Klebsiella pneumoniae serotypes 21 and 24 and Enterobacter cloacae were responsible for an outbreak of polymicrobial bacteremia associated with the receipt of lipid emulsion. Since it is recommended that lipid emulsion be kept refrigerated between uses, we undertook a study to determine the growth characteristics of these organisms in lipid emulsion at 5 and 25 degrees C and to examine the use of alternative measurements (pH and endotoxin) to determine contamination by viable and nonviable microorganisms. The bacteria survived but did not proliferate at 5 degrees C; no endotoxin was detected, and the pH remained unchanged. In contrast, after a 2-h lag phase, all three organisms proliferated rapidly when incubated at 25 degrees C and reached concentrations of greater than or equal to 10(7) CFU/ml at 24 h. A decrease in pH was detected after proliferation to 10(7) CFU/ml. Endotoxin was detected after proliferation reached 10(2) CFU/ml. The amount of endotoxin elaborated by the three organisms differed and ranged from 0.013 ng per 8 X 10(2) CFU/ml to 1.3 ng per 2 X 10(3) CFU/ml at 8 h. Our findings show that these microorganisms do not proliferate at refrigerator temperature in lipid emulsion, but can reach significant levels at room temperature. It is, therefore, important to keep lipid emulsion refrigerated between uses. Furthermore, when lipid emulsion contamination is suspected, endotoxin and pH determinations should be considered as possible adjunctive tests while results of bacterial cultures are pending. The results of the present study are applicable to only selected gram-negative bacteria and may not apply to gram-positive bacteria and fungi. However, these data demonstrate that measurement of pH and detection of endotoxin is quite useful when lipid emulsion contamination occurs with selected gram-negative bacteria.