in the above-mentioned eruption medium, polypeptone, protease peptone, yeast extract, heart extract (bovine heart extract), and mannitol are nutrients, sodium chloride is a pH stabilizer, and sodium thiosulfate and iron ammonium citrate are used to prevent salmonella. Raw material for hydrogen sulfide gas generation, L-lysine hydrochloride is a nutrient that Salmonella can selectively use, bile salts and steel sulfate are inhibitors, and lead acetate combines with hydrogen sulfide gas produced by Salmonella to form spots. However, in this medium, by using bile salts as an inhibitor together with copper sulfate, which has not been used in the past, it is possible to reliably selectively enrich only Salmonella, as well as provide nutrients. By using protease peptone and heart extract as the catalyst, the generation of hydrogen sulfide gas by Salmonella can be favorably promoted and a clear spot can be formed on the detection paper.

Therefore, whereas previously Salmonella and other bacteria were multiplied together using EEM broth and then selectively cultured for Salmonella using selenite medium, with the pretreatment medium of the present invention, only one culture is required. Salmonella can be reliably selectively cultured. In addition, whereas previously Salmonella was detected by direct layer culture and differential culture, with the detection medium of the present invention, it is possible to simply culture on detection paper and examine the spots that appear on the detection paper. One can simply detect Salmonella.

Therefore, according to the present invention, since Salmonella is enriched and cultured using the pretreatment medium and detection medium with excellent selectivity, Salmonella and other hydrogen sulfide-producing bacteria can be cultured twice. Therefore, Salmonella can be detected in a short time. In addition, culture using a detection medium is carried out on a detection paper, and judgment is made by the spots that appear on this detection paper, making it possible to detect Salmonella very simply and at low cost. It is something that can be done.

The present invention will be explained in more detail below.

In the detection method of the present invention, first, a sample containing Salmonella is added to the above-mentioned pretreatment liquid medium and cultured for enrichment. In addition, it is preferable to perform the culture under anaerobic conditions as much as possible.

In this case, there are no limitations on the method of adding the specimen to the medium, but in the case of solid or powdered specimens, it may be added directly, or in the case of wiped specimens, after wiping the specimen with a cotton swab etc., the two swabs etc. are added to the medium. A soaking and washing method can be adopted. Note that there is no particular limit to the amount of sample added.

In addition, the pretreatment liquid medium has the following amounts of each component in 1000 ml of water, and has a p++ of 7.2.
It is particularly preferable to increase Salmonella bacteria.

Namely: Peptone 10g Mannitol 5g Sodium monohydrogen phosphate 6.45 g Potassium dihydrogen phosphate
2g sodium selenite
10g copper sulfate low
Brilliant Grine 0.2 ■ Furthermore, it is particularly preferable to set the culture temperature to 35 to 37°C and the culture time to 18 to 20 hours from the viewpoint of increasing Salmonella bacteria.

In the present invention, next, the pretreatment medium subjected to the enrichment culture is attached to a detection paper impregnated with the liquid detection medium, and further culture is performed. Note that it is desirable to perform the culture under anaerobic conditions as much as possible.

In this case, there is no limitation on the means for attaching the pretreatment medium to the detection paper, but a method of immersing the detection paper in the pretreatment medium and soaking the pretreatment medium into the detection paper is preferably adopted.

Note that there is no particular limit to the amount of the pretreatment medium attached to the detection paper.

In addition, the detection liquid medium to be impregnated into the detection paper is water 100%
It is particularly preferable for the detection of Salmonella that the amounts of each component in 0 ml are as follows and the pH is 6.8. i.e. polypeptone
lOg proteose peptone 5g
Yeast extract 5g Heart extract 2g Mannitol
3g sodium chloride
4g sodium sulfate
4g iron ammonium citrate 1gL
-Lysine hydrochloride 5g bile salt
3g lead acetate
0.2g copper sulfate
0.002g Further, it is particularly suitable for the detection of Salmonella that the culture temperature is 35 to 37°C and the culture time is 20 to 24 hours. As the bile salt, it is preferable to use Bile Salt No. 3 manufactured by Difco.

The present invention detects Salmonella from the spot formed on the detection paper after the above-mentioned culture. In this case, the front and back sides of the detection paper are observed, and if a copper-colored spot is seen, it is determined to be salmonella positive.

Note that the present invention can be used to detect any hydrogen sulfide-producing bacteria belonging to the genus Salmonella.

〔Effect of the invention〕

As explained above, according to the present invention, Salmonella, which is a food poisoning bacterium present in meat and the like, can be detected quickly, conveniently, and inexpensively. In this case, the pretreatment medium and detection medium of the present invention can reliably selectively enrich Salmonella, and the detection paper of the present invention can reliably cause spots caused by Salmonella to appear.

Therefore, the present invention is applicable to funeral homes, poultry slaughterhouses, meat shops,
It is used in industries that handle many foods, such as school lunch centers and lunch box caterers, because it is simple, quick, and inexpensive to constantly inspect food and cooking utensils to see if they are contaminated with salmonids. High value. Also, DHL,
In a live medium such as MLCB, Salmonella and other producing bacteria grow at the same time, so they must be identified using a confirmation medium after separation, but with the detection paper of the present invention, only Salmonella forms copper-colored spots, so Salmonella can be easily detected. Even if separation and identification are omitted, anything that develops color can be determined to be Lumonella without any doubt. However, since the detection by the present invention goes through a pretreatment of selective enrichment using a pretreatment medium, the number of colored spots does not indicate the presence of Salmonella in food, but is only a qualitative test. If you use it,
The present invention is extremely useful in preventing food poisoning, as food and cooking utensils can be frequently checked for Salmonella contamination.

Hereinafter, the effects of the present invention will be specifically illustrated by experimental examples.

[Experiment example]

In order to investigate the performance of the above pretreatment liquid medium and detection paper,
The following experiments were conducted using Salmo nigra and other hydrogen sulfide producing bacteria.

Test strain ■ Salmonella typhimurium
atyphimurium, food poisoning origin method): Hereinafter abbreviated as St ■ Salmonella enteritidis (Salm.
onellaenLeritidis, food poisoning origin); hereinafter abbreviated as Se■ Citro-bacLer freundi
i, Food-derived method) Abbreviated as 2-sided cr ■ Proteus mirabilis (Proteus mlrabilis food-derived strain) Preparation of test bacterial solution The preserved bacterial strain was subcultured three times in ordinary broth to rejuvenate it, and then transferred to ordinary agar. It was cultured on a slant medium and used as a test fungus.

To prepare the test bacteria, St and Se were taken together in normal broth, and Cfc! : Pm was also collected in ordinary broth, and after culturing them at 37°C for 24 hours, Cf
For +Pm, this was used as the test solution, and for St+36, this was used as the test stock solution, and then 10-1 and 1 was added with sterile physiological saline.
A dilution of 0-2 was used in the experiment.

The experimental groups according to the combined bacterial ratio of seven monkeys and one other 2S-producing bacteria are as follows.

St+3e: Cf+Pm Group 1 1 ・ 1 ■ Group 1 ・ 10 Group ■ 1: ] 00 Ingest 1cc of each of the bacterial solutions prepared to have such a bacterial amount ratio in normal broth, and then ingest them in the order shown in Figure 1. The experiment was conducted according to the following.

Experimental Results ■ Comparison of Detection for Test Solutions for Salmonella and Other Hydrogen Sulfide-Producing Bacteria After pre-treating the test solutions for Salmonella and other hydrogen sulfide-producing bacteria with the pretreatment medium of the present invention and the selenite medium, these media were tested for the detection of the present invention. Adsorb a certain amount onto paper and culture at 37°C for 20 hours.
Table 1 shows a comparison of the number of colored spots that appeared on the detection paper.
We obtained the results shown in .

Table 1 (Spot detection status on test paper after bacterial enrichment culture) There is a tendency to decrease as the proportion becomes higher than the amount of caries, but there are 1/100i of Salmonella in other HzS producing cells in the test solution. Spots were observed on the detection paper even in the presence of .

■ In order to compare the selectivity of the pretreatment medium of the present invention and the selenite medium as enrichment culture for pretreatment for identification of bacterial species spotted on the detection paper, bacteria were isolated from the expression spots, and their properties were determined using TSI medium and LIM medium. The inoculated bacteria were confirmed and identified using diagnostic serum for Salmonella. The results are shown in Table 2, and when pretreated with the culture medium of the present invention, all spots expressed on the detection paper in groups 1 to 2 of each bacterial amount mixing ratio were Salmonella.

The number of colored spots was determined by the number of Salmonella bacteria in Cf + Pm.However, in the case of pre-treated with selenite medium, all the expressed spots were Salmonella when the bacterial amount ratio was 1 group.
In the case of the 1:10 bacterial load ratio in group (2), 80% of the expression spots were Salmonella and 20% were other 11□3-producing bacteria.

■In the case of group t:ioo leap ratio, 40% of the expression spots
It can be seen that as the amount of Salmonella and 60% of other H, S-producing bacteria decreases, the number of spots expressed on the detection paper increases as other 11□S-producing bacteria become weaker.

From this, the detection paper of the present invention has higher selectivity when the pretreatment medium of the present invention is used as the pretreatment medium, and the spots on the detection paper do not express H, S-producing bacteria other than Salmonella. confirmed.

[Brief explanation of the drawing]

FIG. 1 is a flowchart showing the culture procedure in an experimental example.

Claims (1)

[Claims] 1. In 1000 ml of water, the following ingredients: 9 to 11 g of peptone, 4.5 to 5.5 g of mannitol, 6 to 7 g of sodium monohydrogen phosphate, 1.5 to 2.5 g of potassium dihydrogen phosphate, selenite A sample containing salmonella was added to a pretreatment liquid medium of pH 7.2±0.2 in which 9 to 11 g of sodium, 0.5 to 1.5 mg of copper sulfate, and 0.1 to 0.3 mg of brilliant green were dissolved, and the sample was heated at 33 to 39°C. In 1
Cultivate for 6 to 26 hours, then add the following ingredients to 1000 ml of water: 9 to 11 g of polypeptone, 4.5 to 5.5 g of proteose peptone, 4.5 to 5.5 g of yeast extract, 1.5 to 2.5 g of heart extract, and Mannitol 2. .5-3.5g Sodium chloride 3.5-4.5g Sodium thiosulfate 3.5-4.5g Iron ammonium citrate 0.5-1.5g L-lysine hydrochloride 4.5-5.5g Bile acids The above detection paper is impregnated with a liquid detection medium of pH 6.8 ± 0.2 in which 2.5 to 3.5 g of salt, 0.1 to 0.3 g of lead acetate, and 1.5 to 2.5 mg of copper sulfate are dissolved. A method for detecting Salmonella, which comprises: attaching a liquid medium for post-culture pretreatment and culturing at 33 to 39°C for 18 to 26 hours, and then detecting Salmonella from spots appearing on the detection paper. . 2. In 1000 ml of water, the following ingredients: 9-11 g of peptone, 4.5-5.5 g of mannitol, 6-7 g of sodium monohydrogen phosphate, 1.5-2.5 g of potassium dihydrogen phosphate, 9-11 g of sodium selenite, copper sulfate A culture medium for Salmonella, characterized in that 0.5 to 1.5 mg and 0.1 to 0.3 mg of brilliantogrin are dissolved therein and the pH is 7.2±0.2. 3. In 1000 ml of water, the following ingredients: 9-11 g of polypeptone, 4.5-5.5 g of proteose peptone, 4.5-5.5 g of yeast extract, 1.5-2.5 g of heart extract, 2.5-3.5 g of mannitol, chloride Sodium 3.5-4.5g Sodium thiosulfate 3.5-4.5g Iron ammonium citrate 0.5-1.5g L-lysine hydrochloride 4.5-5.5g Bile salts 2.5-3. A culture medium for Salmonella, characterized in that 5g of lead acetate, 0.1 to 0.3g of copper sulfate, and 1.5 to 2.5mg of copper sulfate are dissolved therein, and the pH thereof is 6.8±0.2. 4. A Salmonella detection paper impregnated with the Salmonella culture medium according to claim 3.