How to Dilution the Bacteria?

DILUTION of Bacteria

1. Add 1ml of bacterial suspension into 1.5 ml tube (2 tubes)
2. Centrifuge at 12.000 rpm, 3 minutes.
3. Decantation
4. Flash
5. Take the supernatant
6. Add 1 ml of 1× PBS
7. Suspend
8. Repeat step 2-5
9. Add 1 ml of 1× PBS
10. Measurement absorbance with Spectrophotometer
    • turn on the Spectrophotometer → bacterial density → ENTER → OD600 → NEXT
    • Blank: Add 1 ml of 1× PBS into a cuvette → BLANK
    • Add 1 ml of bacterial suspension into a cuvette → SAMPLE  (after step 11)
11. Transfer all the suspension into 15 ml tube (from 2 tubes)
12. Add (….) ml of PBS into 15 ml suspend tube.
    • ↑ depend on the measurement, change according to circumstance
13. Washing the cuvette
    • Add 1 ml of bacteria suspended into a cuvette
    • return into 15 ml tube
    • Add 1 ml of bacterial suspend into a cuvette
14. 1 ml of bacteria suspended in a cuvette → Spectrophotometer→ SAMPLE (step 10)
15. Repeat step 12-13 until OD600 1.009 ∼ 0.991