Table 1.

Antimicrobial Synergy Assay Protocol

Step	Plate	Parameter	Value	Description
1	Antibiotic	Dispense CAMHB	35 μL	Into each well of rows B–P
2	Antibiotic	Antibiotic addition; twofold, horizontal serial dilution	70 μL of highest (antibiotic)	Into each well of row A
3	Assay	Dispense CAMHB	15 μL	Into each well of all columns except 3 and 13
4	Assay	Test BLI compound addition; twofold, vertical serial dilution	30 μL of highest (test BLI)	Into each well of columns 3 and 13
5	Antibiotic into assay	Transfer antibiotic dilutions into assay plates	15 μL into each well of the plate	Each well in the assay plate now contains a unique combination of Test BLI and antibiotic
6		Prepare bacteria to 1E+06 cfu/mL	10 mL of bacterial solution per (1/2) plate	Bacteria prepared externally in a polypropylene tube
7	Assay	Dispense bacteria into plate	30 μL in each well of columns 1–23	If testing two different bacteria on the same plate, place bacteria 1 in columns 1–12 and bacteria 2 in columns 13–23
8	Assay	Dispense highest (antibiotic) into background control column 24	30 μL into each well	Use the same antibiotic solution used in step 2
9	Assay	Incubate	37°C for 18–20 h	Incubate in ample humidification
10	Assay	Read	OD 600	Determine OD 600 using Tecan Safire II
11	Assay	Data analysis	Synergy RunTool	Determination of synergistic effect of antibiotic and test compound

Step Notes

1. Using the 384-well liquid handle (aka Multidrop) add media to Corning 384-well translucent plates for the purpose of antibiotic dilution.

2. Using the Beckman FX serial dilution technique perform serial twofold dilutions down the rows of the same plate used in step 1.

3. Using the 384-well liquid handle (aka Multidrop) add media to Corning 384-well translucent plates for the purpose of test inhibitor dilution.

4. Using the Beckman FX serial dilution technique perform serial twofold dilutions across the columns of the same plate used in step 3.

5. Transfer samples from the antibiotic source plate prepared in steps 1 and 2 into the assay plate prepared in steps 3 and 4.

6. Fresh bacteria is diluted in CAMHB.

7. Using the Multidrop transfer bacteria into appropriate wells; column 24 being the exception.

8. Prepare column 24 as “No Bacteria” control.

9. Plates are covered and placed at 37°C overnight in a humidified chamber.

10. Read the plates using ABS 590 modality.

11. Use the automated Synergy RunTool to import data, analyze, and archive.

 BLI, beta-lactamase inhibitor; CAMHB, cation adjusted Mueller–Hinton broth.