The secondary metabolites produced by certain microorganisms during metabolism can inhibit or kill certain microorganisms at low concentrations, and such substances are called antibiotics. There are three major types of bioassays for antibiotic potency: dilution, turbidimetric, and diffusion. Tube-dish method is one of the diffusion methods, because this method is a direct measurement method using antibiotics to inhibit sensitive bacteria, in line with the actual situation of clinical use, and the sensitivity is very high, do not need special equipment, so it is mostly used. There are many types of antibiotics. In this experiment, the penicillin produced by Penicillium chrysogenum is used as an example to determine its potency. Content from Microbiology Laboratory, Third Edition
Operation method
master disk method (math.)
Principle
The tube-dish method is one of the diffusion methods, in which a standard antibiotic solution of known concentration and a sample solution of unknown concentration are added to a standard stainless steel tube (i.e., Oxford cuplet) and diffusion penetration is performed on the surface of agar containing sensitive test bacteria. The inhibitory effect of the two on the test bacteria is compared and the size of the circle of inhibition is measured to calculate the concentration of the antibiotic. Within a certain concentration range, the concentration of the antibiotic and the diameter of the circle of inhibition on a bi-weekly semi-logarithmic scale (concentration is the logarithmic value, the diameter of the circle of inhibition is the numerical value) become a linear function of the concentration of the antibiotic and the diameter of the circle of inhibition in a standard curve can be derived from the samples of the circle of inhibition of the potency of the standard curve.
Materials and Instruments
Staphylococcus aureus Penicillium chrysogenum Move 1. Preparation of 0.2 mol/L pH 6.0 phosphate buffer solution Accurately weigh 0.8 g of KH2PO4 and 0.2 g of K2HPO4, put them into a 100 ml volumetric flask, dilute them to the scale with distilled water and sterilize them. 2. Preparation of standard penicillin solution Accurately weigh 15-20 mg of benzylpenicillin standard, each mg contains 1667 units (1,667 U/mg, 1 U is 1 international unit, equal to 0.6 μg), dissolve in 0.2 mol/L pH 6.0 phosphate buffer, and then dilute into 10 U/ml penicillin standard solution, and then formulate into penicillin solutions with different concentrations according to Table IX-2, and keep it at 5 ℃. Keep it at 5℃. 3. Preparation of penicillin fermentation broth sample solution Dilute the penicillin fermentation solution with 0.2 mol/L pH 6.0 phosphate buffer, and set aside. 4. Preparation of Staphylococcus aureus bacterial solution Take Staphylococcus aureus strain preserved in the slant of medium I, inoculate it on the slant of medium II, incubate it at 37℃ for 18~20 h, and pass it on for 3~4 times consecutively, then wash it down with 0.85% of saline, and then centrifuge it, and wash the bacterium with saline for 1~2 times, and then dilute it to a certain concentration (about 109 / ml, or measure it with a photoelectrocolorimeter at the wavelength of 650 nm; The transmittance of light is about 20%.) 5. Preparation of antibiotic diffusion plates Take 18 sterilized petri dishes. Add 20 ml of melted medium I, shake well and let it solidify horizontally as the bottom layer. Take medium II melted and cooled to 48~50 ℃, add appropriate amount of the above Staphylococcus aureus bacterial liquid, shake well quickly, add 5 ml of this bacterial medium into each plate, make it evenly distributed on the bottom layer, set it in the horizontal position to solidify, and then place 6 oxford cups in each double-layer plate at equal distance evenly, covered by ceramic tiles for spare use. 6. Preparation of standard curve Take 18 diffusion plates prepared above, add 1 U/ml of standard solution into 3 of the 6 Oxford cups spaced apart on each plate, and form a group of every 3 plates into 6 groups. In the first group of each plate of three empty Oxford cups are added to the standard solution of 0.4 U/ml, so that the six different concentrations of the standard solution were added to the 6 groups of plates (Figure IX-6), each dilution should be replaced with a pipette, each Oxford cup in the addition of 0.2 ml or a dropper with a dropping tip to add samples, the amount of sample added to the level of the mouth of the cup shall prevail, and all covered with a ceramic tile cover 37 ℃ incubated 16~18 h. The sample was then incubated for 16~18 hours. After all the samples were covered with ceramic tile lid, incubate at 37 ℃ for 16~18 h. Measure the diameter of each circle of inhibition accurately, and obtain the average of the diameter of the circle of inhibition of 1 U/ml standard and the diameter of the circle of inhibition of other standards in each group of 3 plates, and then find out the average of the diameter of the circle of inhibition of 10 U/ml standard in the 6 groups, and the difference between the total average value and the average value of the diameter of the circle of inhibition of 10 U/ml standard in each group, that is, the calibration value of each group. For example, if the total mean value of the circle of inhibition diameter of the 6 groups of 1 U/ml standards is 22.6 mm, and the mean value of the circle of inhibition diameter of the 9 1 U/ml standards in the group of 0.4 U/ml is 22.4 mm, the correction should be 22.6-22.4=0.2, if the mean value of the circle of inhibition diameter of the 9 0.4 U/ml standards is 18.6 mm, then the correction should be 18.6+0.2=18.8 mm, and the correction should be 18.6+0.2=18.8 mm. 0.2=18.8 mm, take the concentration as the vertical coordinate, take the corrected diameter of the circle of inhibition as the horizontal coordinate, and plot the standard curve on a bi-weekly semi-logarithmic graph paper. 7. Determination of potency of penicillin fermentation solution Take 3 diffusion plates, add 1 U/ml of standard solution into 3 of the 6 Oxford cups spaced on each plate, and add appropriately diluted sample fermentation broth into each of the other 3 cups, and incubate for 16~18 h after covering with ceramic tile lid. The diameter of each ring of inhibition was measured precisely, and the average of the 9 diameters of the rings of inhibition caused by the standard solution and the sample solution were calculated respectively. The average of the diameters of the rings of inhibition of the sample solution was corrected by obtaining the calibration number according to the method of the standard curve mentioned above, and then the potency of the standard solution was calculated from the standard curve and converted into the number of units per ml of the sample. Caveat Be careful to control the concentration of S. aureus bacterial solution so that it does not affect the size of the inhibition circle. In general, 100 ml of medium II with 3~4 ml of bacterial solution (10~10 ml) is used to control the concentration of Staphylococcus aureus.9In general, it is better to add 3-4 ml of bacterial solution (10 9 / ml) to 100 ml of medium II. For more product details, please visit Aladdin Scientific website.
0.85% sterile saline 50% sterile dextrose Penicillin Sodium Salt Standard
Medium I (Beef Peptone Agar Medium) Medium II (Medium I with 0.5% glucose) Plates Oxford Cups/Standard Stainless Steel Tubes Tile Lids