阅读说明：本技术 一种鼠疫菌显色分离培养基的制备方法 (Preparation method of plague bacterium chromogenic isolation medium ) 是由 谢辉 田富彰 张雪飞 李君� 马龙 熊浩明 祁芝珍 冯建萍 杨晓艳 赵海红 游培 于 2021-02-09 设计创作，主要内容包括：本申请公开了一种鼠疫菌显色分离培养基的制备方法,包括如下步骤：步骤S1：在基础成分中加入蒸馏水,调节其pH值,再进行高压灭菌处理；步骤S2：待步骤S1中的产物冷却至55℃时,加入抑菌成分,配置成指示性显色培养基。本申请的鼠疫菌显色分离培养基的制备方法,以CIN琼脂为基础,添加抑菌成分,制成指示性显色培养基,获得配制方法简单、配制成本低、分离效率更高的鼠疫菌显色分离培养基。鼠疫菌指示性显色培养基的工作原理是使用适当显色底物,在细菌特异性酶的作用下,发色基团游离并附着于菌落上,使菌显示一定的颜色。(The application discloses a preparation method of a plague bacteria chromogenic isolation medium, which comprises the following steps: step S1: adding distilled water into the basic component, adjusting pH, and autoclaving; step S2: and (4) when the product in the step S1 is cooled to 55 ℃, adding bacteriostatic components to prepare an indicative chromogenic culture medium. According to the preparation method of the plague bacteria chromogenic isolation medium, the CIN agar is used as a base, the bacteriostatic component is added to prepare the indicative chromogenic culture medium, and the plague bacteria chromogenic isolation medium which is simple in preparation method, low in preparation cost and higher in separation efficiency is obtained. The operation principle of the indicative chromogenic culture medium for the plague bacteria is that a proper chromogenic substrate is used, and under the action of bacteria specific enzyme, chromogenic groups are dissociated and attached to bacterial colonies, so that the bacteria display certain color.)

1. A preparation method of a plague bacterium chromogenic isolation medium is characterized by comprising the following steps:

step S1: adding distilled water into the basic component, adjusting pH, and autoclaving;

step S2: and (4) when the product in the step S1 is cooled to 55 ℃, adding bacteriostatic components to prepare an indicative chromogenic culture medium.

2. The method for preparing a culture medium for the chromogenic isolation of plague bacteria according to claim 1, wherein in step S2, the bacteriostatic component is 0.5% to 1.5% bile salt.

3. The method for preparing a culture medium for the chromogenic isolation of plague bacteria according to claim 2, wherein in step S2, the bacteriostatic component is 1% bile salt.

4. The method for preparing a culture medium for the chromogenic isolation of plague according to claim 1, wherein in step S1, the basic components comprise the following components:

peptone 20g, yeast 2g, mannitol 12g, K₂HPO₄2g, NaCl 1g, sodium sulfite 0.25g, neutral red 0.03g, crystal violet 0.001g, agar 15g, Hertz digestive juice 200ml and deoxycholic acid 0.36 g.

5. The method for preparing a culture medium for the chromogenic isolation of plague bacteria according to claim 3, wherein in step S2, the bacteriostatic component is 1g of bile salt.

6. The method for preparing a culture medium for the chromogenic isolation of plague according to claim 4, wherein the volume of distilled water added in step S1 is 1000 ml.

7. The method for preparing a chromogenic isolation medium for plague bacteria according to claim 1, wherein the pH value adjusted in step S1 is 7.0 to 7.4.

8. The method for preparing a culture medium for the chromogenic isolation of plague bacteria according to claim 1, wherein in step S1, the pressure of the autoclaving process is 0.15MP, the temperature is 121 ℃, and the time is 30 min.

9. The method for preparing a culture medium for the chromogenic isolation of plague according to any of claims 1 to 8, wherein the method for preparing the hertzian digest used in step S1 is as follows:

adding 3000g of minced beef without fat tendon into 5000ml of tap water, boiling for 30min, cooling to 56 ℃, adding 250g of pig pancreas and 50-60g of anhydrous sodium carbonate, fully mixing uniformly to correct the pH value to be 7.8, adding 80-100ml of chloroform, tightening the bottle mouth, placing in a 37-oscillation incubator for digestion for 7-9 days, and measuring the content of amino nitrogen to reach 800 mg/L.

Technical Field

The application relates to the technical field of culture media, in particular to a preparation method of a plague bacteria chromogenic isolation culture medium.

Background

At present, various molecular biological methods and immunological methods are used for detecting plague bacteria, but the traditional plague bacteriological method is still the 'gold standard' of plague detection, the isolated culture of the plague bacteria plays an important role in the plague prevention and treatment work, if the detected material has low bacteria content and serious putrefaction, or contains plague bacteriophage, only the separation of plague bacteria by common agar is very difficult, a selective sensitive culture medium is needed, simultaneously, the suspicious colony can be selected by the rich experience of professionals, compared with the traditional selective sensitive culture medium, the substances which can inhibit the growth of other mixed bacteria and promote the development of plague bacteria, such as a gentian violet culture medium, a gall tellurium copper violet culture medium and the like, are added into the components of the culture medium, these media can inhibit proteus and escherichia coli in putrefying materials, but the plague colonies are very atypical and still have an effect on isolation of plague.

Chromogenic media have been widely used since their development by CHROMagar, France, 1974 because of their high accuracy, sensitivity and specificity. The culture medium is an important tool for testing microorganisms by using a culture method, and a yersinia selective culture medium (CIN agar) developed abroad is recommended by the ISO committee for use, but cannot be used as a conventional culture medium due to the high price, the intermittent delivery and the failure to supply in time in case of emergency of plague. Therefore, a developing culture medium which is convenient to prepare, free of barrier supply and easy to separate and screen plague bacteria is needed to be researched when plague epidemic situations are in emergency.

Disclosure of Invention

It is an object of the present application to overcome the above problems or to at least partially solve or mitigate the above problems.

According to one aspect of the application, a preparation method of a plague bacteria chromogenic isolation medium is provided, which comprises the following steps:

step S1: adding distilled water into the basic component, adjusting pH, and autoclaving;

step S2: and (4) when the product in the step S1 is cooled to 55 ℃, adding bacteriostatic components to prepare an indicative chromogenic culture medium.

Optionally, in step S2, the bacteriostatic component is 0.5% to 1.5% of bile salt.

Optionally, in step S2, the bacteriostatic component is 1% bile salt.

Optionally, in step S1, the base component includes the following components:

peptone 20g, yeast 2g, mannitol 12g, K₂HPO₄2g, NaCl 1g, sodium sulfite 0.25g, neutral red 0.03g, crystal violet 0.001g, agar 15g, Hertz digestive juice 200ml and deoxycholic acid 0.36 g.

Optionally, in step S2, the bacteriostatic component is 1g gall bladder.

Alternatively, in the step S1, the volume of the added distilled water is 1000 ml.

Optionally, in the step S1, the adjusted pH value is 7.0 to 7.4.

Optionally, in step S1, the pressure of the autoclaving process is 0.15MP, the temperature is 121 ℃, and the time is 30 min.

Alternatively, the hertzian digest used in step S1 may be prepared as follows:

adding 3000g of minced beef without fat tendon into 5000ml of tap water, boiling for 30min, cooling to 56 ℃, adding 250g of pig pancreas and 50-60g of anhydrous sodium carbonate, fully mixing uniformly to correct the pH value to be 7.8, adding 80-100ml of chloroform, tightening the bottle mouth, placing in a 37-oscillation incubator for digestion for 7-9 days, and measuring the content of amino nitrogen to reach 800 mg/L.

According to the preparation method of the plague bacteria chromogenic isolation medium, the CIN agar is used as a base, the bacteriostatic component is added to prepare the indicative chromogenic culture medium, and the plague bacteria chromogenic isolation medium which is simple in preparation method, low in preparation cost and higher in separation efficiency is obtained. The operation principle of the indicative chromogenic culture medium for the plague bacteria is that a proper chromogenic substrate is used, and under the action of bacteria specific enzyme, chromogenic groups are dissociated and attached to bacterial colonies, so that the bacteria display certain color.

The above, as well as additional purposes, advantages, and features of the present application, will become apparent to those of ordinary skill in the art upon examination of the following detailed description of specific embodiments of the application.

Detailed Description

The experimental strains used in the examples of the present application, Yersinia pestis (EV strain), Pseudotuberculosis bacilli, enterocolitis bacilli, and Proteus, Staphylococcus aureus, Escherichia coli, group A streptococci, which are common pathogenic bacteria, were provided by professional bacterial libraries of Qinghai endemic disease prevention institute strains.

The raw materials of the culture medium adopted in the embodiment of the application, namely peptone, yeast, K2HPO4, mannitol, NaCI, sodium sulfite, neutral red, crystal violet, agar, bile salt, deoxycholic acid and chloramphenicol are purchased from Biotech limited company in Qingdao to provide; he's digestive juice is made by self.

Example 1: preparation of chromogenic Medium

80g of peptone, 8g of yeast, 48g of mannitol and K₂HPO₄8g, NaCl 4g, sodium sulfite 1g, neutral red 0.12g, crystal violet 0.004g, agar 60g, Hertz digestive juice 800ml and deoxycholic acid 1.44 g.

Mixing the above components, adding 1000ml distilled water, adjusting pH to 7.4, autoclaving (121 deg.C, 30min), and cooling to 55 deg.C;

the above product was divided into 4 equal parts on average, and 1g of bile salt, 2g of bile salt, 2.5g of chloramphenicol, and 0.5g of chloramphenicol were added to prepare a 1% bile salt chromogenic medium, a 2% bile salt chromogenic medium, a 2.5% chloramphenicol chromogenic medium, and a 0.5% chloramphenicol chromogenic medium, respectively.

Example 2: specific detection of chromogenic Medium

The method comprises the steps of calculating plague bacteria colonies by adopting a plate counting method, preparing Yersinia pestis (EV), Yersinia pseudotuberculosis, Yersinia enterocolitica, staphylococcus aureus, escherichia coli and proteus into bacterial suspensions with the concentration of 1 Meyer unit/ml respectively, inoculating 0.1ml of the bacterial suspensions into a 1% cholate chromogenic culture medium, a 2% cholate chromogenic culture medium, a 2.5% chloramphenicol chromogenic culture medium, a 0.5% chloramphenicol chromogenic culture medium and a 1% hemolytic Hecheng culture medium (control culture medium), uniformly coating by using an L rod, and culturing in a 28 ℃ incubator for 24 hours to observe the growth condition and the colony characteristics of the colonies.

The growth conditions of the tested strains on 4 chromogenic media and 1% hemolytic hertzian media are observed, the plague bacteria, the pseudotuberculosis bacteria and the enterocolitis bacteria have better chromogenic effects on 0.5% chloramphenicol chromogenic media and 1% bile salt chromogenic media, the bacterial colonies are rose red, and the results are shown in table 1. Wherein, "-" indicates no growth, and the comparison of P of 1 group, 3 groups, 4 groups and 5 groups respectively is less than 0.05, and has significant difference; the P of the 2 groups is more than 0.05 compared with the 5 groups, and no significant difference exists.

TABLE 1 growth observation of plague bacteria in various chromogenic media

As can be seen from Table 1, the tested strains did not grow on the 2% cholate chromogenic medium and the 2.5% chloramphenicol chromogenic medium except for the proteobacteria; the plague bacillus grows to be rose red microcolonies on a 0.5 percent chloramphenicol chromogenic medium, the diameter of the colonies cultured for 48 hours is less than or equal to 1mm, the growth is slow, the colony morphology of dysplasia is atypical, the colonies of other yersinia enterocolitica and pseudotuberculosis are red, the growth is poor, the bacteria are small and small in quantity, the staphylococcus aureus does not grow, the escherichia coli grows well, the color of the colonies is yellow, the proteus is a single colony, the bacterium grows without diffusion, and the color of the colonies is faint yellow; 2.1 the plague bacillus grows well in 1% bile salt indicative culture medium, can see typical mature colony after culturing 16h, compare plague bacillus colony count with the hertzian culture medium and do not have statistical difference, the colony is rosy, observe under the mirror and see typical plague bacillus colony form, the surface is coarse, the center is red, there is transparent lace in the periphery, pseudotuberculosis and enterocolitis fungus grow well, the colony color is red, the escherichia coli growth is not inhibited, the colony color is yellow, staphylococcus aureus and proteus growth are inhibited, proteus bacillus is single yellow colony, the non-diffuse growth.

Example 3: sensitivity detection of chromogenic Medium

Sequentially diluting the stock solution of the suspension of the plague bacteria prepared in the example 2 by 10 times, respectively taking 1ml of each of 10-4, 10-5, 10-6 and 10-7 dilution bacterial solutions, sucking 0.1ml of the bacterial solution by a pipette, respectively coating the bacterial solution on each chromogenic medium, culturing at 28 ℃ for 24-48h, counting, observing the growth conditions of the plague bacteria on different culture media, and determining the detection result. Hertzian medium was used as a control and the experiment was repeated 3 times.

The results of the bacterial solutions with different concentrations coated on the chromogenic media are shown in table 2, and the colony counting results show that 10-6 colony counting results show that 1% of cholate chromogenic media plague bacteria have a small amount of bacteria growth, other 3 chromogenic media have no bacteria growth, and have no statistical difference with hertzian media (t ═ 1.569, P >0.05), which proves that the sensitivity of 1% of cholate chromogenic media is higher.

TABLE 25 sensitivity test results for the detection of the media

Example 4: isolation test of plague bacteria

EV and common pathogenic bacteria Escherichia coli, Proteus and Staphylococcus aureus equal amount are mixed, 0.1ml is added into 5 kinds of culture medium, placed in 28 ℃ incubator for 24h to observe colony growth, plague bacteria are separated and tested for plague phage lysis, and the herd's culture medium is used as control for repeated detection for 3 times.

And (3) mixing all the test strains, adding 0.1ml of the mixed test strains into 4 culture media, and culturing for 24h and 48h to observe the growth condition of the tested strains and the separation effect of the plague bacteria. The 2.5% chloramphenicol chromogenic medium and the 2% cholate chromogenic medium can inhibit the growth of plague bacteria and cannot be used as chromogenic selection media suitable for plague bacteria separation, the 0.5% chloramphenicol chromogenic medium can inhibit the diffuse growth of proteobacteria, has an inhibiting effect on staphylococcus aureus, has no inhibiting effect on escherichia coli, is slow in plague bacteria growth, is still in a primary colony form after 48 hours, and the tiny plague bacteria are covered by the escherichia coli which grows well after 72 hours and cannot be selected and separated; 1% bile salt chromogenic medium proteus diffuse growth is inhibited, be single colony, staphylococcus aureus does not grow in this culture medium, the escherichia coli colony is yellow, and the plague bacterium among the yersinia, pseudotuberculosis bacterium and enterocolitis fungus colony are rose red, grow well in 1% bile salt chromogenic medium, very easily separated, select suspicious colony and walk plague phage lysis verification test, the test bacterial strain grows well on the control culture medium, plague bacterium is difficult for selecting.

In the 4 kinds of plague bacteria chromogenic medium prepared in the embodiment 1, basic nutritional ingredients refer to basic ingredients in a CIN agar formula, hertzian nutrient solution is added to stimulate the growth of plague bacteria, different bacteriostatic agents are selected at the same time, and an ideal medium which has good growth of plague bacteria and is inhibited as much as possible is screened by observing the colony counts of the plague bacteria and the mixed bacteria in different bacteriostatic agents and different bacteriostatic concentrations.

The test strains are all observed to be inhibited on a chloramphenicol chromogenic medium, the chloramphenicol is a broad-spectrum antibiotic and can inhibit gram-positive bacteria and gram-negative bacteria, and experiments show that the plague bacteria do not grow on the chloramphenicol chromogenic medium, because the chloramphenicol and crystal violet are combined to inhibit the growth of the plague bacteria, the chloramphenicol chromogenic medium is not suitable for the separation culture of the plague bacteria.

Because a certain concentration of bile salt has high selectivity, namely no inhibition effect on bacteria in enterobacteriaceae and inhibition effect on other common pathogenic bacteria, bile salts with different concentrations are used as antibacterial ingredients and are divided into2 kinds of different concentrations prepare into the chromogenic medium, the experimental result shows, the Staphylococcus aureus does not grow on 2% bile salt chromogenic medium, the plague bacterium grows slowly, this is because the bile salt concentration is too high to have certain inhibitory action to the plague bacterium, the colony 48h still presents the primary growth form, 1% bile salt chromogenic medium plague bacterium grows well, the statistical analysis and the herd's medium have no significant difference (p is different from herd's medium)>0.05), the diameter of the bacterial colony is 1.4mm-2.5mm for 16-24h, the bacterial colony is rose red, the surface of the bacterial colony visible under the mirror is rough, the periphery has rose red transparent laces, the bacterial colony is characteristic of typical plague bacteria, although the escherichia coli grows well on the culture medium, the color of the bacterial colony is faint yellow, the bacterial colony is easily distinguished from the plague bacteria, the proteus bacillus grows into a single bacterial colony, the diffusibility growth is avoided, and the isolated culture of the plague bacteria is not influenced, so the 1% bile salt chromogenic culture medium has stronger specificity and selectivity. Furthermore, the medium sensitivity test results of example 3 also showed that the 1% bile salt chromogenic medium had high sensitivity and was diluted 10 in bacterial suspension^-6The bacteria grow.

In conclusion, the experimental results prove that the 1% bile salt chromogenic medium plague bacteria have good chromogenic effect, have higher detection efficiency than the Hersche medium, have the characteristics of easy observation of results, strong specificity, high sensitivity, economy and the like, have shorter separation time, can improve the detection rate of the plague bacteria, and are very suitable for large-scale use in field inspection and monitoring of plague sources.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which this application belongs.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.