1. Add 20uL Opsonization Buffer (OB) to shaded wells 1

2. Add 30uL serum sample to duplicate wells 2

3. Transfer 10uL serum from H to G 3

4. Continue 1:3 dilutions up the plate to A Discard excess 10uL volume from A 4

5. 5 500 PFU Streptococci pneumoniae in 10uL OB added to all wells Orbital shaker 30 minutes at room temperature Prepare HL-60 cells differentiated HL-60 cells at 17 million cells per ml in OB diluted 4:5 with active baby rabbit complement Prepare control HL-60 cells differentiated HL-60 cells at 17 million cells per ml in OB diluted 4:5 with heat inactivated complement

6. Add 50uL HL-60 cells with heat inactivated complement to row 1 6

7. 7 Add 50uL HL-60 cells with active complement to rows 2-12

8. 8 Orbital shaker 37oC 45 minutes Transfer plate to wet ice to stop phagocytic activity Spot 10uL volumes to THB agar Overlay with TTC containing overlay agar Incubate 37oC overnight in CO2 incubator Count colonies