1. Bring test reagents to room temperature (22-28 C) approximately 30 minutes prior to use.
2. Maintain proper incubation temperature:
Lower temperature can decrease OD values.
Higher temperatures can increase OD values.
Evaporation in wells can cause edging effect.
3. The optimal temperature for incubation is 22-28 C.
4. Check temperature against calibrated thermometer.
5. Strict adherence to time must be maintained.
6. Check calibration of timers.
7. Record time of incubation.
8. Read plate with specified time limits of adding stop solution.