Bioprocess Analytics — Estimating Biomass by Optical Density (OD): Which practice is correct to obtain reliable biomass concentration from spectrophotometric readings during fermentation?

Introduction:
Optical density (OD) at a wavelength such as 600 nm is a fast, non-destructive proxy for biomass concentration in fermentation. However, OD must be used carefully to stay within the spectrophotometer linear response range. This question checks the correct laboratory practice for trustworthy biomass estimates.

Given Data / Assumptions:

• Standard cuvette path length and properly zeroed instrument with blank medium.
• Cell suspensions exhibit scattering, and linearity breaks down at high turbidity.
• Goal is a quantitative estimate of biomass concentration from OD.

Concept / Approach:

Beer–Lambert law strictly applies to absorbing solutes at low concentrations. For cell suspensions, multiple scattering and shading cause nonlinearity at higher OD. Therefore, samples must be diluted so that measured OD falls in an empirically linear window, often OD600 less than about 0.3 to 0.4 for many instruments and organisms.

Step-by-Step Solution:

Verification / Alternative check:

Cross-check OD-based estimates with gravimetric dry cell weight or cell counting to confirm calibration. Consistency across dilutions indicates measurements are within the linear range.

Why Other Options Are Wrong:

B is incorrect because OD correlates with cell concentration, not surface area. C ignores the necessity of dilution to maintain linearity. D is circular and does not describe an actual measurement. E is false because cell suspensions deviate from ideal Beer–Lambert behavior at high turbidity.

Common Pitfalls:

Relying on undiluted readings at high OD, failing to mix after dilution, or using mismatched blanks can all distort biomass estimates.

Final Answer:

Dilute samples so that OD stays in the linear range (for example, OD600 less than about 0.3 to 0.4) before recording absorbance