Determination of Antibiotic Potency by Tube and Disk Method

Determination of Antibiotic Potency by Tube and Disk Method

[Purpose and Requirements]
1. Understand the principle of the tube-dish method.
2. Master the technique and calculation method of the two-dose method to determine the antibiotic titer unit.
[Principle]
The bioassay of antibiotics is based on the antibacterial efficacy of antibiotics on microorganisms as a measure of potency, which has the advantages of being consistent with the application principle, less dosage and high sensitivity. The tube-dish method is one of the agar diffusion methods, which has been widely adopted by the pharmacopoeia of various countries as a legal antibiotic bioassay method.
When antibiotics are diffused in the bacterial layer medium, a natural gradient of antibiotic concentration from high to low will be formed, that is, the concentration of the diffusion center is high and the concentration of the edge is low. Therefore, when the antibiotic concentration reaches or is higher than the MIC (minimum inhibitory concentration), the test bacteria are inhibited and cannot reproduce, thus showing a transparent inhibition zone. According to the derivation of the law of diffusion, the logarithm of the total amount of antibiotics has a linear relationship with the square of the diameter of the inhibition zone.
[Experimental Materials]
1. Standards and samples of tetracycline antibiotics.
2. Test bacteria: Sarcina lutea.
3. Culture medium: 150ml LB agar in a 250ml conical flask, 50ml LB agar in a 150ml conical flask, and 20ml in a 50ml conical flask with a medium for S. luteus suspension.
4. Sterilized items: Petri dish, Oxford cup (inner diameter: 6.0±0.1mm, outer diameter: 7.8±0.1mm, height: 10.0±0.1mm), dropper, 1ml pipette, pH6.0 phosphate buffer, bend Tip tweezers, 15×150mm test tube, tile lid.
5. Others: Analytical balance, volumetric flask, glass plate, spirit level, vernier caliper.
[Experiment implementation]
1. Prepare the standard solution
2. Prepare the sample solution
3. Prepare the bacterial suspension.
4. Prepare the plate:
(1) Take 6 sets of sterile petri dishes, add about 20ml of LB medium respectively, and set them on a horizontal glass plate to solidify as the bottom layer.
(2) Take another 50ml of LB medium cooled to about 50°C, add 1ml of the test bacteria suspension, shake it up quickly, use a 10ml broken pipette to take 6ml of the mixed culture medium and pour it on the bottom plate as a bacterial layer. Place on a horizontal glass plate to solidify.
5. Mark the bottom of the medium plate accordingly and place 4 Oxford cups equidistant from each Petri dish using angled tweezers.
6. Use a dropper to add the high and low concentration standard and sample solutions into the Oxford cup respectively, and replace the lids when the cup is full.
7. Put the petri dish into the incubator smoothly and incubate at 37℃ for 16-18h.
8. Measure the diameter of the inhibition zone with a vernier caliper.

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