Sensitivity and Precision of Bioluminescent Techniques for Enumeration of Bacteria in Skim Milk

Sensitivity and precision of the ATP assay were improved when a chilled Enterobacter cloacae skim milk culture was activated at 30°C for 30 min and the ATP was extracted with boiling Tris/EDTA buffer (1:10 culture/buffer ratio). Of three luciferase-luciferin preparations evaluated, Monitoring reagent (a purified preparation) yielded the highest light output that reached a peak 60 s after injection of the sample and remained stable thereafter. Skim milk quenches the light output and necessitates the use of an internal ATP standard for the ATP assay. Skim milk samples containing between 12.5 and 200 pg of ATP were assayed with a mean precision of 3.1% (coefficient of variation; CV). Lower ATP concentrations could be determined with poorer precision. The lower limit of precise (7% CV) E. cloacae enumeration in skim milk was 1.6 × 10⁵/ml.