This study compared the acridine orange and Gram stains of an external smear of intravascular catheter-tip segments with the culture results obtained 24 to 48 h later. Both staining techniques were randomly assigned to be performed either before (group A) or after (group B) rolling on the agar plate. Of the 425 catheter tips processed, 25.7% were significantly colonized and 6.1% were from patients with catheter-related bloodstream infections (CRBSIs). The yield of group A was superior to that of group B for the prediction of colonization (sensitivity, 94.3%/69.6%; specificity, 92.4%/96.2%; positive predictive value (PPV), 80.6%/86.7%; negative predictive value (NPV), 98.0%/90.0%; and full concordance, 91.9%/89.3%). The values for the prediction of CRBSIs were as follows for group A and group B: sensitivity, 100%/69.2%; specificity, 96.3%/100%; PPV, 86.7%/100.0%; NPV, 100%/93.9%; and full concordance, 97.0%/94.7%. Catheter-tip staining before culture is an easy-to-perform, cheap, and effective procedure to anticipate catheter colonization and rule out CRBSIs.