Comparison of four different methods of smear preparation for Gram staining of positively flagged automated blood culture bottles

Introduction: Gram staining of smears prepared from positively flagged blood culture bottles plays a vital role because its accurate interpretation helps the clinicians to modify empirical therapy as well as to perform the direct susceptibility test, which reduces the turnaround time by 24 h to initiate pathogen-directed antimicrobial therapy. However, the interpretation of direct Gram stain from blood culture bottles has always been at risk of errors in interpreting. Aim: The present study was conducted to find the agreement between the blood culture broth Gram stain finding of four different methods (conventional, water wash, blood film, and drop and rest) of smear preparation with the culture smear Gram stain result and also to compare the quantity of charcoal and resin particles present in the Gram stained smears made from four different smear preparation methods. Method and Result: Smears were made from 500 positively flagged automated blood culture bottles using the four different methods and then compared for agreement with the culture smear Gram stain using kappa statistics and were found to be maximum for the blood film method (0.637), followed by drop and rest (0.570), water-wash (0.496), and conventional method (0.475). The 3+ grading for resin/charcoal was found in 184 (36.80%) smears, 169 (33.80%), 93 (18.60%), and 26 (5.20%) smears prepared by water-wash, conventional, drop and rest, and blood film method, respectively. The interference caused by resin/charcoal was maximum when smears were prepared by the conventional method resulting in high inaccurate interpretation. Conclusion: In this study, the agreement between direct smear preparation methods from positively flagged blood culture broth with the culture smear Gram stain was found to be maximum for the blood film method, followed by drop and rest, waterwash, and conventional method.It was also observed that the least amount of resin/charcoal was encountered when the direct smears were prepared using the blood film method.Hence, we found that the blood film method helps in interpreting the Gram stained smears from positively flagged blood culture broth more accurately compared to other methods