How to use Oxford cup for antibacterial test/drug sensitivity test (cup and saucer method)
There are two main methods of antibacterial/drug susceptibility testing, namely diffusion method and dilution method. Among them, the diffusion method is a manual test method. By testing the size of the inhibition zone of the drug paper or Oxford cup on the solid medium, it is judged whether the bacteria are sensitive to the drug. The dilution method includes the test tube dilution method and the micro-dilution method. The minimum inhibitory concentration (MIC) is judged by testing the growth of bacteria in the medium containing different concentrations of drugs.

Due to its simple operation and low cost, the diffusion method has been adopted by most laboratories and grassroots units. The diffusion method includes the paper method, the Oxford cup method and the punch method.

1. Experimental principle

1. Put the medium plate in the incubator. On the one hand, the test bacteria (indicator bacteria) begin to grow and multiply; on the other hand, the antibiotic spreads on a spherical surface and forms a decreasing gradient concentration. The closer you are to the cup, the greater the antibiotic concentration will be. The farther away the antibiotic concentration decreases.

2. The growth of bacteria within the inhibitory concentration range around the Oxford Cup is inhibited, forming a transparent inhibition zone.

3. The size of the inhibition zone reflects the sensitivity of the test bacteria to the tested drug (drug sensitivity test) or the degree of inhibition of the drug to the indicator bacteria (bacteriostatic test).

4. The higher the antibiotic concentration, the greater the inhibition zone.

5. At the edge of the inhibition zone, the concentration of the antibacterial substance in the agar medium is the minimum inhibitory concentration (MIC) of the indicator bacteria in the bacterial suspension.

6. The diameter of the zone of inhibition is negatively correlated with the minimum inhibitory concentration of the drug on the tested bacteria, that is, the larger the zone of inhibition, the smaller the MIC.
2. Experimental materials

Medium: Beef extract peptone solid medium

Strains: Escherichia coli (E. coli), Staphylococcus aureus (Staphylococcus aureus), Bacillus subtilis (Bacillus subtilis)

Drugs: Amoxicillin, Astragalus Polysaccharides,

Others: Oxford cup, petri dish, alcohol cotton, alcohol lamp, spreader, pipette, 1mL, 200uL , constant temperature incubator, ultra-clean workbench, inoculation loop

Three, experimental operation

1. Pour the plate: heat the sterilized agar medium until it melts completely, pour it into a petri dish, about 20ml per dish, and solidify.

2. Marking: strain, placement of Oxford cup, medicine and its concentration

3. Preparation of bacterial suspension: Wash the bacterial lawn in the test tube with normal saline and dilute it.

4. Bacterial liquid coating: suck 1mL of bacterial liquid into the surface of the plate, and spread the bacterial liquid evenly with a spreader

5. Place the Oxford cup: Place the Oxford cup vertically on the surface of the culture medium and gently pressurize it to make it contact with the culture medium without gaps.

6. Add the drug solution to be tested: add the drug solution of different dilutions or the sample to be tested into the cup.

7. Incubation: After topping up, incubate at 37 ℃ for 16-18 h.

8. Result report: Measure the diameter of the inhibition zone with a millimeter ruler, refer to the standard in the table to interpret the results, and report according to sensitivity (S), intermediate (I), and drug resistance (R)

The area around the Oxford Cup without visible growth is regarded as the inhibition zone, and the susceptibility of bacteria to antibacterial drugs is judged according to the diameter of the inhibition zone.

Bacteriostatic test result judgment standard: diameter of inhibition zone (mm)

＞20 extremely sensitive

15-20 high sensitivity

Sensitive in 10-14

＜10 low sensitivity

0 insensitive

Four, matters needing attention

1. Operate in the ultra-clean workbench or beside the alcohol lamp

2. Pour the plate well, it must be flat

3. Only when the Oxford cup is upright can the antibacterial substances in the cup spread to the surroundings evenly.

4. Place the Oxford cups evenly on the culture medium and arrange them properly to prevent overlapping of the suppression circles. One can be placed in the center of the plate and 5-6 at the outer periphery.

The factory sells Oxford cups directly. All Oxford cups are precision CNC automatic processing to ensure accuracy. It is not the kind of steel pipe cutting on the market. The 2 heads of the Oxford cup are flat and smooth. All four corners are CNC chamfered to ensure that each Oxford cup is poured. The angles are the same size.