CN115491407A - Burkholderia cepacia chromogenic medium and application thereof - Google Patents
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Abstract
The invention relates to a Burkholderia cepacia chromogenic medium and application thereof, belonging to the field of microorganisms, wherein the components of the medium comprise peptone, glucose, sodium chloride, agar, gentamicin sulfate, a chromogenic agent, a cosolvent and deionized water, the pH value of the medium is 7.2-7.4, and the medium is applied to the chromogenic reaction and separation of the Burkholderia cepacia.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Burkholderia cepacia chromogenic medium and application thereof.
Background
Burkholderia cepacia (burkholderia cepacia) is a gram-negative bacterium widely present in water, soil, plants and the human body. Plant pathologist burkholder in the United states of 1949 first found that it can cause stem rot in onion, known as Pseudomonas cepacia, which was assigned to Burkholderia in 1992, known as Burkholderia cepacia.
The Burkholderia cepacia is gram-negative bacilli widely existing in soil and water, does not cause diseases to healthy human bodies, is usually planted in the lung of cystic fibrosis patients, and has high lethality once infected. Burkholderia cepacia is also an inspection item for import and export cosmetics and import and export oral cleaning products.
The growth of Burkholderia cepacia has low nutritional requirements, can grow on various culture media and grows well. At present, BCSA agar and Macconk agar are commonly used as culture media for detecting Burkholderia cepacia, but selective enrichment pretreatment is required, the detection period is long, the characteristics of bacterial colonies are not obvious, a series of biochemical identification is required after suspected bacteria are cultured, the steps are complicated, and the detection difficulty is relatively high.
The chromogenic medium is a novel medium which is used for combining specific enzymes generated by the metabolism of microorganisms with corresponding chromogenic substrates in the medium and reacting for chromogenic reaction, and is used for quickly and accurately detecting the microorganisms.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a Burkholderia cepacia chromogenic medium and application thereof.
The invention researches a color development culture medium of Burkholderia cepacia by screening components of the culture medium and regulating and controlling the pH value of the culture medium by applying the action principles of molecular biology, microbiology, chemistry, basic carbon-nitrogen source inorganic salt and the like, and provides a quicker and simpler inspection scheme for the Burkholderia cepacia.
The technical scheme adopted by the invention is as follows:
the Burkholderia cepacia chromogenic culture medium is characterized in that each liter of the Burkholderia cepacia chromogenic culture medium contains 15-25g of peptone, 3-10g of glucose, 5-10g of sodium chloride, 10-15g of agar, 0.1-0.5g of color developing agent and the balance of deionized water, and the pH value is adjusted to 7.2-7.4 by sodium hydroxide.
A preparation method of a Burkholderia cepacia chromogenic culture medium comprises the following steps:
(1) Weighing 15-25g of the peptone, 3-10g of the glucose, 5-10g of the sodium chloride, 10-15g of the agar, 0.01-0.1g of the gentamicin sulfate and 0.1-0.5g of the color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting pH to 7.2-7.4 with sodium hydroxide;
(4) Placing the conical flask on an electric heating sleeve, slowly heating while stirring with a glass rod to prevent the culture medium from being burnt during heating, boiling for 1-5 minutes after all the components are completely dissolved, sterilizing, closing the electric heating sleeve, and plugging a bottle stopper to prevent pollution;
(5) And (3) when the temperature in the step (4) is cooled to 40-50 ℃, opening the bottle stopper under aseptic condition, adding 0.01-0.1g of gentamicin sulfate, mixing uniformly, pouring into a disposable aseptic plate, and solidifying to obtain the Burkholderia cepacia chromogenic medium.
The application of the Burkholderia cepacia chromogenic medium is characterized in that: the method is applied to the color development and separation of Burkholderia cepacia.
The application method of the chromogenic medium for the Burkholderia cepacia is characterized by comprising the following steps: dissolving and diluting a clinically collected sample, coating the sample on the Burkholderia cepacia chromogenic culture medium by using a sterile coating rod, putting the sample into an incubator at 30-35 ℃, and observing the result after aerobic culture is carried out for 18-24 hours. The results obtained with this method of use are: burkholderia cepacia is reddish, and other bacteria do not grow or develop color.
The invention has the beneficial effects that:
(1) According to the invention, the basic nutrition required by the Burkholderia cepacia is ensured according to the proper proportion of the nutritional components, and meanwhile, the broad-spectrum antibacterial agent gentamycin sulfate is added in a proper amount, so that most of mixed bacteria can be inhibited or killed without influencing the growth of the Burkholderia cepacia;
(2) The Burkholderia cepacia chromogenic medium and the application thereof can enable the cultured Burkholderia cepacia to develop color and achieve the effects of counting and separating, simplify the steps of detection, separation and identification, and provide a simpler, more convenient and effective scheme for accurately detecting the Burkholderia cepacia;
(3) The invention has simple components, convenient operation and low cost, and is beneficial to popularization and application;
(4) The growth of Burkholderia cepacia is facilitated, the mutation rate is low, and the counting is convenient.
Drawings
FIG. 1 is a graph showing the effects of culture in the medium according to example 1 of the present invention
FIG. 2 is a graph showing the effects of culture in the medium according to example 2 of the present invention
FIG. 3 is a graph showing the effects of the culture in the medium according to example 3 of the present invention
FIG. 4 is a graph showing the effect of the specificity test of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. In the following examples, reagents and instruments are not specified by manufacturers, and are all conventional products commercially available from normal sources.
A Burkholderia cepacia chromogenic medium and a preparation method thereof are as follows:
1. the formula of the culture medium is as follows:
each liter of culture medium comprises 15-25g of peptone, 3-10g of glucose, 5-10g of sodium chloride, 10-15g of agar, 0.01-0.1g of gentamicin sulfate and 0.1-0.5g of color developing agent, and the pH value is 7.2-7.4.
2. Preparation of culture Medium
(1) Weighing 15-25g of peptone, 3-10g of glucose, 5-10g of sodium chloride, 10-15g of agar and 0.1-0.5g of color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting pH to 7.2-7.4 with sodium hydroxide;
(4) Placing the conical flask on an electric heating sleeve, slowly heating while stirring with a glass rod to prevent the culture medium from being burnt during heating, boiling for 1-5 minutes after all the components are completely dissolved, sterilizing, closing the electric heating sleeve, and plugging a bottle stopper to prevent pollution;
(5) And (3) when the temperature in the step (4) is cooled to 40-50 ℃, opening the bottle stopper under aseptic condition, adding 0.01-0.1g of gentamicin sulfate, mixing uniformly, pouring into a disposable aseptic plate, and solidifying to obtain the Burkholderia cepacia chromogenic medium.
3. Culturing
After a clinically collected sample is dissolved and diluted, 0.1mL of the sample is transferred to a Burkholderia cepacia chromogenic culture medium by a liquid transfer gun, the sample is uniformly coated by a sterile coating rod, the sample is placed into an incubator at the temperature of 30-35 ℃, and the result is observed after aerobic culture for 18-24 hours, wherein the Burkholderia cepacia is reddish, and other bacteria do not grow or develop color.
Example 1
1. Preparation of culture Medium
(1) Weighing 20g of peptone, 5g of glucose, 5g of sodium chloride, 14g of agar and 0.32g of color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting the pH value to 7.3 by using sodium hydroxide;
(4) Placing the conical flask on an electric heating sleeve for slowly heating, stirring with a glass rod while heating to prevent the culture medium from being burnt, boiling for 3 min for sterilization after all components are completely dissolved, closing the electric heating sleeve, plugging a bottle stopper, and preventing pollution;
(5) And (3) when the temperature in the step (4) is cooled to 45 ℃, opening the bottle stopper under aseptic condition, adding 0.01g of gentamycin sulfate, mixing uniformly, pouring into a disposable sterile plate, and solidifying to obtain the Burkholderia cepacia chromogenic medium.
2. Culturing
After a clinically collected sample is dissolved and diluted, 0.1mL of the sample is transferred to a Burkholderia cepacia chromogenic culture medium by a liquid transfer gun, the sample is uniformly coated by a sterile coating rod, the sample is placed into an incubator at the temperature of 30-35 ℃, and the result is observed after aerobic culture for 18-24 hours, wherein the Burkholderia cepacia is reddish, and other bacteria do not grow or develop color.
Example 1 the effect of the chromogenic Burkholderia cepacia medium is shown in FIG. 1.
As can be seen from the results of the present example, the culture medium of the present invention has the fastest growth speed and the deepest redness of the Burkholderia cepacia, can achieve the effects of color development, counting and separation in a shorter time, is convenient for visual observation and counting, and achieves the purpose of counting and separating the Burkholderia cepacia through color development.
Example 2
1. Preparation of culture Medium
(1) Weighing 15g of peptone, 8g of glucose, 10g of sodium chloride, 12g of agar and 0.25g of color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting the pH value to 7.2 by using sodium hydroxide;
(4) Placing the conical flask on an electric heating sleeve for slowly heating, stirring with a glass rod while heating to prevent the culture medium from being burnt, boiling for 4 minutes after all components are completely dissolved for sterilization, closing the electric heating sleeve, plugging a bottle stopper, and preventing pollution;
(5) And (5) when the temperature in the step (4) is cooled to 45 ℃, opening the bottle stopper under aseptic condition, adding 0.02g of gentamicin sulfate, mixing uniformly, pouring into a disposable sterile plate, and solidifying to obtain the color development culture medium for the Burkholderia cepacia.
2. Cultivation of
After a clinically collected sample is dissolved and diluted, 0.1mL of the sample is transferred to a Burkholderia cepacia chromogenic culture medium by a liquid transfer gun, the sample is uniformly coated by a sterile coating rod, the sample is placed into an incubator at the temperature of 30-35 ℃, and the result is observed after aerobic culture for 18-24 hours, wherein the Burkholderia cepacia is reddish, and other bacteria do not grow or develop color.
Example 2 the effect of the chromogenic Burkholderia cepacia medium is shown in FIG. 2.
As can be seen from the results of this example, in the medium of the present invention, burkholderia cepacia rapidly grows and turns red, and compared with example 1, the size of the colony is consistent, the color produced is lighter, the effects of color development, counting and separation can be achieved in a short time, and counting and separation of Burkholderia cepacia are facilitated.
Example 3
1. Preparation of culture Medium
(1) Weighing 25g of peptone, 10g of glucose, 8g of sodium chloride, 13g of agar and 0.15g of color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting the pH value to 7.4 by using sodium hydroxide;
(4) Placing the conical flask on an electric heating sleeve for slowly heating, stirring with a glass rod while heating to prevent the culture medium from being burnt, boiling for 4 minutes after all components are completely dissolved for sterilization, closing the electric heating sleeve, plugging a bottle stopper, and preventing pollution;
(5) And (3) when the temperature in the step (4) is cooled to 45 ℃, opening the bottle stopper under aseptic condition, adding 0.03g of gentamycin sulfate, mixing uniformly, pouring into a disposable sterile plate, and solidifying to obtain the Burkholderia cepacia chromogenic medium.
2. Cultivation of
After a clinically collected sample is dissolved and diluted, 0.1mL of the sample is transferred to a Burkholderia cepacia chromogenic culture medium by a liquid transfer gun, the sample is uniformly coated by a sterile coating rod, the sample is placed into an incubator at the temperature of 30-35 ℃, and the result is observed after aerobic culture for 18-24 hours, wherein the Burkholderia cepacia is reddish, and other bacteria do not grow or develop color.
Example 3 the effect of the chromogenic Burkholderia cepacia medium is shown in FIG. 3.
As can be seen from the results of this example, in the medium of the present invention, burkholderia cepacia grew rapidly and appeared red, and compared with examples 1-2, the colony was smaller, and the color appeared was close to that of example 2, which can achieve the effects of color development, counting and separation, and is advantageous for counting and separation of Burkholderia cepacia.
Specificity test of the invention:
respectively diluting Burkholderia cepacia, enterococcus faecalis, streptococcus pneumoniae, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and salmonella typhi with sterile normal saline, respectively inoculating the diluted bacterial liquid into a Burkholderia cepacia chromogenic culture medium, putting the Burkholderia cepacia chromogenic culture medium into an incubator at 30-35 ℃, aerobically culturing for 18-24 hours, and observing the result.
TABLE 1 specific color development test results of Burkholderia cepacia color development medium
The results of the specificity experiments are shown in FIG. 4.
In conclusion, in examples 1-3, the chromogenic medium for Burkholderia cepacia of the present invention was used to achieve the chromogenic effect of Burkholderia cepacia and to achieve the counting and separation effect, wherein the chromogenic separation effect of example 1 is the best, the detection, separation and identification steps are simplified, and a simpler, more efficient and effective scheme is provided for the accurate detection of Burkholderia cepacia.
It is to be understood that the embodiments described are merely a few examples of the invention and not all examples. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Claims (6)
1. A Burkholderia cepacia chromogenic medium is characterized in that: the Burkholderia cepacia chromogenic medium contains 15-25g of peptone, 3-10g of glucose, 5-10g of sodium chloride, 10-15g of agar, 0.01-0.1g of gentamicin sulfate, 0.1-0.5g of color developing agent, 2-4ml of cosolvent and the balance of deionized water per liter, and the pH value is 7.2-7.4.
2. A method for preparing the chromogenic medium for Burkholderia cepacia according to claim 1, wherein:
(1) Weighing 15-25g of the peptone, 3-10g of the glucose, 5-10g of the sodium chloride, 10-15g of the agar and 0.1-0.5g of the color developing agent;
(2) Adding the components in the step (1) into a clean conical flask, adding 1000mL of deionized water, and stirring for dissolving;
(3) Adjusting pH to 7.2-7.4 with sodium hydroxide;
(4) Heating and stirring the mixed solution in the conical flask, boiling and sterilizing for 1-5 minutes;
(5) And (3) cooling to 40-60 ℃, adding 0.01-0.1g of gentamicin sulfate, mixing uniformly, and aseptically filling the culture medium into a disposable sterile plate to obtain the Burkholderia cepacia chromogenic culture medium.
3. The Burkholderia cepacia chromogenic medium according to claim 1, wherein the color developer is ethyl carbazole.
4. The chromogenic medium for Burkholderia cepacia according to claim 1, wherein: the cosolvent is N, N-Dimethylformamide (DMF) or dimethyl sulfoxide.
5. The use of the chromogenic medium for Burkholderia cepacia according to claim 1, wherein: the method is applied to the color development and separation of Burkholderia cepacia.
6. A method of using the chromogenic medium for Burkholderia cepacia according to claim 1, wherein: dissolving and diluting a clinically collected sample, coating the sample on the Burkholderia cepacia chromogenic culture medium by using a sterile coating rod, putting the sample into an incubator at 30-35 ℃, and observing the result after aerobic culture is carried out for 18-24 hours.
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| US20040110258A1 (en) * | 2002-09-10 | 2004-06-10 | Kayser Kevin J. | Method for metabolizing carbazole in petroleum |
| US20120052528A1 (en) * | 2009-02-25 | 2012-03-01 | The University Of Tokyo | Method for producing selective medium and use thereof |
| CN103396971A (en) * | 2013-08-22 | 2013-11-20 | 牛赡光 | Burkholderia cepacia and application thereof |
| WO2016151092A1 (en) * | 2015-03-25 | 2016-09-29 | Universite De Fribourg | Selective culture medium for polymyxin-resistant, gram-negative bacteria |
| WO2018134487A1 (en) * | 2017-01-18 | 2018-07-26 | Fondation Mediterranee Infection | Selective multipurpose culture medium for isolating and selecting colistin-resistant and vancomycin-resistant bacteria |
| RU2668406C1 (en) * | 2017-06-27 | 2018-09-28 | Ольга Владимировна Кондратенко | Method of primary sowing of biomaterial, divided from the lower respiratory ways of patients with cystic fibrosis |
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