Brief introduction of drug resistance test

Directory 1 overview 2 Classification of drug sensitivity test 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 automatic instrument method 3 experimental steps 3.654 38+0 experimental materials 3. 1./kloc .2 bacteria 3. 1.3 instrument hole method 3.4 results observation 3.5 standard 3.6 factors affecting drug sensitivity 3.6. 1 culture medium 3.6.2 bacterial inoculation amount 3.6.3 drug concentration 3.6.4 culture time 4 application of drug sensitivity test This is a redirection project, and * * * enjoys the content of drug sensitivity test. For the convenience of reading, the drug sensitivity test below has been automatically replaced by drug resistance test. You can click here to restore the original appearance, or you can use the remarks to show the drug resistance test in 1. The purpose is to understand the sensitivity (or tolerance) of pathogenic microorganisms to various antibiotics, so as to guide the rational selection of antibiotics in clinical microbiology experiments. An antibiotic is called a "sensitive" antibiotic if it can inhibit and kill pathogenic bacteria in a small dose. Otherwise it is called "insensitivity" or "drug resistance". In order to know which antibiotics pathogens are sensitive to, use drugs rationally and reduce blindness, drug sensitivity tests should be carried out frequently. The general method is as follows: take samples containing pathogenic bacteria from the infected parts of patients, inoculate them on suitable culture medium and cultivate them under certain conditions; At the same time, stick a piece of paper with a certain amount of antibiotics on the surface of the culture medium (or put a certain amount of antibiotic solution in it with a stainless steel ring), and observe the results after a certain period of culture. Because of the different sensitivity of pathogenic bacteria to various antibiotics, there are "empty circles" of different sizes around the medicine paper to inhibit the growth of pathogenic bacteria, which is called bacteriostatic circle. The size of bacteriostatic circle is directly proportional to the sensitivity of pathogenic bacteria to various antibiotics. So we can choose antibiotics according to the test results. In recent years, the automatic drug sensitivity test instrument has been introduced, which makes the test faster and more accurate. At present, the abuse of antibiotics leads to the increase of drug-resistant bacteria, and even the long-term use of broad-spectrum antibiotics kills normal microorganisms in the body, losing the mutual inhibition of microorganisms, thus causing a large number of rare or common non-pathogenic bacteria to multiply, causing the so-called "secondary infection" to occur frequently and artificially causing treatment difficulties. Therefore, it is very important to advocate the use of drug sensitivity test and adhere to rational drug use.

2 Classification of drug sensitivity test 2. 1 disk diffusion method This method is to stick a filter paper containing quantitative antibacterial drugs on the surface of agar inoculated with test bacteria, and the drugs in the paper diffuse in the agar. With the increase of diffusion distance, the concentration of antibacterial drugs decreased logarithmically, thus forming a concentration gradient around the paper. At the same time, the strains in the bacteriostatic concentration range around the paper can't grow, but the strains outside the second bacteriostatic range can grow, thus forming a transparent bacteriostatic circle around the paper. The diameter of bacteriostatic circle of different bacteriostatic drugs may be different because of the diffusion speed of drugs in agar. The size of bacteriostatic circle can reflect the sensitivity of test bacteria to drugs, which is negatively correlated with the MIC of drugs to test bacteria.

2.2 Dilution drug sensitivity test can be used to quantitatively detect the in vitro activity of antibacterial drugs against certain bacteria, which can be divided into agar dilution method and broth dilution method. In the experiment, the concentration of antibacterial drugs is usually diluted by the ratio of times (lg2), and the lowest drug concentration that can inhibit the growth of the bacteria to be tested is the minimum inhibitory concentration (MIC). The detection concentration range of specific antibacterial drugs should include the concentration that can detect the explanatory inflection point (sensitivity, neutrality and drug resistance) of bacteria, and should also include the MIC of reference strains for quality control.

2.2. 1 agar dilution method Firstly, agar dilution plates containing antibacterial drugs were prepared. Inoculate the strains 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: common nutrient agar medium can be purchased from biochemical reagent stores, and different media can be selected for drug sensitivity test of different bacteria, such as common nutrient agar or McPherson medium for drug sensitivity test of Escherichia coli. Salmonella can choose serum culture medium.

3. 1. 1 Drug sensitivity test paper is purchased or made by ourselves (see experimental preparation for details).

3. 1.2 Drug sensitivity of bacteria to be tested

3. 1.3 instrument inoculation ring, alcohol lamp, punch, Oxford cup, pipette and emitter.

3.2 Experimental Preparation of Yao Min Tablets: Purchase or homemade.

Preparation method: Take Xinhua 1 qualitative filter paper, and punch it into circular pieces of paper with a diameter of 6 mm with a punching machine. Put 50 round pieces of paper into a clean and dry empty penicillin bottle, and wrap the bottle mouth with a single layer of kraft paper. After autoclaving at 15 lbs. 1520min, and put it in an incubator or oven at 37℃ for several days to completely dry it.

Preparation of antibacterial paper: Add 0.25ml of liquid medicine into a penicillin bottle containing 50 pieces of paper, and turn the paper over so that each piece of paper is fully saturated with liquid medicine. When turning the paper, the paper should not be mashed. At the same time, write down the name of the medicine on the bottle mouth, put it in an incubator at 37℃ overnight, dry it and cover it. If possible, it can be dried in vacuum. Keep away from moisture and store in a dark and dry place. The validity period is 36 months.

Preparation of liquid medicine (used for the test of commercial drugs): prepare liquid medicine according to the therapeutic dose ratio of commercial drugs; For example, Bai Bing Xiao, a commercial drug, can treat 0.0 1% of drinking water according to the specified amount and prepare liquid medicine according to this ratio. 10mg can be added to10ml water and mixed evenly. This diluent is a liquid medicine used for drug sensitivity test.

3.3 Experimental Operation Method 3.3. 1 Drug Sensitive Tablets Method In the "super clean table", an appropriate amount of bacterial culture was picked out by the inoculation ring sterilized by alcohol lamp flame, and the bacteria were coated on the culture medium of the Petri dish by the scribing method. Specific ways; Using aseptic inoculation ring, take a proper amount of bacteria and smear them on four points on the edge of the culture dish, and label the bacteria from each point to 1/2 of the culture dish. Then, find the second dotted line to 1/2 of the plate, and draw lines in turn until the bacteria are evenly distributed on the plate. (In addition, the bacteria to be tested can be selected, made into bacterial suspension in a small amount of physiological saline, and coated on the surface of the flat culture medium with sterile cotton swabs. A uniform and dense coat is required)

Stop tweezers after alcohol lamp flame sterilization, and take drug sensitive tablets and stick them on the surface of plate culture medium. In order to make the drug sensitive tablets stick to the culture medium tightly, you can gently press the drug sensitive tablets with tweezers. In order to accurately observe the results, it is required that the drug sensitive tablets can be regularly distributed on the plate culture medium; Generally, you can stick one piece in the center of the dish and several pieces at the periphery at the same distance (seven pieces can be attached to the periphery). Remember the name of each drug sensitive tablet.

Petri dish culture medium was cultured in an incubator at 37℃ for 24 hours, and the effect was observed.

3.3.2 Oxford Cup In the "ultra-clean table", a proper amount of bacterial culture is selected from the inoculation ring sterilized by flame (alcohol lamp), and the bacteria are coated on the plate culture medium by the scribing method. Specific ways; Using aseptic inoculation ring, take a proper amount of bacteria and smear them on four points on the edge of the culture dish, and label the bacteria from each point to 1/2 of the culture dish. Then, find the second dotted line to 1/2 of the plate, and draw lines in turn until the bacteria are evenly distributed on the plate. (In addition, the bacteria to be tested can be selected, made into bacterial suspension in a small amount of physiological saline, and coated on the surface of the flat culture medium with sterile cotton swabs. The coating is required to be uniform and dense. )

Sterilized stainless steel tubules (circular tubules with an inner diameter of 6nm and an outer diameter of 8nm and a height of 10nm, both ends of which should be smooth, and glass tubes and porcelain tubes can also be used) are placed on the culture medium, and gently pressed to make them contact with the culture medium without gaps, and the names of various drugs are marked on the tubules. Each plate can accommodate 46 small tubes. After equal minutes, drop a certain amount of various liquid medicines into each small test tube to prevent overflow. Culture at 37℃ for 8 18 hours, and observe the results.

Petri dish culture medium was cultured in an incubator at 37℃ for 24 hours, and the effect was observed.

3.3.3 Punching Method This method is simple, low cost and easy to operate, and is suitable for the detection of commercial drugs.

In the "ultra-clean table", a proper amount of bacterial culture is picked from the inoculation ring sterilized by flame (alcohol lamp), and the bacteria are streaked and coated on the culture medium of the Petri dish. Specific ways; Using aseptic inoculation ring, take a proper amount of bacteria and smear them on four points on the edge of the culture dish, and label the bacteria from each point to 1/2 of the culture dish. Then, find the second dotted line to 1/2 of the plate, and draw lines in turn until the bacteria are evenly distributed on the plate. (In addition, the bacteria to be tested can be selected, made into bacterial suspension in a small amount of physiological saline, and coated on the surface of the flat culture medium with sterile cotton swabs. The coating is required to be uniform and dense. )

Sterilized stainless steel tubes (4 mm in outer diameter, 3 mm in hole diameter and hole spacing, smooth at both ends of the tubes, and glass tubes and porcelain tubes are also acceptable) are punched on the culture medium, and the culture medium in the holes is picked out with a needle and sealed with a flame, so that the culture medium and the flat plate can be fully integrated (to prevent the leakage of liquid medicine and affect the results).

Sample addition: add samples according to different liquid medicines until the samples are full and do not overflow.

Petri dish culture medium was cultured in an incubator at 37℃ for 24 hours, and the effect was observed.

3.4 Results On the agar plate coated with bacteria, antibacterial drugs spread around the agar, and its concentration gradually decreased, so the growth of bacteria was inhibited within a certain distance around the paper. After overnight culture, a bacteriostatic circle was formed. The larger the inhibition zone, the greater the sensitivity of the strain to this drug; Otherwise, the smaller. If there is no bacteriostatic circle, it means that the strain is resistant to this drug. Its diameter is directly related to drug concentration and streak bacteria concentration.

3.5 To judge the results of standard drug sensitivity test, the diameter of bacteriostatic circle should be used as the standard to judge the sensitivity.

Table 1 drug sensitivity test standard

Sensitivity of inhibition zone diameter (mm)

Over 20, extremely sensitive

15~20 high sensitivity

10~ 14 Zhong Min

Low sensitivity below 10

0 is insensitive

Reference table of drug sensitivity test standards

1, polymyxin inhibition zone; More than 9 mm is highly sensitive, 6-9 mm is low sensitive, and no bacteriostatic zone is insensitive.

3.6 factors affecting drug sensitivity 3.6. 1 culture medium should be prepared according to the nutritional needs of test bacteria. When pouring plates, the thickness should be appropriate (about 56mm), not too thin. Generally, it is appropriate to pour 1820ml culture medium into a Petri dish with a diameter of 90 mm. The antagonistic substances of antibacterial drugs, such as calcium and magnesium ions, should be avoided in the culture medium, which can reduce the antibacterial activity of aminoglycosides, while thymine and p-aminobenzoic acid (PABA) can antagonize the activities of sulfonamides and TMP.

3.6.2 Inoculation amount of bacteria should be constant; if it is too much, the bacteriostatic circle will become smaller, and strains that can produce enzymes will destroy the antibacterial activity of drugs.

3.6.3 Drug concentration The concentration and total amount of drugs directly affect the results of bacteriostatic test, so it needs to be prepared accurately. Commercially available drugs should be prepared in strict accordance with their recommended therapeutic doses.

3.6.4 The general culture time is 37℃ 865438 08 hours. Some antibacterial drugs spread slowly as polymyxin. The flat culture medium containing antibacterial drugs can be put in the refrigerator at 4℃ for 2~4 hours to make the antibacterial drugs pre-diffuse, and then put in the incubator at 37℃ for culture, which can delay the growth of bacteria and get a larger bacteriostatic circle.

Application of drug sensitivity test