Measuring optical density (OD) using a spectrophotometer is often problematic if the physics behind the method is not understood. For example, the relationship is between biomass concentration and OD, not cell number and OD. This is because larger cells absorb and scatter more light. Furthermore, the relationship between OD and biomass concentration is not linear, it only approximates to linearity at low OD values (typically up to 0.3 - 0.4, depending on the spectrophotometer).
Most spectrophotometers do not give you an accurate reading above an OD600 of 0.9-1. So, you could prepare a dilution series of the microbial suspension in 10% steps and plot OD vs. % to determine the approximately linear range of the instrument. If the initial biomass concentration is too high to show the relationship with sufficient resolution, then you need to go down to e.g. 2.5, 5.0 and 7.5 %.
Other things to watch are:
the quality of the cuvette (plastic ones are quite variable if tested empty or with filtered broth or water, glass or quartz are better, especially if a matched pair are available);
the cleanliness of the cuvette (hold up to the light and make sure the optical faces are clean);
the absence of gas bubbles (if gas bubbles are attached to the inside of an optical face, gently tap the base of the cuvette on the bench to dislodge them - taking care not to spill any of the contents!
As with any technique, attention to detail and an understanding of the method are essential for obtaining reliable and reproducible results.
Souce: NovoPro 2018-02-25