OBJECTIVE To determine the risk of bacterial growth in single use 50% dextrose vials punctured multiple times and stored in various hospital environments. METHODS Three groups of three 50% dextrose vials were stored in our hospital intensive care unit at either ambient light or in a darkened drawer at room temperature or refrigerated at 4°C. One vial in each group was punctured either once, once weekly or once daily for 28 days and samples taken for bacterial culture every 7 days until completion of the project. A fourth group of three vials were inoculated with several species of bacteria and stored in our microbiology laboratory under the environmental conditions described above with cultures performed every 7 days for 28 days. In addition, the water activity of 50% dextrose was determined using commercial laboratory equipment. RESULTS Scant growth of Escherichia coli and Enterobacter agglomerans was detected in cultures performed on day 7, but not subsequent time points, from the inoculated refrigerated vials. The vial punctured once daily for 28 days and stored under refrigerated conditions showed growth of Bacillus subtilis on day 28. All remaining bottles had no bacterial growth at any time point or environmental condition. The water activity of 50% dextrose was 093 at 24°C and 092 at 4°C. CONCLUSIONS Bacterial growth in 50% dextrose vials was uncommon even when inoculated with pathogens. Bacterial growth only occurred in refrigerated storage conditions. The water activity of 50% dextrose is not low enough to inhibit all bacterial and fungal growth.