Several methods for determining the minimum inhibitory concentration (MIC) of antibacterial drugs
1.1. Constant broth dilution method
1.1.1. Preparation of antibacterial drug storage solution The concentration of antibacterial drug storage solution should not be lower than 1000μg / ml (such as 1280μg / ml) or 10 times the highest measured concentration. Antibacterial drugs with low solubility can be slightly lower than the above concentration. Antibacterial drugs were purchased directly from manufacturers or related institutions. The required amount of antibacterial drug solution or powder dose can be calculated by formula. For example, it is necessary to prepare 100 ml of antibiotic stock solution with a concentration of 1280 μg / ml. The antibiotic used is a powder, and the effective force of the drug is 750 μg / mg. Use an analytical balance to accurately weigh 182.6 mg of antibiotic powder. Calculate the required amount of diluent according to the formula: (182.6 mg × 750μg / ml) /1280μg/ml=107.0ml, then dissolve 182.6 mg antibiotic powder in 107.0ml diluent. The solvents and diluents used in the preparation of antibacterial drug storage solutions are shown in Table 5. The prepared antibacterial drug storage solution should be stored in the environment below -60 ℃, and the storage period should not exceed 6 months.
1.1.2. The concentration range of antimicrobial drugs used in drug susceptibility testing According to the operating standards of the NCCLS antimicrobial sensitivity test, the drug concentration range should include resistance, intermediary and sensitivity cut-off values, with the exception of special circumstances.
1.1.3. The medium NCCLS recommends the use of Mueller-Hinton (MH) broth, pH 7.2 ~ 7.4. Aerobic bacteria and facultative anaerobic bacteria grow well in this medium. When testing the susceptibility of staphylococcus to oxacillin, 2% (W / V) sodium chloride should be added to the broth to prepare the required amount of MH broth according to the manufacturer's requirements. HTM broth is used for Haemophilus bacteria, and MH broth containing 2% to 5% dissolved horse blood is used for Streptococcus pneumoniae and other streptococci.
1.1.4. Preparation of the inoculum There are two methods to prepare the inoculum. One is the bacterial growth method. Use the inoculating ring to pick up 3-5 colonies with similar morphology and inoculate 4-5ml of hydrolyzed casein (MH) meat. In the soup, incubate at 35 ℃ for 2-6h. After enrichment, the logarithmic growth phase bacterial solution was adjusted to 0.5 Mie turbidimetric standard with physiological saline or MH broth, containing about 1 ~ 2 × 108CFU / ml. The second is the direct colony suspension preparation method. For some fastidious bacteria, such as Haemophilus influenzae, Neisseria gonorrhoeae and Streptococcus and methicillin-resistant Staphylococcus strains, it is recommended to directly take colonies for 18-24 hours Prepare a bacterial suspension with a standard of 0.5 Mie turbidity. The above bacterial suspension was diluted 1: 100 with MH broth and set aside. Note that the prepared inoculum should be inoculated within 15 minutes, and a portion of the inoculum should be taken on non-selective agar plates for subculture to check the purity of the inoculum.
1.1.5. Preparation of diluted antibacterial drugs and inoculation of bacterial solution Take 13 sterile test tubes (13 × 100mm) in a row, except for the first tube to add 1.6ml of MH broth, the other tube to add 1ml of MH broth, Add 0.4ml of antibacterial drug stock solution (such as 1280μg / ml) to the first tube and mix it, then draw 1ml to the second tube, and then mix and then draw 1ml to the third tube, so as to continuously dilute to the 11th tube, and from Pipette 1ml into the 11th tube and discard it. The 12th tube is the growth control without drug. At this time, the drug concentration in each tube was 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg / ml. Then add 1ml each of the above prepared inoculum to each tube, so that the final bacterial concentration of each tube is about 5 × 105CFU / ml. The drug concentrations in the first to eleventh tubes were 128, 64, 32, 16, 8, 4, 2, 1, 05, 0.25, and 0.125 μg / ml, respectively.
1.1.6. Incubation Plug the inoculated dilution tube into a stopper and incubate in a 35 ° C ordinary air incubator for 16-20 hours; Haemophilus and Streptococcus in an ordinary air incubator for 20-24 hours; for possible nail resistance Oxycillin staphylococcus and vancomycin-resistant enterococci should be incubated continuously for 24h.
1.1.7. Judgment and interpretation of results Before reading and reporting the MIC of the tested strain, the bacterial growth of the growth control tube should be checked for good health, and the subculture of the inoculum should also be checked to determine whether it is contaminated, quality control Whether the strain's MIC value is within the quality control range. Observed with the naked eye, the lowest concentration of the drug without bacterial growth is the MIC of the test bacteria. The end point of broth dilution method for trimethoprim or sulfa drugs is 80% of the bacterial growth tube drug concentration that is 80% less than the positive growth control tube.
According to the cut-off point standard recommended by NCCLS, determine resistance (R), susceptible (S) or intermediate (I). S indicates that the infection caused by the tested strain can be effectively treated with the usual dose of the antibacterial drug, except for contraindications. R means that the bacteria cannot be inhibited by the concentration of the common dose of antibacterial drugs in tissue fluid or blood, or belong to a specific drug resistance mechanism (such as β-lactamase), so the clinical treatment effect is not good. I refers to the blood or tissue fluid concentration of MIC close to the drug, and the curative effect is lower than that of sensitive bacteria. It also means that the tested strain can be suppressed by increasing the dose (such as β-lactam drugs), or at the site where the drug is physiologically concentrated (such as urine). In addition, the intermediary also acts as a "buffer domain" to prevent large technical factors from getting out of control and causing a large misinterpretation.
1.2. Micro broth dilution method
1.2.1. Preparation of antimicrobial drugs and culture medium with the same method of broth dilution
1.2.2. Prepare the MIC plate for aseptic operation, add the antibacterial drug solutions of different concentrations after multiple dilutions to the sterilized 96-well polystyrene plates, add the drug solution from the 1st to the 11th well, 10μl per well, No drug was added as the growth control in the 12th well. After freeze-drying, it was sealed, and stored below -20 ℃ until use.
1.2.3. Preparation of inoculum The bacterial suspension prepared by the growth method or the direct bacterial suspension method with a concentration equivalent to 0.5 McLaren's turbidity standard is diluted with MH broth 1: 1000, and 100 μl is added to each well. After sealing, place it in a 35 ℃ common air incubator and incubate for 16 ~ 20h to judge the result. When testing for Haemophilus and Streptococcus, the incubation time is 20 to 24 hours, and the incubation time for the susceptibility test of staphylococcus and enterococci to oxacillin and vancomycin must be 24 hours. At this time, the drug concentrations in the first to eleventh wells were 128, 64, 32, 16, 8, 4, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml, respectively.
1.2.4. As a result, the minimum drug concentration that completely inhibits the growth of bacteria in the small hole is MIC. The test of significant bacterial growth in the positive control well (ie without antibiotics) is meaningful. When a single jumping hole appears in the micro broth dilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If there are multiple holes, the results should not be reported and the test needs to be repeated. Generally for Gram-negative bacilli, the MIC measured by the micro-broth dilution method is the same as or lower by one dilution (1 well or 2 times) as measured by the macro broth dilution method.
2. Agar dilution method Agar dilution method is to add different doses of antibacterial drugs to quantitative MH agar melted and cooled to about 50 ° C to prepare plates containing different decreasing concentrations of antibacterial drugs, inoculate test bacteria, and observe bacteria after incubation For the growth situation, the minimum drug concentration in the agar plate that inhibits bacterial growth is MIC. The advantage of this method is that the MIC determination of multiple strains of bacteria can be performed on a single plate at the same time, the results are reliable, and it is easy to find contaminated bacteria; the disadvantage is that it takes time and effort to prepare a drug-containing agar plate.
2.1. The medium is prepared using MH agar, which is prepared according to the product instructions, pH 7.2 ~ 7.4. Neisseria gonorrhoeae uses GC agar base plus 1% additive; other streptococci use MH agar containing 5% (V / V) sheep blood (when testing sulfonamides, use dissolved horse blood).
2.2. Preparation of drug-containing agar plates According to the experimental design, the antibacterial drugs with different concentrations that have been diluted multiple times are added to MH agar that has been heated and dissolved, and equilibrated in a 45 ~ 50 ℃ water bath. The thickness of agar is 3 ~ 4mm. The drug agar plate is usually prepared in a ratio of 1: 9, and the drug concentration range is selected according to needs. The prepared drug-containing agar plate should be packed in a sealed plastic bag and stored in a refrigerator at 2-8 ° C for 5 days.
2.3. Preparation of inoculum and inoculation prepared a bacterial suspension with a concentration equal to 0.5 McLay standard turbidity tube, and then diluted 1:10, sucked the prepared bacterial solution (about 1 ~ 2μl) on the surface of the agar plate with a multi-point inoculator The number of bacteria per point is about 104CFU, forming plaques with a diameter of 5 ~ 8mm. After inoculation, incubate at 35 ℃ for 16 ~ 20h (methicillin-resistant staphylococcus and vancomycin-resistant enterococci should be incubated for 24h), and observe the results. Neisseria and Streptococcus bacteria were incubated in 5% carbon dioxide and Helicobacter pylori in a microaerobic environment.
2.4. Judgment of Results Place the plate on the surface of a dark, non-reflective object to judge the end point of the test, and the minimum drug concentration to inhibit the growth of bacteria is MIC. Slight bacterial growth can be seen on agar plates containing trimethoprim or sulfonamide, and the lowest drug concentration that inhibits bacterial growth by more than 80% compared with the growth control is taken as the end point concentration.
If there are more than two colonies growing on agar plates with drug concentrations higher than the end point level, or if the low concentration drug agar plates are not long and the high concentration drug agar plates are growing, the culture purity should be checked or the test repeated.
3. E-test (E-test) E-test refers to the concentration gradient agar diffusion test, the principle is basically the same as the diffusion method, that is, the concentration of a continuous gradient of antimicrobial drug diffuses from the plastic strip to the agar, and inhibits bacteria around the strip The growth of the tested bacteria in the concentration range is inhibited, thereby forming a transparent bacteriostatic circle. The E test combines the principles and characteristics of the dilution method and the diffusion method, and also makes up for some of the deficiencies of the two. The MIC of the antibacterial drug against the test bacteria can be directly and quantitatively measured like the dilution method.
3.1. Preparation and inoculation of culture medium and bacterial solution are the same as the paper diffusion method.
3.2. The E test strip is attached to the paper diffusion method. The scale of the E test strip is facing upwards, and it cannot be reversed. Once it touches the agar, it cannot be moved again. Six E test strips can be placed in a dish with a diameter of 150 mm, and only one can be placed for 90 mm.
3.3. The incubation time and temperature are the same as the paper diffusion method.
3.4. Results After reading and incubation, an oval bacteriostatic circle can be formed around the test strip. The reading scale on the test strip at the intersection of the bacteriostatic circle and the test strip is the MIC of the antibacterial drug against the test bacteria. Please refer to the supplier's product manual for issues that should be noted when reading.
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