Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The quadruple checkerboard is a technique that studies all possible combinations that can be obtained by combining four drugs, and it is important to find a treatment for resistant microorganisms, mainly MDRs or also known as multi-drug resistant microorganisms. This technique is not labor intensive and the results can be obtained the following day or after the incubation period is over. In addition to this, it can study all possible combinations and not only specific and very limited combinations that can be obtained by combining these four drugs and all, all in one experiment.

In addition to this, this technique can be used in multiple disciplinaries and not only in microbiology and even in microbiology. It can study all possible combinations between different antimicrobial agents and not only antibacterial agents. In addition to the microbiology part, it can be used in order to study combinations between anti-cancer drugs, between plant extracts and drugs, and even between molecular effective molecular treatments panels.

Preparation place 4 96, well plates next to each other in order to form a square using a tape, tape their bottom together. Repeat this step in order to obtain four panels each containing four plates and name them a one, A two, A three, and a Four. Add 50 Microliters Mueller.

Hint and broth to the wells between column two and column 11 in the 1696, well plates of the four Panels add 200 microliters Mueller Hinton broth to the well H 12 serving as the negative control. Well in the 1696 well plates of the four panels add one 50 microliters Mueller Hinton broth to the wells A one and H one serving as positive control wells in the 1696 well plates of the four panels drug one cefotaxime serial dilution to a conical tube at 15 ml. Sterile distilled water.

Calculate the volume to be removed from the stock solution of the drug following the formula C one V one equals C two V two. Remove the calculated volume from the 15 ml sterile distilled water and add the same volume of the drug stock solution To the conical tube pipette 50 Microliters of the prepared drug solution into each well in column 11 and column 12, except for the well H 12, start the serial dilution by removing 50 microliters from column 11 and putting it into the corresponding wells in column 10, and then from 10 until reaching column two where the 50 microliters taken from column two will be discarded. Repeat the serial dilution for each of the 1696 well plates of the four panels.

Drug two amika serial dilution to a conical tube at 10 ml. Sterile distilled water. Calculate the volume to be removed from the stock solution of the drug.

Remove the calculated volume from the 10 ml sterile distilled water and add the same volume of the drug stock solution to The conical tube. Take a separate 96 well plate add a hundred microliters sterile Mueller Hinton broth to the wells between rose G and B.In this plate, add hundred microliters of the previously prepared drug. Two, solution to the wells Of row G.Dilute serially From row G to row B by taking hundred microliters from each well in RO G.And finally discarding the a hundred microliters from the wells of row B.Repeat these steps in order to prepare eight separate 96 well plates transfer of drug two to the four panels pipette 50 microliters of drug two from the wells between rows G and B into the corresponding wells in each plate in the four panels, one prepared 96 well plate contains a hundred microliters of drug two.

That is enough for two plates in one panel drug. Three live of loin addition to four different conical tubes at 14 ml sterile distilled water. Calculate the volume to be removed from the stock solution of the drug.

Remove the calculated volume from the 14 ml sterile distilled water from each tube and add the corresponding volume of the drug. After preparing the required concentrations of the third drug, take 50 microliters and add it to the corresponding wells between rows B and G and columns two and 12 in the corresponding plate. In each panel where C one corresponds to the four P one plates and the four panels C two corresponds to the four P two plates and the four panels C3 corresponds to the four P three plates and the four panels and C four corresponds to the four P four plates and the four panels drug four trimethoprim sulfamethoxazole addition to a conical tube add 14 ml.

Strive distilled water. Calculate the volume to be removed from the stock solution of the drug. Remove the calculated volume from the 14 ml sterile distilled water of each tube and add the required volume of the drug.

After preparing the required concentrations of the fourth drug, take 50 microliters and add it to the corresponding wells between rows B and G and columns two and 12. In the four plates in the corresponding panel where C one corresponds to panel one C two corresponds to panel two C3, corresponds to panel three and C four corresponds to panel four. Preparation and addition of bacterial inoculum equal I-E-S-B-L using a sterile loop transfer.

One colony of the bacterial isolate equals previously cultured on a plate into two ml sterile Mueller Hinton broth and vortex Check for the turbidity where it should be 0.5 McFarland using a density add A TML sterile Mueller Hinton broth to sterile urine cup add bacterial inoculum from the 0.5 McFarland inoculum to the urine cup following the formula C one V one equals C two V two where V one equals 800 microliters pipe pipette 50 microliters of the inoculum solution and to each well except H 12, which is the sterility control, Well incubate the panels at 37 degrees Celsius overnight. This figure represents panel A one. The color dwells are the wells that contain bacterial growth, and the black arrows represent the wells on the growth.

No growth interface in plate one, plate two and plate three. We can see that there is inhibition in row D containing one over eight MIC of Amikacin in addition to several sub mics of cefotaxime and Levofloxacin in addition to one over eight MIC of Trimethoprim sulfamethoxazole in plate four. We notice that no bacterial growth is seen in the quadrant containing the combination.

This is because in this plate we have the MIC of Levofloxacin that will inhibit totally the growth. Figure one B represents panel two. In this panel, we can see that inhibition of bacterial growth is also seen in RD, which contains sub ics of cefotaxime and Levofloxacin in addition to one over eight MIC of Amikacin and one over four MIC of trimethoprim sulfamethoxazole figure one C represents panel A three.

In this figure, we can see that the inhibition of bacterial growth is seen in row C and row D containing several sub mics of cefotaxime and Levofloxacin, and addition to half MIC of trimethoprim sulfamethoxazole, and one over four MIC of Amikacin in row C and one over two MIC of Amikacin in row D figure 1D represents panel A four. In this panel we have one MIC of Trimethoprim sulfamethoxazole in combination with several concentrations of Cefotaxime, amikacin, and Levofloxacin. In this figure, we can see that the bacterial growth is totally inhibited in the quadrant containing the combination where this inhibition is the effect of the one MIC of Trimethoprim sulfamethoxazole in sheet FIC, all in the Excel file.

The table will show the final sound FIC, with the interpretation of the obtained value. In conclusion, the Q checkerboard technique studies all possible combinations between four drugs in a single experiment. The results are obtained the following day and are interpreted in an easy and fast way using an Excel template.