Application effect of using a selective sensitive medium for detecting Yersinia pestis in spoiled materials

Abstract

The present invention provides an application effect of using a sensitive culture medium to detect Yersinia pestis in corrupt materials, belonging to the technical field of isolation and culture of Yersinia pestis. The culture medium for isolating and culturing Yersinia pestis from corrupt materials provided by the present invention comprises the following raw materials: 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol and neutral red. The neutral red indicator in the culture medium of the present invention produces red aggregates in an acidic environment and adheres to the colonies, making the colonies of Yersinia pestis show red. It has a strong inhibitory effect on Proteus, Escherichia coli and Gram-positive bacteria, is suitable for testing animal materials contaminated by corruption and miscellaneous bacteria, has high specificity and strong sensitivity, and the Yersinia pestis develops into typical mature colonies after 24 hours of culture. Moreover, the grown colonies of Yersinia pestis are typical and easy to select, which has practical significance for the isolation of Yersinia pestis from field materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of isolation and culture of Yersinia pestis, and particularly relates to the application effect of using a sensitive culture medium to detect Yersinia pestis in putrid materials. Background Art

[0002] In the monitoring of plague foci, when animal plague is in the late epidemic or quiescent period, due to the weakened or completely lost virulence of Yersinia pestis, it cannot cause the death of animals, but exists in the animal body. Due to the reduced amount of bacteria, it is difficult to isolate Yersinia pestis according to general conventional culture media. Therefore, a particularly sensitive enrichment culture medium must be required to isolate Yersinia pestis during cultivation. Moreover, natural Yersinia pestis is generally isolated from self-killed animals. Due to the putrefaction of self-killed animals, it is difficult to isolate Yersinia pestis (plague bacteria) from a contaminated material due to the influence of Proteus and Gram-positive bacteria. Therefore, developing a selective culture medium that can isolate Yersinia pestis from putrid materials and improve the isolation rate of Yersinia pestis is a key technology for field plague monitoring sites and laboratory research. The existing selective culture media for Yersinia pestis mainly include gentian violet culture medium. In actual work, the effect of gentian violet culture medium in isolating Yersinia pestis from contaminated (putrid) materials is not ideal. There is an urgent need in this field to provide a culture medium that can well isolate Yersinia pestis from putrid materials. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a culture medium with high specificity and strong sensitivity that can well isolate Yersinia pestis from putrid materials.

[0004] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a culture medium for isolating and culturing Yersinia pestis from putrid materials, and the culture medium comprises the following raw materials: 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol, and neutral red.

[0006] Preferably, the bile salts are bile salts No. 3.

[0007] Preferably, the 0.1% hemolytic Hiss medium is composed of beef digestive juice, water, peptone, Hiss agar, lactose, and defibrinated rabbit blood, and the volume-weight ratio of the above raw materials is 200 ml: 800 ml: 8 - 10 g: 13 - 14 g: 10 - 11 g: 0.1 - 0.2 ml.

[0008] Preferably, the volume-weight ratio of 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol, and neutral red in the culture medium is 1000 ml: 0.36 - 0.43 g: 0.0004 - 0.0005 g: 4.7 - 5.5 g: 0.0115 - 0.0125 g: 0.8 - 1 g: 0.02 - 0.03 g.

[0009] Preferably, the preparation method of the 0.1% hemolytic Hiss medium comprises the following steps: mixing beef digestive juice, water, peptone, agar, and lactose, adjusting the pH to 7.2 - 7.4, cooling after sterilization, and adding defibrinated rabbit hemolysis.

[0010] The present invention also provides a preparation method of the above-mentioned culture medium, which comprises the following steps: mixing 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol, and neutral red to obtain the product.

[0011] The present invention also provides an application of the above-mentioned culture medium in the isolation and culture of Yersinia pestis.

[0012] The present invention also provides an application of the above-mentioned culture medium in the isolation and culture of Yersinia pestis from corrupt materials.

[0013] The present invention also provides an application of the above-mentioned culture medium in plague monitoring.

[0014] Advantages of the present invention:

[0015] The culture medium for isolating and culturing Yersinia pestis from corrupt materials provided by the present invention has better effects than the gentian violet medium commonly used in the art for isolating and culturing plague bacteria. In the culture medium of the present invention, the neutral red indicator produces red aggregates in an acidic environment and adheres to the colonies, making the plague bacteria colonies show red. It has strong inhibitory effects on Proteus, Escherichia coli, and Gram-positive bacteria, and is suitable for testing animal materials contaminated by corruption and miscellaneous bacteria. It has high specificity and strong sensitivity, and the plague bacteria develop into typical mature colonies after 24 hours of culture. Moreover, the grown plague bacteria colonies are typical and easy to select, which has practical significance for the isolation of plague bacteria from field materials. Description of the drawings

[0016] Figure 1 Shows the growth of plague bacteria in selective medium and Hiss medium, where Figure A shows the growth of plague bacteria in Medium 1; Figure B shows the growth of plague bacteria in 1% hemolytic Hiss medium. Detailed implementation manners

[0017] The present invention provides a culture medium for isolating and culturing Yersinia pestis from corrupt materials, and the culture medium comprises the following raw materials: 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol and neutral red.

[0018] In the present invention, the 0.1% hemolytic Hiss medium preferably consists of beef digestive juice, water, peptone, Hiss agar, lactose and defibrinated rabbit blood, and the volume-weight ratio of the above raw materials is preferably 200 ml: 800 ml: 8 - 10 g: 13 - 14 g: 10 - 11 g: 0.1 - 0.2 ml, more preferably 200 ml: 800 ml: 10 g: 14 g: 10 g: 0.1 ml. The present invention has no special limitation on the specific sources of the various raw materials in the culture medium for isolating and culturing Yersinia pestis from corrupt materials. In the present invention, the bile salts are preferably bile salt No. 3.

[0019] In the culture medium of the present invention, the volume-weight ratio of 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol and neutral red is preferably 1000 ml: 0.36 - 0.43 g: 0.0004 - 0.0005 g: 4.7 - 5.5 g: amoxicillin 0.0115 - 0.0125 g: 0.8 - 1 g: 0.02 - 0.03 g, more preferably 1000 ml: 0.4 g: 0.0005 g: 5 g: 0.0125 g: 1 g: 0.03 g. In the present invention, the preparation method of the 0.1% hemolytic Hiss medium preferably comprises the following steps: mixing beef digestive juice, water, peptone, agar and lactose, adjusting the pH to 7.2 - 7.4, sterilizing and then cooling, and adding defibrinated rabbit hemolysis. The present invention has no special limitation on the specific reagent and specific method for adjusting the pH value, and the conventional reagents and methods for adjusting the pH value in the art can be used. In the present invention, the cooling is preferably cooling to about 50 °C. The present invention has no special limitation on the specific sterilization method.

[0020] The present invention also provides a preparation method of the above culture medium, preferably comprising the following steps: mixing 0.1% hemolytic Hiss medium, sodium dodecyl sulfate, erythromycin, bile salts, amoxicillin, mannitol and neutral red to obtain the product. The present invention has no special limitation on the addition sequence of the above raw materials.

[0021] The present invention also provides an application of the above culture medium in isolating and culturing Yersinia pestis, in isolating and culturing Yersinia pestis from corrupt materials or in plague monitoring.

[0022] The following combines examples to elaborate on the technical solutions provided by the present invention in detail, but they cannot be construed as limiting the protection scope of the present invention.

[0023] In the following examples, unless otherwise specified, all are conventional methods.

[0024] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0025] Example 1

[0026] Mix 13 g of Herellea agar, 200 ml of beef digestive juice, 800 ml of water, 8 g of peptone, and 11 g of lactose, adjust the pH to 7.2, sterilize at 121 °C under high pressure for 30 min, cool to about 50 °C, add 0.1 ml of defibrinated rabbit blood, 3.6 g of sodium dodecyl sulfate, 0.4 mg of erythromycin, 4.7 g of bile salt No. 3, 11.5 mg of amoxicillin, 0.8 g of mannitol, and 0.02 g of neutral red after cooling. After mixing evenly, pour into petri dishes to obtain the culture medium of the present invention.

[0027] Example 2

[0028] Mix 14 g of Herellea agar, 200 ml of beef digestive juice, 800 ml of water, 10 g of peptone, and 10 g of lactose, adjust the pH to 7.4, sterilize at 121 °C under high pressure for 30 min, cool to about 50 °C, add 0.1 ml of defibrinated rabbit blood, 4.3 g of sodium dodecyl sulfate, 0.5 mg of erythromycin, 5.5 g of bile salt No. 3, 12.5 mg of amoxicillin, 1 g of mannitol, and 0.03 g of neutral red after cooling. After mixing evenly, pour into petri dishes to obtain the culture medium of the present invention.

[0029] Example 3

[0030] Mix 14 g of Herellea agar, 200 ml of beef digestive juice, 800 ml of water, 10 g of peptone, and 10 g of lactose, adjust the pH to 7.3, sterilize at 121 °C under high pressure for 30 min, cool to about 50 °C, add 0.1 ml of defibrinated rabbit blood, 4 g of sodium dodecyl sulfate, 0.5 mg of erythromycin, 5 g of bile salt No. 3, 12.5 mg of amoxicillin, 1 g of mannitol, and 0.03 g of neutral red after cooling. After mixing evenly, pour into petri dishes to obtain the culture medium of the present invention, denoted as Medium No. 1.

[0031] Example 4

[0032] Using 0.1% hemolytic Hiss medium (prepared by mixing 14 g of Hiss agar, 200 ml of beef digestive juice, 800 ml of water, 10 g of peptone, and 10 g of lactose, adjusting the pH to 7.2, autoclaving at 121 °C for 30 min, cooling to about 50 °C, adding 0.1 ml of defibrinated rabbit blood after cooling, mixing well and pouring into petri dishes to obtain 0.1% hemolytic Hiss medium) as the control medium (basic medium), and gentian violet medium and 1% sodium citrate selective medium (designated as medium No. 2) as the other two selective media (positive control groups), the growth ability, medium sensitivity, and specificity of Yersinia pestis and other miscellaneous bacteria were detected using the selective medium of Example 3 (medium No. 1).

[0033] Prepare bacterial suspensions from each experimental strain (Yersinia pestis EV strain, Yersinia pseudotuberculosis, Yersinia enterocolitica of the genus Yersinia) and common miscellaneous bacteria in field decaying materials (Proteus, Staphylococcus aureus, Escherichia coli, Group A streptococcus) (all provided by the professional laboratory for plague bacteria), dilute them to a bacterial suspension of 1000 bacteria / ml according to the turbidimetric tube, take 0.1 ml and spread it on 4 kinds of media, incubate in an incubator at 28 °C for 248 h, repeat the experiment 3 times, and observe the bacterial growth. (When performing the medium sensitivity test, the dilution degrees of plague bacteria are 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 ).

[0034] Gentian violet medium: Dissolve 0.1 g of gentian violet in 2 ml of absolute ethanol, add distilled water to 90 ml, autoclave at 121 °C for 30 min, cool to 50 °C after autoclaving and add 10 ml of fresh blood, mix well and take 10 ml and add it to 1 L of 0.1% hemolytic Hiss medium to prepare a 1 / 100,000 gentian violet hemolytic agar medium, and store it at 4 °C for later use.

[0035] 1% sodium citrate selective medium (medium No. 2): Add 1 g of deoxycholate, 1 g of sodium citrate, and 1 g of ferric citrate to the 0.1% hemolytic Hiss medium, and store it at 4 °C for later use.

[0036] The results are shown in Table 1, Table 2 and Figure 1 as follows.

[0037] Table 1 Growth of plague bacteria on 4 kinds of media

[0038]

[0039] As can be seen from Table 1, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica in Medium 1 grew well, all forming rose-colored colonies. The plague bacteria were fully developed, with a light red center in the colony (see Figure 1 ). Staphylococcus aureus and Streptococcus did not grow, the growth of Escherichia coli was inhibited, and the colony count was low. The diffuse growth of Proteus was inhibited, showing single colonies. In Medium 2, the growth of Escherichia coli was visible, while the growth of other experimental strains was inhibited. The plague bacteria grew on the gentian violet medium, with the colonies slightly raised, semi-transparent and slightly bluish-gray. Under microscopic observation, the colony morphology of the plague bacteria was not typical and the growth was slow. The growth of Proteus was inhibited on the gentian violet medium, showing single colonies, and Escherichia coli covered the entire plate.

[0040] Table 2 Sensitivity test results of Yersinia pestis in various media

[0041]

[0042] As can be seen from Table 2, the sensitivity of the plague bacteria in the gentian violet medium and Medium 2 was poor. When the concentration of the plague EV bacterial solution was 10 -7 , no growth of the plague bacteria was observed. In Medium 1, bacteria were still growing when the concentration was 10 -8 , and its sensitivity was consistent with that of Herellea medium.

[0043] Example 5

[0044] Isolation and culture of plague bacteria from putrefied animal materials

[0045] A total of 5 pieces of wild animal materials (marmots, dogs) were collected. Among them, only the femoral remains were left in Marmot No. 1 and No. 4. The viscera of Marmot No. 2, No. 3, and Dog No. 5 were highly putrefied, but the heart, liver, spleen, and lungs were still retained, and each viscus had varying degrees of ulceration. The putrefied animal viscera materials were respectively inoculated into the four media of Example 4 and cultured in an incubator at 28 °C for 24 h to observe the growth and isolation of the plague bacteria.

[0046] Suspicious plague bacteria colonies were selected on each medium and confirmed as plague bacteria through the plague phage experiment. The colony count referred to "GB 4789.2-2016, Determination of total number of colonies".

[0047] The results are shown in Table 3. In Table 3, "-" indicates no bacterial growth.

[0048] Table 3 Results of detecting putrefied materials with different media

[0049]

[0050] The isolation of plague bacteria is the premise and core of all plague prevention and control work. For any plague host animals or corresponding samples brought back from the plague monitoring site, plague professionals will make every effort to isolate plague bacteria and preserve the plague bacteria as long as it is still possible. The basis for isolating plague bacteria is to select a suitable culture medium and be able to quickly select the target bacteria. The traditional basic culture medium for isolating plague is Herxheimer's medium. On Herxheimer's medium, small gray-white colonies can be seen under a low-power microscope. Under the microscope, the center of the colony can be seen to be bulging, with yellow-brown rough particles on the surface, and the edges are irregular and thin and transparent lace-like, which is the typical mature colony morphology of plague bacteria. This requires professionals to rely on rich experience and solid professional knowledge to identify plague bacteria and determine plague bacteria based on plague bacteriophage lysis experiments. However, in actual work, due to the influence of different factors, the morphology of plague bacteria is diverse; there are plague bacteria that are not lysed by bacteriophages in nature, so it is easy to miss the diagnosis only by judging from the colony morphology and phage lysis experiments.

[0051] The present invention tests the sensitivity and specificity of three selective culture media, namely, the selective culture medium (medium No. 1) of Example 3, the gentian violet culture medium, and the 0.1% sodium citrate selective culture medium (medium No. 2). It can be seen that among the three selective culture media, the gentian violet culture medium and medium No. 1 can meet the basic detection requirements for the growth and selection of plague bacteria, and medium No. 1 can detect 10 -7 The sensitivity was higher than that of the gentian violet medium, and there was no statistical difference with the basic medium (P value and statistical method). The plague strain grew to maturity after culturing at 28°C for 24 hours in medium No. 1. The colonies were rose red and had the typical structure of plague bacteria (large lace borders and rough central particles). The growth of Proteus, Escherichia coli and Gram-positive bacilli was inhibited. The results showed that the medium had good specificity.

[0052] Gentian violet medium is a commonly used selective medium for isolating Yersinia pestis. It can inhibit Proteus and Gram-positive bacteria. However, Escherichia coli grows well on this medium and inhibits the normal growth of Yersinia pestis. Therefore, the gentian violet selective medium is not ideal for culturing Yersinia pestis from severely contaminated materials in the wild. It is only suitable for laboratory animal infection tests or the isolation of Yersinia pestis when the contamination is not severe. For example, in experiments such as the virulence determination of Yersinia pestis, Yersinia pestis grows poorly on Medium No. 2, and the colonies are grayish-white, which are not easily distinguishable from other miscellaneous bacteria, so it is not suitable for the isolation and culture of Yersinia pestis. When the three selective media are applied to the detection of animal epidemics, the coincidence rate of the detection results between Medium No. 1 and the gentian violet medium is 100%. Due to the corruption of wild animal materials, only the femoral remains of Marmota himalayana No. 01 and No. 04 are left. Proteus diffused in each organ of the control medium, covering a little Yersinia pestis, resulting in difficult isolation. The application of Medium No. 1 selective culture can inhibit the diffuse growth of Proteus, and the rose-colored Yersinia pestis grows well and is very easy to select. Yersinia pestis can also be detected on the gentian violet medium, but the growth is slow, and it is still in the primary colony stage after 24 hours of culture. The culture of dog organs shows the presence of phage and mixed bacteria contamination. Yersinia pestis cannot be selected on Herellea medium. Usually, an anti-phage serum medium is needed to isolate Yersinia pestis, but it cannot be prepared in the wild. The present invention applies Medium No. 1 selective sensitive medium. Yersinia pestis matures in 24 hours, the colonies are rose-colored, with typical structures, can inhibit the growth of miscellaneous bacteria, and Yersinia pestis is easy to select.

[0053] Example 6

[0054] The difference from Example 3 is that the addition amounts of amoxicillin are 5.5, 7.5, 9.5, 11.5, 13.5, and 15.5 mg respectively, and the rest are the same as in Example 3. The plague EV strain of the genus Yersinia was made into a bacterial suspension, diluted to a bacterial suspension of 1000 bacteria / ml according to the turbidimetric tube, and 0.1 ml was taken and spread on the medium of Example 3 and the medium of this example, and cultured in an incubator at 28°C for 248 hours. The experiment was repeated 3 times to observe the growth of Yersinia pestis. The results are shown in Table 4. Colony counting refers to "GB 4789.2-2016, Determination of total number of colonies".

[0055] Table 4 Effects of different concentrations of amoxicillin on the growth of Yersinia pestis and miscellaneous bacteria

[0056]

[0057] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium for isolating and culturing Yersinia pestis from corrupt materials, characterized in that, after mixing 14 g of Hektoen agar, 200 ml of beef digestive juice, 800 ml of water, 10 g of peptone, and 10 g of lactose, adjusting the pH to 7.3, autoclaving at 121 °C for 30 min, adding 0.1 ml of defibrinated rabbit blood, 4 g of sodium dodecyl sulfate, 0.5 mg of erythromycin, 5 g of bile salt No. 3, 12.5 mg of amoxicillin, 1 g of mannitol, and 0.03 g of neutral red after cooling, and mixing evenly to obtain the culture medium.

2. The application of the culture medium according to claim 1 in isolating and culturing Yersinia pestis, characterized in that, the application is for non-disease diagnosis purposes.

3. The application of the culture medium according to claim 1 in isolating and culturing Yersinia pestis from corrupt materials, characterized in that, the application is for non-disease diagnosis purposes.

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