This standard method is recognized by EPA, which is labeled Method 5210B in the Standard Methods for the Examination of Water and Wastewater. In order to obtain BOD, dissolved oxygen (DO) concentrations in a sample must be measured before and after the incubation period, and appropriately adjusted by the sample corresponding dilution factor. This analysis is performed using 300 mL incubation bottles in which buffered dilution water is dosed with seed microorganisms and stored for 5 days in the dark room at 20 °C to prevent DO production via photosynthesis. The bottles have traditionally been made of glass, which required cleaning and rinsing between samples. A SM 5210B approved, disposable, plastic BOD bottle is available which eliminates this step. In addition to the various dilutions of BOD samples, this procedure requires dilution water blanks, glucose glutamic acid (GGA) controls, and seed controls. The dilution water blank is used to confirm the quality of the dilution water that is used to dilute the other samples. This is necessary because impurities in the dilution water may cause significant alterations in the results. The GGA control is a standardized solution to determine the quality of the seed, where its recommended BOD concentration is 198 mg/L ± 30.5 mg/L. For measurement of carbonaceous BOD (cBOD), a nitrification inhibitor is added after the dilution water has been added to the sample. The inhibitor hinders the oxidation of ammonia nitrogen, which supplies the nitrogenous BOD (nBOD). When performing the BOD test, it is conventional practice to measure only cBOD because nitrogenous demand does not reflect the oxygen demand from organic matter.  This is because nBOD is generated by the breakdown of proteins, whereas cBOD is produced by the breakdown of organic molecules.
BOD is calculated by:
*Unseeded :
*Seeded: 
where:
: is the dissolved oxygen (DO) of the diluted solution after preparation (mg/L)
: is the DO of the diluted solution after 5 day incubation (mg/L)
: is the decimal dilution factor
: is the DO of diluted seed sample after preparation (mg/L)
: is the DO of diluted seed sample after 5 day incubation (mg/L)
: is the ratio of seed volume in dilution solution to seed volume in BOD test on seed