Contents 1 Overview 2 Classification of drug susceptibility testing 2.1 Disk diffusion method 2.2 Dilution method 2.2.1 Agar dilution method 2.2.2 Broth dilution method 2.3 Antibiotic concentration gradient method 2.4 Automated instrument method 3 Experimental steps 3.1 Experiment Materials 3.1.1 Drug susceptibility test paper 3.1.2 Bacteria 3.1.3 Instrument 3.2 Experimental preparation 3.3 Experimental operation method 3.3.1 Drug susceptibility tablet method 3.3.2 Oxford cup method 3.3.3 Punch method 3.4 Result observation 3.5 Judgment criteria 3.6 Influencing drugs Factors for susceptibility results 3.6.1 Culture medium 3.6.2 Bacterial inoculum 3.6.3 Drug concentration 3.6.4 Culture time 4 Application of drug susceptibility testing This is a redirect entry that shares the content of drug susceptibility testing. For the convenience of reading, the drug sensitivity test below has been automatically replaced by the drug resistance test. You can click here to restore the original appearance, or use the note method to display 1 Overview
The drug resistance test is referred to as the drug susceptibility test (or drug sensitivity test) ). Microbiological tests aimed at understanding the sensitivity (or tolerance) of pathogenic microorganisms to various antibiotics to guide the rational selection of antibiotic drugs in clinical practice. If an antibiotic can inhibit and kill pathogenic bacteria at a very small dose, the pathogenic bacteria are said to be "sensitive" to the antibiotic. On the contrary, it is called "insensitive" or "resistant". In order to understand which antibiotics pathogenic bacteria are sensitive to, so as to use drugs rationally and reduce blindness, drug susceptibility testing should often be performed. The general method is: take specimens containing pathogenic bacteria from the patient's infected site, inoculate them on an appropriate culture medium, and culture them under certain conditions; at the same time, stick pieces of paper stained with a certain amount of various antibiotics on the surface of the culture medium ( Or use a stainless steel ring with a quantitative amount of antibiotic solution inside, and observe the results after incubating for a certain period of time. Since pathogenic bacteria have different sensitivities to various antibiotics, "empty circles" of different sizes formed around the drug paper to inhibit the growth of pathogens are called inhibition zones. The size of the inhibition zone is directly proportional to the sensitivity of pathogenic bacteria to various antibiotics. Therefore, antibiotics can be selected in a targeted manner based on the test results. In recent years, automated drug susceptibility testing instruments have come out, making the testing faster and more accurate. The current abuse of antibiotics has led to the increase of drug-resistant bacteria. Even the long-term use of broad-spectrum antibiotics in large quantities kills normal microorganisms in the body and loses the mutual restraint of microorganisms, causing some rare or common non-pathogenic bacteria to multiply in large numbers, causing so-called "Second infection" occurs frequently, causing artificial difficulties in treatment. Therefore, it is important to advocate the use of drug susceptibility testing and adhere to rational drug use. 2 Classification of drug susceptibility tests 2.1 Disk diffusion method
This method is to stick a filter paper containing a quantitative amount of antibacterial drugs on the surface of agar that has been inoculated with test bacteria. The drug in the paper diffuses in the agar. As the diffusion distance increases, the concentration of the antimicrobial agent decreases logarithmically, forming a concentration gradient around the paper. At the same time, strains within the inhibitory concentration range around the paper cannot grow, while strains outside the inhibitory range can grow, thus forming a transparent inhibitory zone around the paper. The diameters of the inhibitory zones of different antibacterial drugs vary. It may be affected by the diffusion rate of the drug in agar. The size of the inhibition zone can reflect the sensitivity of the test bacteria to the drug and is negatively correlated with the MIC of the drug against the test bacteria. 2.2 Dilution method
The dilution method drug susceptibility test can be used to quantitatively test the in vitro activity of antibacterial drugs against a certain bacteria, and is divided into agar dilution method and broth dilution method. During experiments, the concentration of antibacterial drugs is usually diluted twice (lg2). The lowest drug concentration that can inhibit the visible growth of the bacteria to be tested becomes the minimum inhibitory concentration (MIC). The test concentration range of a specific antibacterial drug should include the ability to detect bacteria. The concentration of the interpretive breakpoint (susceptible, intermediate and resistant) should also include the MIC of the quality control reference strain. 2.2.1 Agar dilution method
First prepare an agar dilution plate containing antibacterial drugs.
Then inoculate the strain to be tested, and then interpret the results. 2.2.2 Broth dilution method 2.3 Antibiotic concentration gradient method 2.4 Automated instrument method 3 Experimental steps 3.1 Experimental materials
Ordinary nutrient agar culture medium: It can be purchased at a biochemical reagent store to conduct drug susceptibility tests of different bacteria. Choose different culture media. For example, for drug susceptibility testing of E. coli, you can choose ordinary nutrient agar or Maifenkai culture medium. Serum culture medium can be used to treat Salmonella.
3.1.1 Drug susceptibility test paper
Purchase or make your own (see experimental preparation for details) 3.1.2 Bacteria
Bacteria to be tested for drug susceptibility 3.1.3 Instruments
Inoculation loop, alcohol lamp, hole punch, Oxford cup, pipette, dropper 3.2 Experimental preparation
Preparation of drug-sensitive tablets: purchased or homemade
Preparation method: Take Xinhua No. 1 qualitative filter paper and punch it into small circular paper pieces with a diameter of 6 mm using a puncher. Take 50 round paper pieces and put them into a clean and dry empty penicillin bottle, and wrap the bottle mouth with a single layer of kraft paper. After high-pressure sterilization of 15 pounds for 1520 minutes, place it in a 37°C incubator or oven for several days to completely dry.
Preparation of antibacterial medicine paper sheets: Add 0.25 ml of the medicine solution into the above-mentioned penicillin bottle containing 50 paper pieces, and turn the paper pieces so that each paper piece is fully soaked with the medicine liquid. Do not turn the paper pieces when turning them. Paper smashed. At the same time, record the name of the drug on the mouth of the bottle, place it in a 37°C incubator overnight, seal it tightly after drying, and vacuum dry it if possible. Do not get wet and store in a dark and dry place. It is valid for 36 months.
Preparation of medicinal solution (for testing of commercial drugs): Prepare the medicinal solution in proportion to the therapeutic dose of the commercial drug; for example, the commercial drug Baibaixiao can be treated with drinking water according to its stated therapeutic dose of 0.01%. To prepare a medicinal solution according to this ratio, add 10 mg to 10 ml of water and mix well. This diluent is the drug solution used for drug sensitivity testing. 3.3 Experimental operation method 3.3.1 Drug-susceptible tablet method
In the "clean bench", use an inoculating loop that has been sterilized by an alcohol lamp (alcohol lamp) to pick up an appropriate amount of bacterial culture and divide it by streaking. Bacteria are spread onto plates. Specific method: Use a sterilized inoculation loop to take an appropriate amount of bacteria and apply them to four points on the edge of the plate, starting from each point and spreading the bacteria to 1/2 of the plate. Then, find the second dotted line to 1/2 of the plate, and draw lines in sequence until the bacteria are evenly distributed on the plate. (Also: You can pick the bacteria to be tested and make a bacterial suspension in a small amount of physiological saline, and use a sterilized cotton swab to apply the bacterial suspension to be tested on the surface of the culture medium. The coating is required to be uniform and dense)
After the tweezers are sterilized by the alcohol lamp flame, stop for a moment, take the drug-sensitive piece and stick it to the surface of the culture medium on the plate. In order to make the drug-sensitive tablet and the culture medium closely adhere to each other, you can use tweezers to gently press the drug-sensitive tablet a few times. In order to achieve accurate observation results, it is required that the drug-sensitive tablets can be regularly distributed on the culture medium; generally, one piece can be affixed to the center of the dish, and several pieces can be affixed at equal distances around the periphery (generally seven pieces can be affixed to the periphery). Each drug can be Remember the name of the sensitive film.
Place the plate culture medium in a 37°C incubator for 24 hours and observe the effect. 3.3.2 Oxford cup method
In the "clean bench", use an inoculating loop that has been sterilized by an alcohol lamp (alcohol lamp) to pick up an appropriate amount of bacterial culture and spread the bacteria onto the plate in a streaking manner. on the culture medium. Specific method: use a sterilized inoculation loop to take an appropriate amount of bacteria and apply them to four points on the edge of the plate, starting from each point and spreading the bacteria to 1/2 of the plate. Then, find the second dotted line to 1/2 of the plate, and draw lines in sequence until the bacteria are evenly distributed on the plate. (Also: You can pick the bacteria to be tested and make a bacterial suspension in a small amount of physiological saline. Use a sterilized cotton swab to apply the bacterial suspension to be tested on the surface of the culture medium. The coating is required to be uniform and dense.) < /p>
Use sterile operation to place the sterilized stainless steel tube (a round tube with an inner diameter of 6 nm, an outer diameter of 8 nm, and a height of 10 nm. Both ends of the tube should be smooth, and glass tubes and porcelain tubes can also be used). On the base, press gently so that there is no gap between it and the culture medium, and mark the name of various drugs on the small tube. Each plate can hold 46 small tubes. After waiting for a few minutes, drop a certain amount of various medicinal liquids into each small tube to prevent them from overflowing. Incubate at 37°C for 818 hours and observe the results.
Place the plate culture medium in a 37°C incubator for 24 hours and observe the effect. 3.3.3 Punch method
This method is relatively simple, low cost, easy to operate, and more suitable for the detection of commercial drugs.
In the "clean bench", use an inoculating loop that has been sterilized by an alcohol lamp (alcohol lamp) to pick up an appropriate amount of bacterial culture and spread the bacteria onto the plate culture medium in a streaking manner. Specific method: Use a sterilized inoculation loop to take an appropriate amount of bacteria and apply them to four points on the edge of the plate, starting from each point and spreading the bacteria to 1/2 of the plate. Then, find the second dotted line to 1/2 of the plate, and draw lines in sequence until the bacteria are evenly distributed on the plate. (Also: You can pick the bacteria to be tested and make a bacterial suspension in a small amount of physiological saline. Use a sterilized cotton swab to apply the bacterial suspension to be tested on the surface of the culture medium. The coating is required to be uniform and dense.) < /p>
Use sterile operation to place the sterilized stainless steel small tube (outer diameter is 4 mm, hole diameter and hole spacing are 3 mm, both ends of the tube should be smooth, glass tubes or porcelain tubes can also be used), Punch a hole in the culture medium, pick out the culture medium in the hole with a needle, and seal the bottom with a flame so that the culture medium can fully integrate with the plate (to prevent leakage of the medicine solution and affect the results).
Add samples: Add samples according to different liquids, and add the samples until they are full but not overflowing.
Place the plate culture medium in a 37°C incubator for 24 hours and observe the effect. 3.4 Observation of results
On an agar plate coated with bacteria, the antibacterial drug diffuses around in the agar, and its concentration decreases in a gradient, so the growth of bacteria within a certain distance around the paper is inhibited. An inhibitory zone is formed after overnight culture. The larger the inhibitory zone, the greater the sensitivity of the bacteria to this drug. On the contrary, the smaller it is, if there is no inhibition zone, it means that the bacteria are resistant to this drug. Its diameter is directly related to the concentration of drugs and the concentration of streaked bacteria. 3.5 Judgment criteria
The results of the drug susceptibility test should be based on the diameter of the inhibition zone as the criterion for determining sensitivity.
Table 1. Criteria for drug sensitivity test
Diameter of inhibition zone (mm) Sensitivity
More than 20, extremely sensitive
15~ 20 Highly sensitive
10~14 Moderately sensitive
Below 10, Hypoallergenic
0 Not sensitive
Reference table for drug sensitivity test criteria< /p>
1. The inhibitory zone of polymyxin; if it is above 9 mm, it is highly sensitive, if it is 6-9 mm, it is hyposensitive, and if there is no inhibitory zone, it is insensitive. 3.6 Factors affecting drug susceptibility results 3.6.1 Culture medium
should be prepared according to the nutritional needs of the test bacteria. When pouring the plate, the thickness should be appropriate (about 56mm), not too thin. Generally, for a 90mm diameter petri dish, it is appropriate to pour 1820ml of culture medium. Substances that are antagonistic to antibacterial drugs should be avoided in the culture medium. For example, calcium and magnesium ions can reduce the antibacterial activity of aminoglycosides. Thymidine and p-aminobenzoic acid (PABA) can antagonize the activity of sulfa drugs and TMP. 3.6.2 Bacterial inoculum amount
The bacterial inoculum amount should be constant. If it is too much, the inhibition zone will become smaller, and enzyme-producing strains may destroy the antibacterial activity of the drug. 3.6.3 Drug concentration
The concentration and total amount of the drug directly affect the results of the antibacterial test and need to be accurately prepared. Commercial drugs should be formulated strictly in accordance with their recommended therapeutic dosage. 3.6.4 Culture time
The general culture temperature and time is 37°C for 818 hours. Some antibacterial drugs diffuse slowly, such as polymyxin. The plate culture medium with the antibacterial drugs placed there can be placed at 4°C first. Keep it in the refrigerator for 2 to 4 hours to pre-diffuse the antibacterial drugs, and then culture it in a 37°C incubator. This can delay the growth of bacteria and obtain a larger inhibition zone. 4 Application of drug susceptibility testing