Large volumes of metalworking fluids (MWFs) are used in manufacturing industries for cooling and lubrication of metal pieces and tools during machining. MWFs accumulate microbial growth through continuous recirculation and reuse. We studied the progression of microbial contamination for 6 months after dumping, cleaning and recharging (DCR) of a large semi-synthetic MWF system managed with several biocides. Fresh, uncontaminated fluid was added to the system after extensive cleaning. The following samples were collected and analyzed: pre-DCR fluid (before system cleaning); neat fluid diluted to 6% with water; in use MWF 12 h and 1, 3 and 6 months post-DCR. Samples were analyzed for total microorganism concentrations by direct counting using fluorescence microscopy and by plate counting on various media (R2A, BHI, Middlebrooks and rose bengal under aerobic conditions). In addition, PCR was performed for the detection of mycobacteria. There was a rapid progression in the total bacterial counts as determined by fluorescence microscopy: 5.7 x 10(7) cells/ml in the pre-DCR used fluid, no measurable bacteria in the neat fluid, 6.9 x 10(6) cells/ml after 12 h and 2.2 x 10(6), 3.6 x 10(8) and 6.1 x 10(8) cells/ml after 1, 3 and 6 months, respectively. On average, only 0.2% of the direct count organisms were quantified on R2A cultures. PCR showed the presence of mycobacteria in the used MWF at 3 and 6 months. Mycobacteria were also identified from cultures on Middlebrooks and R2A. This study demonstrates that standard methods for cleaning MWF systems are inadequate since residual bacteria in the system can rapidly repopulate the newly charged MWF.