CN102517198B - Device for testing or screening microorganisms in capillary culture method - Google Patents

Abstract

The invention discloses a device for detecting or screening microorganisms by a capillary culture method, which comprises a capillary, and the capillary is a straight or bent pipe with a diameter less than 0.5 cm and a length greater than 1 cm. Further, it also includes a detection tube, and one end of the capillary extends into the detection tube. The detection tube is provided with an air-tight or air-tight sealing cover, and one end of the capillary passes through the sealing cover and extends into the detection tube. The side wall of the detection tube is provided with a plane wall to facilitate close contact with the inner or outer wall of the enrichment container of the sample to be tested to prevent falling off or loss. A magnet is arranged inside or outside the side wall of the detection tube. The device for detecting or screening microorganisms by capillary culture method of the present invention can be used for detecting or screening microorganisms by capillary culture method, and the detection or separation time of most bacterial genera is shortened from 36 to 48 hours in the prior art to 12 to 24 hours. It has the advantages of fast, efficient, time-saving and labor-saving, good specificity, and strong sensitivity.

Description

A device for detecting or screening microorganisms by capillary culture method

technical field

The invention relates to a device for detecting or screening microorganisms by capillary culture method.

Background technique

At present, in the process of microbial inspection at home and abroad, the isolation and cultivation methods are mainly pre-enrichment, enrichment, and selective plate isolation and cultivation. In the 1970s, the invention of the chromogenic medium based on the basic principle of enzyme substrate color Greatly improved the specificity and sensitivity of pathogenic bacteria isolation and culture. The basic principle is selective enrichment, the streaking is dispersed into a single bacterial body, and the single bacterial body grows into a single colony and decomposes the chromogenic substrate to develop color, which requires two steps, and requires a large amount of medium and experienced manual streaking technology, took 36-48 hours. As we all know, in the separation process of chemical substances and biological macromolecules, the separation method based on chromatography or electrophoresis as the basic principle is fast, time-saving, and has high sensitivity, specificity and accuracy. However, due to the insolubility of the microorganisms, partitioning is not possible, so it is not possible to use chromatography. Moreover, because microorganisms are too large to perform effective conventional electrophoresis, some scientists have explored the effect of high-efficiency capillary electrophoresis, and the results show that it is effective in separating pathogenic cocci, such as staphylococcus and streptococcus and E. coli, but is limited by growth. Affected by various factors such as environment, conditions, and growth period, the same microorganism will show relatively large differences in charge-to-mass ratio, resulting in low reproducibility, which greatly limits its applicability.

So is it possible to think of a way to indirectly simulate the principle of color layers? Just like digital analog signals solve the problems of digital signals. According to literature analysis, the catheter is filled with semi-solid agar, and Salmonella is inoculated at one end, and Salmonella with flagella can be dissociated at the other end of the capillary; of chemotaxis. Therefore whether it is possible to design the motility or chemotaxis of microorganisms as the basic power (the power in the simulated chromatographic chromatography), and the selective bacteria inhibitor as the selective basic substance (the solid phase partitioning agent in the simulated chromatographic chromatography), to The basic method is that semi-solid agar is used as the carrier (liquid-phase partitioning agent in simulated chromatography), that is, put one end of the capillary into the sample enrichment solution, and the mixed bacteria pass through the semi-solid medium equipped with a relatively strong selective bacteria inhibitor. The solid agar capillary, as the mixed bacteria grow forward, will be subjected to repeated inhibitory selection, and finally the miscellaneous bacteria will be completely inhibited. Theoretically speaking, the target bacteria will be completely released to the other end of the capillary. The result of continuous redistribution in the chromatography, so as to realize the rapid and efficient separation of target bacteria. And from theoretical analysis, a large part of microorganisms have flagella and motility; other microorganisms can use their chemotaxis, so the dynamics can be guaranteed; According to the analysis of color selectivity, it can screen out the selective bacteria inhibitory substances with relatively strong specificity for most pathogenic microorganisms. Therefore, the capillary selective semi-solid culture method can theoretically separate most microorganisms. So far, there have been no reports on detection or screening of microorganisms using capillary culture.

Contents of the invention

For the above-mentioned prior art, the present invention provides a device for detecting or screening microorganisms by capillary culture method, which can be used for detecting or screening microorganisms, and the detection or separation time of most bacterial genera is changed from 36 to 48 hours in the prior art It is shortened to 12 to 24 hours, and has the advantages of fast, efficient, time-saving and labor-saving, good specificity, and strong sensitivity.

The present invention is achieved through the following technical solutions:

A device for detecting or screening microorganisms by capillary culture method, comprising a capillary tube, the capillary tube is a straight or bent tube with a diameter less than 0.5 cm and a length greater than 1 cm.

The material of the capillary is one of glass, plastic, synthetic resin or silica gel, and the material is required to be hard and water-tight, and air-permeable or air-tight is selected according to the aerobic conditions of the target microorganisms. It is preferred to use a transparent material, which is easy to observe the culture medium The filling effect is also convenient for the end point judgment. The most preferred high pressure resistant hard non-toxic transparent material.

The capillary is a straight or bent tube with a diameter of 0.5-2 mm and a length of 2-20 cm.

The device for detecting or screening microorganisms by capillary culture method also includes a detection tube, and one end of the capillary tube extends into the detection tube.

The detection tube is provided with an air-tight or air-tight sealing cover (plug), and one end of the capillary passes through the sealing cover (plug) and extends into the detection tube.

The side wall of the detection tube is provided with a plane to adhere to the wall, so as to be conveniently attached to the inner or outer wall of the enrichment container of the sample to be tested to prevent falling off or loss, and the plane is fixed to the enrichment container with adhesive tape or magnetic strip during detection on the inner or outer wall.

A magnet is arranged inside or outside the side wall of the detection tube. When in use, the magnet can fix the detection tube by cooperating with iron-containing items or other magnets to prevent falling off or loss.

When in use, fill the medium into the capillary (the medium is a semi-solid medium or a liquid medium or a solid medium; the medium contains nutrients for the growth of the target microorganism; the medium contains or does not contain selective miscellaneous bacteria Inhibitor or/and target microbial chromogen), and then one end of the filled capillary is connected to the sample to be tested or the diluent or enrichment solution of the sample to be tested, or: the sample to be tested is connected to one end of the capillary (When there is a detection tube, add culture medium to the detection tube at the same time, and insert the other end of the capillary into the culture medium of the detection tube), after culturing for 0<t≤96h, perform one of the following operations:

① When the medium contains selective bacteria inhibitors, directly observe the other end of the capillary to determine whether there is bacterial growth. If there is bacterial growth, it proves that the target microorganism is contained in the sample to be tested. If not, vice versa;

②When the culture medium contains selective miscellaneous bacteria inhibitors, directly observe the other end of the capillary to determine whether there is bacterial growth. If there is bacterial growth, it proves that the target microorganism is contained in the sample to be tested, and the conventional method of inoculating the bacteria into the target microorganism Further separation and purification or culture on the selection medium, if not available, and vice versa;

③When the culture medium contains the target microorganism chromogen, directly observe the other end of the capillary to judge whether there is a specific color reaction, if there is a specific color reaction, it proves that the sample to be tested contains the target microorganism, if not, vice versa;

④ When the culture medium does not contain the target microorganism chromogen, with or without the selective miscellaneous bacteria inhibitor, observe whether there is bacterial growth at the other end of the capillary. In the semi-solid medium or liquid medium or solid medium (in the detection tube) of the chromogenic agent, after culturing, observe whether there is a specific color reaction, if there is a specific color reaction, it proves that the sample to be tested contains the target microorganism , if not, vice versa; if there is no bacterial growth, it proves that the sample to be tested does not contain the target microorganism;

⑤ When the culture medium does not contain the target microorganism chromogen, with or without the selective miscellaneous bacteria inhibitor, observe whether there is bacterial growth at the other end of the capillary, if there is bacterial growth, inoculate the bacteria into the conventional selective culture of the target microorganism Purify or culture on the base;

⑥When the medium contains or does not contain selective miscellaneous bacteria inhibitors or/and target microorganism chromogenic reagents, the other end can also be connected to a set of liquid or semi-solid biochemical identification medium (in the detection tube), and the target Microorganisms are identified, or the other ends of multiple capillaries are respectively connected to a liquid or semi-solid biochemical identification medium to form a set of biochemical identification experiments.

The capillary is a straight or bent pipe with a pipe diameter less than 0.5cm and a pipe length greater than 1cm: the pipe diameter is less than 0.5cm, the pipe diameter is relatively small, the filling amount is small, the medium is saved, and it is easy to fill by siphon. The influence of lateral diffusion on the mid-cut surface is small; the length of the tube is greater than 1cm, and the length of the tube is selected based on the specific target microorganisms and the ability of the selective inhibitor to separate target microorganisms and miscellaneous bacteria through experimental verification to achieve better selective separation. The best solution is to separate the target microorganisms in a short time; the shape of the tube can be bent to a certain extent according to the convenience of the experiment, so as not to affect the experimental results.

The filling capillary adopts aseptic siphon method or perfusion method.

The target microorganisms are bacteria with or without flagella.

The nutrients for the growth of the target microorganisms are selected from one or more of carbon sources, nitrogen sources, growth factors and inorganic salts that can support or promote the growth of microorganisms.

The selective chromogenic agent of the target microorganism is a specific biochemical acid-base indicator, a biochemical identification reagent or/and an enzyme substrate chromogenic agent of the target microorganism, and its function is to display the specific biochemical reaction of the target microorganism through color reaction. Biochemical identification culture selects items according to the biochemical characteristics of the target bacteria. Generally, a target microorganism requires a set of biochemical reaction identification, so the biochemical identification medium needs to form a set of multiple tubes, and some biochemical substrates can be completed in one tube with several reactions.

The selective miscellaneous bacteria inhibitor is a reagent that has no inhibitory effect on target microorganisms and has an inhibitory effect on non-target microorganisms, or: a reagent that has an inhibitory effect on target microorganisms that is less than that of non-target microorganisms, or: an agent that has an inhibitory effect on target microorganisms A reagent whose growth-promoting effect is relatively greater than the growth effect on non-target microorganisms; in specific applications, select the appropriate selective miscellaneous bacteria inhibitor according to the target microorganisms.

The selective miscellaneous bacteria inhibitor is selected from the group consisting of bile salt, sodium deoxycholate, iodine, potassium iodide, brilliant green, malachite green, selenite, cystine, sodium chloride solution with a mass concentration of more than 1%, acridine pyriflavin, nalidixic acid, lauryl sulfate, lithium chloride, crystal violet, neutral red, eosin, methylene blue, vancomycin, novobiocin, amphotericin, cephalosporins, cefoperazone, One or more of polymyxin, sulfadiazine, rifampicin, dyes and antibiotics.

Preferably, the medium is a semi-solid medium, such as a semi-solid medium composed of a selective chromogenic agent for target microorganisms, nutrients, 0.1%-1.0% (mass fraction) agar and water.

The enrichment solution contains nutrients capable of supporting or promoting the growth of target microorganisms, with or without the addition of selective miscellaneous bacteria inhibitors. The sample solution with a volume ratio of 10:1 or more of the enrichment solution and the sample to be tested is preferred. The enrichment solution contains nutrients, and the addition or non-addition of selective miscellaneous bacteria inhibitors is determined according to the survival of the target microorganisms in the sample. If the survival status of the target microorganisms is relatively good, a selective miscellaneous bacteria inhibitor can be added. If the survival status of the target microorganisms in the sample is not good, no selective miscellaneous bacteria inhibitor should be added, and the nutrient recovery should be mainly used.

For the access method, if the sample is liquid or semi-solid, it can be directly inserted. The access method is preferably continuously inserted throughout the cultivation process. According to the conditions of the target microorganism and the sample, if the contamination is serious or the content of the target microorganism is relatively high, it can be inserted One end of the capillary selective semi-solid medium touches the sample to be tested or the dilution or enrichment solution of the sample to be tested. The so-called point contact means that the capillary is in contact with the liquid and then leaves it for a short time. It can also be touched indirectly, that is, through cotton The rod, the bacterium-taking loop (needle) and other effective tools first touch the sample to be tested or the dilution or enrichment solution of the sample to be tested, and then touch one end of the capillary selective semi-solid medium.

The culture time is 3<t≤96h, preferably 12<t≤24h. The culture temperature and time are selected according to the growth characteristics of the target microorganisms, such as 36°C or 42°C for Salmonella, and 30°C for Listeria monocytogenes. The culture time is generally 3 to 96 hours. During the cultivation process, the mixed bacteria pass through the semi-solid agar equipped with a relatively strong inhibitor of miscellaneous bacteria. As the mixed bacteria grow forward or/and move forward, the miscellaneous bacteria will be subjected to repeated inhibitory selection, and the target The microorganisms maintain normal or abnormal forward growth or/and movement, similar to the process of continuous redistribution of chemical components in the chromatogram, and finally the target microorganisms will be released to the other end of the capillary, thereby achieving rapid and efficient separation of the target bacteria.

The device for detecting or screening microorganisms by capillary culture method of the present invention can be used for detecting or screening microorganisms by capillary culture method, and the detection or separation time of most bacterial genera is shortened from 36 to 48 hours in the prior art to 12 to 24 hours. It has the advantages of fast, efficient, time-saving and labor-saving, good specificity, and strong sensitivity.

Description of drawings

Fig. 1 is the structural representation (straight tube, no detection tube) that is used for capillary culture method detection or screening microorganism device of the present invention;

Fig. 2 is a schematic diagram of the structure of the device for detecting or screening microorganisms by capillary culture method of the present invention (elbow, with detection tube).

Among them, 1. Capillary tube; 2. Detection tube; 3. Sealing cover; 4. Plane sticking to the wall; 5. Magnet.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and the accompanying drawings.

A device for detecting or screening microorganisms by capillary culture method, comprising a capillary 1, as shown in Figure 1, the capillary is a straight tube with a diameter of 1mm and a length of 10cm.

The material of the capillary 1 is glass.

When in use, fill the medium into the capillary 1, and then insert one end of the filled capillary 1 into the sample to be tested or the dilution or enrichment solution of the sample to be tested, or: insert the sample to be tested into the capillary 1 After culturing 0<t≤96h, judge whether there is bacterial growth or color reaction at the other end of the capillary 1, and then judge whether the target microorganism is contained in the sample to be tested.

Another structure of the present invention is: a device for detecting or screening microorganisms by capillary culture, comprising a capillary 1 and a detection tube 2, as shown in Figure 2, the capillary 1 is an elbow with a diameter of 1mm and a length of 10cm , the material is glass; the detection tube 2 is provided with a sealing cover 3, and one end of the capillary 1 passes through the sealing cover and stretches into the detection tube 2, and the side wall of the detection tube 2 is provided with a plane sticking wall 4 (the detection tube 2 is cylindrical , similar to a centrifuge tube), so that it can be tightly attached to the packaging bag of the sample to be tested to prevent falling off. The side wall of the detection tube 2 is provided with a magnet 5. When in use, the magnet 5 can fix the detection tube 2 by cooperating with iron-containing articles or other magnets to prevent falling off.

When in use, fill the medium into the capillary 1 and the detection tube 2 (the filled medium is filled according to the demand, and the composition is not consistent), and then connect one end of the filled capillary 1 to the sample to be tested or the sample to be tested or: insert the sample to be tested into one end of capillary 1, and connect the other end of capillary 1 into the culture medium of detection tube 2, and after culturing 0<t≤96h, judge that capillary 1 Whether there is bacterial growth or color reaction at the other end, or further judge whether there is bacterial growth or color reaction on the culture medium in the detection tube 2, and then determine whether the target microorganism is contained in the sample to be detected.

The specific application examples of the device for capillary culture method detection or screening microorganisms of the present invention are as follows:

Example 1 A capillary selective semi-solid method for testing Salmonella

Proceed as follows:

1. Selection of capillary: Select several glass capillaries with inner diameter of 0.5mm, 1mm, 2mm and length of 2, 5, 10, 20cm.

2. Preparation of selective semi-solid medium: take 900mL after heating and dissolving 10.0g of peptone, 5.0g of beef extract, 3.0g of sodium chloride, 45.0g of calcium carbonate, 4.0g of agar powder and 1000mL of distilled water, and add 100mL of 50% ( Mass fraction, the same below) sodium thiosulfate solution, 20.0mL 20% iodine solution (containing 25% potassium iodide), 2.0mL 0.5% brilliant green aqueous solution, 50.0mL 10% ox bile salt solution, mix and sterilize to obtain.

3. Preparation of liquid and semi-solid chromogenic medium: CHROMAGAR Salmonella chromogenic medium (Zhengzhou Bosai Biotechnology Co., Ltd., batch number 1004) was prepared by adding deionized water according to the proportion in the instructions to prepare a 200mL suspension, and magnetic stirring was dissolved for 15min. After standing still for 15min, take 100mL of supernatant, which is the chromogenic agent and nutrient solution of the target bacteria, and is a liquid chromogenic medium (after heating and sterilizing, divide into 1mL tubes for later use); Add 0.2 g of agar to the chromogenic medium, heat, dissolve and sterilize to obtain a semi-solid chromogenic medium.

4. Preparation of semi-solid biochemical identification medium: The trisaccharide iron in the Salmonella biochemical identification medium set (S005, Beijing Land Bridge Technology Co., Ltd.) is a semi-solid medium, so it can be used directly without treatment. The rest of the biochemical tubes are the target microbial biochemical reaction substrate, biochemical reaction indicator, and nutrient mixture (except trisaccharide iron), and 4 mg of agar is added to 1 mL of each of the other tubes, heated to dissolve and sterilized. Since trisaccharide iron is a semi-solid medium, the trisaccharide iron agar in this set can be used directly without treatment.

5. Filling of the capillary: insert the sterile capillary into the melted medium prepared in the above steps 2-4, observe the upper edge of the capillary siphon, fill the whole capillary, let it stand at room temperature until it solidifies, and fill it with the selective semi-solid medium. Put 0.5mm, 1mm, 2mm inner diameter, 5, 10, 20cm long capillary tubes into several pieces, semi-solid chromogenic medium and semi-solid biochemical identification medium into 0.5mm, 1mm, 2mm inner diameter, 2cm long capillary tubes respectively.

6. Preparation of sample enrichment night: Add about 10 ¹ CFU/mL Salmonella to 100mL fresh milk as a sample, take 25g of the test sample and add it to 225mL buffered peptone water medium (Beijing Land Bridge Technology Co., Ltd., the following implementations The finished medium used in the enrichment solution in the example is produced by the company and used within the validity period).

7. Select any 18 capillary selective semi-solid media prepared in step 5, insert one end into the sample enrichment solution, and continue until the entire cultivation process. Put 15 of them into a group of 3, and insert the other end of each group into the prepared capillary semi-solid chromogenic medium, liquid chromogenic medium, capillary semi-solid biochemical identification medium, and Salmonella biochemical identification medium set product (S005, Beijing Land Bridge Technology Co., Ltd.), in the nutrient broth, the remaining 3 roots were not inserted into any medium.

8. Cultivation: The capillary-selective semi-solid system prepared in step 7 was placed in a constant temperature and humidity incubator at 36° C. for 24 hours. It was observed that the ends of the semi-solid chromogenic medium of 3 capillaries and the liquid chromogenic medium of 3 tubes were all purple-red, and it was preliminarily determined that Salmonella was detected in the sample. Observe and record the results of capillary semi-solid biochemical identification medium and Salmonella biochemical identification medium set product. The identification shows that the detected bacteria are Salmonella. The results are shown in Table 1:

Table 1

Collect the cultured nutrient broth and use it as a bacterial liquid or store it; the other end of the selective semi-solid of the 3 capillaries that are not inserted into any medium after cultivation can be seen in a turbid state with obvious bacterial growth, and Salmonella grows, touch or use to take bacteria The loop was inoculated on the Salmonella chromogenic plate and the nutrient agar plate and cultivated for 24 hours. The purple-red colonies on the Salmonella chromogenic plate were proved to be Salmonella. The colonies on the nutrient agar plate were collected and preserved.

Example 2 A capillary selective semi-solid chromogenic culture method for testing Listeria monocytogenes, the steps are as follows:

1. Selection of capillary: Select several glass capillaries with inner diameter of 0.5mm, 1mm, 2mm and length of 2, 5, 10, 20cm.

2. Preparation of selective semi-solid medium: according to the formula tryptone 5.0g, polyvalent peptone 5.0g, yeast extract 5.0g, sodium chloride 20.0g, potassium dihydrogen phosphate 1.35g, disodium hydrogen phosphate 12.0g, agar Mix 4.0g of powder with 1000mL of distilled water, heat to dissolve and autoclave 200mL, add 0.40mL of 1% nalidixic acid and 0.50mL of 1% acridine yellow to obtain the product.

3. Preparation of liquid and semi-solid chromogenic medium: CHROMAGAR Listeria chromogenic medium (Zhengzhou Bosai Biotechnology Co., Ltd., batch number 975) was prepared by adding deionized water according to the proportion of the instructions to prepare a 200mL suspension, and magnetic After stirring and dissolving for 15 minutes, take 100 mL of the supernatant after standing for 15 minutes, and use it as a liquid chromogenic medium (after heating and sterilizing, divide into 1 mL tubes for later use); take 50 mL of liquid chromogenic medium and add 0.4 g to 2 g of agar to dissolve and sterilize , to obtain a semi-solid chromogenic medium.

4. Preparation of semi-solid biochemical identification medium: The trisaccharide iron in the Listeria biochemical identification medium set product (S014, Beijing Land Bridge Technology Co., Ltd.) is a semi-solid medium, so it can be used directly without treatment. The rest of the biochemical tubes are the target microbial biochemical reaction substrate, biochemical reaction indicator, and nutrient mixture. Add 4.0 mg of agar to 1 mL of each tube, heat, dissolve and sterilize.

5. Filling of the capillary: insert the sterile capillary into the melted medium prepared in the above steps 2-4, observe the upper edge of the capillary siphon, fill the whole capillary, let it stand at room temperature until it solidifies, and fill it with the selective semi-solid medium. Put 0.5mm, 1mm, 2mm inner diameter, 5, 10, 20cm long capillary tubes into several pieces, semi-solid chromogenic medium and semi-solid biochemical identification medium into 0.5mm, 1mm, 2mm inner diameter, 2cm long capillary tubes respectively.

6. Preparation of sample enrichment night: about 10 ¹ CFU/mL of Listeria monocytogenes was added to 100 mL of fresh milk as a sample, and 25 g of the test sample was added to 225 mL of LB1 medium.

7. Select any 18 capillary selective semi-solid media prepared in step 5, insert one end into the sample enrichment solution, and continue until the entire cultivation process. 15 of them are divided into 3 groups, and the other end of each group is inserted into the prepared capillary semi-solid chromogenic medium, liquid chromogenic medium, capillary semi-solid biochemical identification medium, mononuclear cell hyperplasia Listeria Bacteria biochemical identification medium set product (S0014, Beijing Land Bridge Technology Co., Ltd.), nutrient broth, the remaining 3 were not inserted into any medium.

8. Cultivation: the capillary selective semi-solid system prepared in step 7 was placed in a constant temperature and humidity incubator at 30° C., and cultivated for 24 hours. Observing that one end of the semi-solid chromogenic medium of 3 capillaries and 3 liquid chromogenic medium were blue, it was preliminarily determined that Listeria monocytogenes was detected in the sample. Observe and record the results of capillary semi-solid biochemical identification medium and Listeria monocytogenes biochemical identification medium set product. The identification shows that the detected bacteria is Listeria monocytogenes. The results are shown in Table 2 :

Table 2

The cultured nutrient broth was collected and used or stored as a bacterial liquid; the other end of the selective semi-solid of the 3 capillaries that were not inserted into any medium after culture showed obvious bacterial growth turbidity, and Listeria monocytogenes grew. Touch or inoculate on the Listeria monocytogenes chromogenic plate and nutrient agar plate for 24 hours by touching or using a bacterial loop to inoculate. Blue colonies appear on the chromogenic plate of Listeria monocytogenes, which proves monocytogenes hyperplasia For Listeria, collect colonies on nutrient agar plates for preservation.

Example 3 A capillary selective semi-solid chromogenic culture method for testing Staphylococcus aureus

Proceed as follows:

1. Selection of capillary: Select several glass capillaries with an inner diameter of 0.5-5 mm and a length of 2, 5, 10, and 20 cm.

2. Preparation of selective semi-solid medium: tryptone 17.0g, plant peptone 3.0g, sodium chloride 100.0g, dipotassium hydrogen phosphate 2.5g, sodium pyruvate 10.0g, glucose 2.5g, agar powder 4.0g according to the formula Mix with 1000mL of distilled water, heat to dissolve and autoclave for later use.

3. Capillary filling: insert the capillary selected in step 1 into the molten medium prepared in step 2, observe the upper edge of the capillary siphon, fill the entire capillary, and let it stand at room temperature until it solidifies.

4. Preparation of sample enrichment night: Add about 10 ¹ CFU/mL of Staphylococcus aureus to 100 mL of fresh milk as a sample, take 25 g of the test sample and add it to 225 mL of 7.5% sodium chloride broth.

5. Preparation of chromogenic medium: CHROMAGAR Staphylococcus aureus chromogenic medium (Zhengzhou Bosai Biotechnology Co., Ltd., batch number 792) was prepared by adding deionized water according to the ratio in the instructions to make a 200mL suspension, and dissolved by magnetic stirring for 15min. After standing for 15 minutes, take 100 mL of the supernatant and heat-sterilize it, and then dispense 1 mL into each tube for later use.

6. Insert one end of the prepared capillary semi-solid selection medium into the sample enrichment solution, and the other end into 1mL chromogenic medium.

7. Cultivation: Put the system prepared in step 6 into a constant temperature and humidity incubator at 36° C. for 24 hours.

8. All experimental chromogenic solutions turned red, and it was preliminarily determined that Staphylococcus aureus was detected in the sample.

Example 4 A capillary selective semi-solid chromogenic culture method for testing Vibrio parahaemolyticus

Proceed as follows:

1. Selection of capillary: Select several glass capillaries with an inner diameter of 0.5-5 mm and a length of 2, 5, 10, and 20 cm.

2. Preparation of selective semi-solid medium: According to the formula of peptone 10.0g, sodium chloride 30.0g, agar powder 4.0g and distilled water 1000mL, heat to dissolve and autoclave for later use.

3. Preparation of liquid chromogenic medium: CHROMAGAR Vibrio parahaemolyticus chromogenic medium (Zhengzhou Bosai Biotechnology Co., Ltd., batch number 938) was prepared into 200mL suspension by adding deionized water according to the ratio in the instructions, and dissolved by magnetic stirring After 15 minutes, after standing still for 15 minutes, take 100 mL of the supernatant and heat-sterilize it, and then divide into 1 mL tubes for later use.

4. Preparation of semi-solid chromogenic medium: add 0.4 g of agar to the liquid chromogenic medium prepared in step 5, heat, dissolve and sterilize, to obtain.

5. Filling of the capillary: insert the capillary selected in step 1 into the melted medium prepared in steps 2 and 5, observe the upper edge of the capillary siphon, fill the whole tube of the capillary, and let it stand at room temperature until it solidifies. Selective semi-solid medium Pack several 0.5mm, 1mm, and 2mm inner diameters, 5, 10, and 20cm long capillaries, and put several 0.5mm, 1mm, and 2mm inner diameters, 2cm long capillaries for the semi-solid chromogenic medium.

6. Preparation of samples for enrichment: Add about 10 ¹ CFU/mL of Vibrio parahaemolyticus to 100 mL of fresh milk as a sample, take 25 g of test samples and add 225 mL of 3% sodium chloride alkaline peptone water.

7. Select any 9 capillary selective semi-solid media prepared in step 5, insert one end into the sample enrichment solution, and continue until the entire cultivation process. 6 of them were divided into 3 groups, and the other end of each group was inserted into the capillary semi-solid chromogenic medium and liquid chromogenic medium prepared above, respectively, and the remaining 3 were not inserted into any medium.

8. The capillary selective semi-solid system prepared in step 7 was placed in a constant temperature and humidity incubator at 36° C. for 24 hours. It was observed that the ends of the semi-solid chromogenic medium of 3 capillaries and the liquid chromogenic medium of 3 tubes were all purple-red, and it was preliminarily determined that Vibrio parahaemolyticus was detected in the sample. After culturing, the other end of the 3 capillary selective semi-solid tubes without inserting any culture medium can be seen in the turbid state of obvious bacterial growth, with the growth of Vibrio parahaemolyticus, touch or inoculate it on the Vibrio parahaemolyticus chromogenic plate and Cultivate on nutrient agar plate for 24 hours, purple-red colonies appear on the chromogenic plate of Vibrio parahaemolyticus, which proves to be Vibrio parahaemolyticus, and collect the colonies on the nutrient agar plate for preservation.

Example 5 A capillary selective semi-solid chromogenic culture method for detection of Enterobacter sakazakii

Proceed as follows:

1. Selection of capillary: Select several glass capillaries with an inner diameter of 0.5-5 mm and a length of 2, 5, 10, and 20 cm.

2. Preparation of selective semi-solid medium: according to the formula tryptone 20.0g, sodium chloride 34.0g, lactose 5.0g, potassium dihydrogen phosphate 2.75g, dipotassium hydrogen phosphate 2.75g, sodium dodecylsulfonate 0.1 g. Proportion of 4.0g agar powder and 1000mL distilled water, after heating and dissolving and autoclaving, add 10.0mg vancomycin for later use.

3. Preparation of liquid chromogenic medium: DFI Enterobacter sakazakii chromogenic medium (CM1055, British OXOID company, batch number 929066) was prepared into a 200mL suspension by adding deionized water according to the proportion in the instructions, and dissolved by magnetic stirring for 15min. After standing for 15 minutes, take 100 mL of the supernatant and heat-sterilize it, then dispense 1 mL into each tube for later use.

4. Preparation of semi-solid chromogenic medium: add 0.4 g of agar to the liquid chromogenic medium prepared in step 5, heat, dissolve and sterilize, to obtain.

5. Filling of the capillary: insert the capillary selected in step 1 into the melted medium prepared in steps 2 and 5, observe the upper edge of the capillary siphon, fill the whole tube of the capillary, and let it stand at room temperature until it solidifies. Selective semi-solid medium Pack several 0.5mm, 1mm, and 2mm inner diameters, 5, 10, and 20cm long capillaries, and put several 0.5mm, 1mm, and 2mm inner diameters, 2cm long capillaries for the semi-solid chromogenic medium.

6. Preparation of sample enrichment night: Add about 10 ¹ CFU/mL of Enterobacter sakazakii to 100 mL of fresh milk as a sample, take 25 g of the test sample and add to 225 mL mLST broth medium.

7. Select any 9 capillary selective semi-solid media prepared in step 5, insert one end into the sample enrichment solution, and continue until the entire cultivation process. 6 of them were divided into 3 groups, and the other end of each group was inserted into the capillary semi-solid chromogenic medium and liquid chromogenic medium prepared above, respectively, and the remaining 3 were not inserted into any medium.

8. The capillary selective semi-solid system prepared in step 7 was placed in a constant temperature and humidity incubator at 36° C. for 24 hours. It was observed that one end of the 3 capillary semi-solid chromogenic medium and 3 liquid chromogenic medium were blue-green, and it was preliminarily determined that Enterobacter sakazakii was detected in the sample. After culturing, the other end of the 3 capillary selective semi-solid tubes without any culture medium can be seen in the turbid state of bacterial growth, with the growth of Enterobacter sakazakii, which can be touched or inoculated on the Enterobacter sakazakii chromogenic plate and nutrient agar with a bacterial loop After culturing on the plate for 24 hours, blue-green colonies appeared on the Enterobacter sakazakii chromogenic plate, which proved to be Enterobacter sakazakii, and the colonies on the nutrient agar plate were collected for preservation.

Example 6 A capillary selective semi-solid chromogenic culture method for detection of Escherichia coli O157: H7/NM

Proceed as follows:

1. Selection of capillary: Select several glass capillaries with an inner diameter of 0.5-5 mm and a length of 2, 5, 10, and 20 cm.

2. Preparation of selective semi-solid medium: according to the formula tryptone 20.0g, sodium chloride 5.0g, lactose 5.0g, potassium dihydrogen phosphate 1.5g, dipotassium hydrogen phosphate heptahydrate 4.0g, bile salt No. 3 1.12g , Agar powder 4.0g and distilled water 1000mL proportioning, after heating and dissolving and autoclaving, add 20.0mg novobiocin for later use.

3 Preparation of liquid chromogenic medium: CHROMAGAR Escherichia coli O157:H7/NM chromogenic medium (Zhengzhou Bosai Biotechnology Co., Ltd., batch number 426) was prepared by adding deionized water according to the proportion of the instructions to prepare a 200mL suspension , magnetically stirred and dissolved for 15 minutes, and after standing for 15 minutes, take 100 mL of the supernatant, heat-sterilize it, and dispense 1 mL into each tube for later use.

4. Preparation of semi-solid chromogenic medium: add 0.4 g of agar to the liquid chromogenic medium prepared in step 5, heat, dissolve and sterilize, to obtain.

5. Filling of the capillary: insert the capillary selected in step 1 into the melted medium prepared in steps 2 and 5, observe the upper edge of the capillary siphon, fill the whole tube of the capillary, and let it stand at room temperature until it solidifies. Selective semi-solid medium Pack several 0.5mm, 1mm, and 2mm inner diameters, 5, 10, and 20cm long capillaries, and put several 0.5mm, 1mm, and 2mm inner diameters, 2cm long capillaries for the semi-solid chromogenic medium.

6. Preparation of sample enrichment night: Add about 10 ¹ CFU/mL of Escherichia coli O157:H7/NM to 100 mL of fresh milk as a sample, take 25 g of the test sample and add it to 225 mL of mEC-n broth medium .

7. Select any 9 capillary selective semi-solid media prepared in step 5, insert one end into the sample enrichment solution, and continue until the entire cultivation process. 6 of them were divided into 3 groups, and the other end of each group was inserted into the capillary semi-solid chromogenic medium and liquid chromogenic medium prepared above, respectively, and the remaining 3 were not inserted into any medium.

8. The capillary selective semi-solid system prepared in step 7 was placed in a constant temperature and humidity incubator at 36° C. for 24 hours. It was observed that one end of the semi-solid chromogenic medium of 3 capillaries and 3 liquid chromogenic medium were all red, and it was preliminarily determined that Escherichia coli O157:H7/NM was detected in the sample. After culturing, the other end of the selective semi-solid of the 3 capillaries without any culture medium can be seen in the turbid state of bacterial growth, with Escherichia coli O157:H7/NM growing, touch or inoculate Escherichia coli with a loop Bacteria O157: H7/NM chromogenic plate and nutrient agar plate were cultured for 24 hours, and Escherichia coli O157: H7/NM chromogenic plate showed red colonies, which proved to be Enterobacter sakazakii, and the colonies on the nutrient agar plate were collected for preservation.

Example 7 Comparison of qualitative detection of Salmonella using capillary selective semi-solid culture method and GB4789.4-2010 microbial inspection method

The experiment was carried out with simulated samples using the standard addition recovery method, and the inoculum amount was about 10 ¹ , 10 ² , 10 ³ cfu Salmonella. The experiment was carried out in strict accordance with the above method and the GB4789.4-2010 microbial inspection method, and the results are shown in Table 3:

table 3

Example 8 Comparison of qualitative detection of Listeria monocytogenes using capillary selective semi-solid culture method and GB4789.30-2010 microbiological testing method

The experiment was carried out with simulated samples using the standard recovery method, and the inoculum amount was about 10 ¹ , 10 ² , 10 ³ cfu Listeria monocytogenes. The experiment was carried out in strict accordance with the above method and the GB4789.30-2010 microbial inspection method. The results are as follows Table 4:

Table 4

Claims (5)

1. A device for detecting microorganisms by capillary culture method, characterized in that: it comprises a capillary, which is filled with selective semi-solid medium or semi-solid chromogenic medium or semi-solid biochemical identification medium in the capillary: the sterile capillary Insert the melted culture medium to fill the whole capillary tube, let it stand at room temperature until it solidifies, fill the selective semi-solid medium with 0.5mm, 1mm, 2mm inner diameter, 5, 10, 20cm long capillary tubes, semi-solid color development The culture medium and the semi-solid biochemical identification medium are filled with several capillaries of 0.5mm, 1mm and 2mm inner diameter and 2cm long respectively; Wherein, the selective semi-solid medium is prepared by the following method: according to the formula, 10.0g of peptone, 5.0g of beef extract, 3.0g of sodium chloride, 45.0g of calcium carbonate, 4.0g of agar powder and 1000mL of distilled water are heated and dissolved Take 900mL, add 100mL 50% sodium thiosulfate solution, 20.0mL 20% iodine solution, 2.0mL 0.5% brilliant green aqueous solution, 50.0mL 10% ox bile salt solution, mix and sterilize to get it; the percentages are mass percentages; The semi-solid chromogenic medium is prepared by the following method: adding CHROMAGAR Salmonella chromogenic medium to deionized water to prepare a 200mL suspension, magnetically stirring and dissolving for 15min, then standing for 15min and then taking the supernatant 100mL. The supernatant is the chromogenic agent and nutrient solution of the target bacteria, which is a liquid chromogenic medium; add 0.2 g of agar to 50 mL of liquid chromogenic medium, heat, dissolve and sterilize to obtain a semi-solid chromogenic medium; The semi-solid biochemical identification medium is a Salmonella biochemical identification medium set product; The microorganisms detected are Salmonella. 2. A device for detecting microorganisms by capillary culture method, characterized in that: it comprises a capillary, which is filled with selective semi-solid medium or semi-solid chromogenic medium: the capillary is inserted into the culture medium of the melting state, so that the capillary After filling the whole tube, let it stand at room temperature until solidification, fill the selective semi-solid medium with inner diameters of 0.5mm, 1mm, and 2mm, and several long capillaries of 5, 10, and 20cm, and fill the semi-solid chromogenic medium with 0.5mm, 1mm , 2mm inner diameter, several 2cm long capillaries; Wherein, the selective semi-solid medium is prepared by the following method: according to the proportion of 10.0 g of peptone, 30.0 g of sodium chloride, 4.0 g of agar powder and 1000 mL of distilled water, heat, dissolve and autoclave for later use; The semi-solid chromogenic medium is prepared by the following method: adding CHROMAGAR Vibrio parahaemolyticus chromogenic medium to deionized water to prepare a 200mL suspension, magnetically stirring and dissolving for 15min, then standing for 15min and then taking the supernatant After heat sterilization of 100mL of solution, add 0.4g of agar to dissolve and sterilize by heat; The detected microorganism is Vibrio parahaemolyticus. 3. A device for detecting microorganisms by capillary culture, characterized in that: it comprises a capillary, which is filled with a selective semi-solid medium or a semi-solid chromogenic medium: the capillary is inserted into the culture medium in a melting state, so that the capillary After filling the whole tube, let it stand at room temperature until solidification, fill the selective semi-solid medium with inner diameters of 0.5mm, 1mm, and 2mm, and several long capillaries of 5, 10, and 20cm, and fill the semi-solid chromogenic medium with 0.5mm, 1mm , 2mm inner diameter, several 2cm long capillaries; Wherein, the selective semi-solid medium is prepared by the following method: tryptone 20.0g, sodium chloride 34.0g, lactose 5.0g, potassium dihydrogen phosphate 2.75g, dipotassium hydrogen phosphate 2.75g, ten Dialkyl sodium sulfonate 0.1g, agar powder 4.0g and distilled water 1000mL proportion, after heating and dissolving and autoclaving, add 10.0mg vancomycin for later use; The semi-solid chromogenic medium is prepared by the following method: add DFI Enterobacter sakazakii chromogenic medium to deionized water to prepare a 200mL suspension, stir and dissolve for 15 minutes, and take the supernatant after standing for 15 minutes After 100mL of heat sterilized, add 0.4g of agar to heat to dissolve and sterilize, to obtain; The microorganism detected is Enterobacter sakazakii. 4. A device for detecting microorganisms by capillary culture method, characterized in that: it comprises a capillary, filled with selective semi-solid medium or semi-solid chromogenic medium in the capillary: the capillary is inserted into the medium in the melting state, so that the capillary After filling the whole tube, let it stand at room temperature until solidification, fill the selective semi-solid medium with inner diameters of 0.5mm, 1mm, and 2mm, and several long capillaries of 5, 10, and 20cm, and fill the semi-solid chromogenic medium with 0.5mm, 1mm , 2mm inner diameter, several 2cm long capillaries; Wherein, the selective semi-solid medium is prepared by the following method: tryptone 20.0g, sodium chloride 5.0g, lactose 5.0g, potassium dihydrogen phosphate 1.5g, dipotassium hydrogen phosphate heptahydrate 4.0g , No. 3 bile salt 1.12g, agar powder 4.0g and distilled water 1000mL proportioning, after heating and dissolving and autoclaving, add 20.0mg novobiocin for later use; The semi-solid chromogenic medium is prepared by the following method: adding CHROMAGAR Escherichia coli O157:H7/NM chromogenic medium to deionized water to prepare a 200mL suspension, magnetic stirring and dissolving for 15min, and standing After 15 minutes, take 100 mL of the supernatant and heat-sterilize it, then add 0.4 g of agar to heat-dissolve and sterilize it to obtain the product; The microorganism detected is Escherichia coli O157:H7/NM. 5. A device for detecting microorganisms by capillary culture, characterized in that: it comprises a capillary and a detection tube, the capillary is an elbow with a diameter of 1 mm and a length of 10 cm, and the material is glass; the detection tube is provided with a sealing cover, and the capillary One end of the test tube is inserted into the detection tube through the sealing cover, and the side wall of the detection tube is provided with a flat wall; the side wall of the detection tube is provided with a magnet; the capillary and the detection tube are filled with any one of claims 1-4. the culture medium.