https://doi.org/10.4081/mm.2025.14213
Optimizing Gram stain interpretation: a comparison of four smear preparation methods for Gram staining of positively automated blood culture bottles
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Published: 2 March 2026
Background: rapid and accurate Gram stain interpretation of positively flagged blood culture broths is essential for early identification of bloodstream infections and timely initiation of empirical therapy. However, artifacts caused by resin and charcoal particles in culture media can compromise diagnostic clarity. This study evaluates and compares four smear preparation methods - conventional, water wash, blood film, and drop and rest - for optimizing Gram stain interpretation.
Methods: a prospective study was conducted on 100 positively flagged blood culture bottles. Each sample was used to prepare smears using all four techniques- conventional, water wash, blood film and drop & rest method. These were evaluated for agreement with the final culture-based Gram stain results. Interfering resin/charcoal particles were graded on a standardized scale. Kappa (κ) statistics were applied to assess concordance.
Results: among 100 samples, the blood film method showed the highest agreement with culture smears (63 samples, κ=0.26), followed by conventional (62, κ=0.24), drop and rest (61, κ=0.22), and water wash (59, κ=0.18). Gram-positive cocci in pairs and Gram-negative bacilli were most accurately detected using the blood film method (10 and 17 samples, respectively). Heavy resin/charcoal deposits were most common with water wash (41 samples) and least with blood film (6 samples). The blood film method also produced the highest number of deposit-free samples (29), indicating superior clarity.
Conclusions: among the four smear preparation methods studied across 100 positive blood culture samples, the blood film method demonstrated the highest diagnostic agreement and the least interference from resin and charcoal particles. It is recommended as the most effective and reliable technique for direct Gram staining in clinical microbiology laboratories.
Downloads
1. Adler H, Baumlin N, Frei R. Evaluation of acridine orange staining as a replacement of subcultures for BacT/ALERT-positive, gram stain-negative blood cultures. J Clin Microbiol 2003;41:5238-9. DOI: https://doi.org/10.1128/JCM.41.11.5238-5239.2003
2. BACT/ALERT® FAN® PLUS Gram Stain; 2018. Available from: https://www.biomerieuxuniversity.com/ bact alert fan plus gram stain.html.
3. Behera B, Mathur P, Gupta B, et al. Blood culture Gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma. Indian J Med Microbiol 2010;28:138-42. DOI: https://doi.org/10.4103/0255-0857.62491
4. BMS Diagnostics. BD BACTEC™ FX: State-of-the-art blood culture system. Available from: http://www.bmsd.com.my/files/editor_files/file/2_February_2013.pdf
5. Fiori B, D’Inzeo T, Di Florio V, et al. Performance of two resin containing blood culture media in detection of bloodstream infections and in direct matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) broth assays for isolate identification: Clinical comparison of the BacT/Alert plus and Bactec plus systems. J Clin Microbiol 2014;52:3558-67. DOI: https://doi.org/10.1128/JCM.01171-14
6. Kristóf K, Pongrácz J. Interpretation of Blood Microbiology Results - Function of the Clinical Microbiologist. EJIFCC 2016;27:147-55.
7. Mackie TJ, McCartney JE. Mackie & McCartney Practical Medical Microbiology. 14th ed. Churchill Livingstone; New York, USA; 1996. Available from: https://archive.org/details/Mackie14Ed
8. Nain J, Deepashree R, Tamang P, et al. Comparison of four different methods of smear preparation for Gram staining of positively flagged automated blood culture bottles. J Curr Res Sci Med 2018;4:98-103.
9. Rand KH, Tillan M. Errors in interpretation of Gram stains from positive blood cultures. Am J Clin Pathol 2006;126:686-90. DOI: https://doi.org/10.1309/V4KE2FPM5T8V4552
10. Samuel LP, Balada-Llasat JM, Harrington A, Cavagnolo R. Multicenter Assessment of Gram Stain Error Rates. J Clin Microbiol 2016;54:1442-7. DOI: https://doi.org/10.1128/JCM.03066-15
11. Søgaard M, Nørgaard M, Schønheyder HC. First notification of positive blood cultures and the high accuracy of the gram stain report. J Clin Microbiol 2007;45:1113-7. DOI: https://doi.org/10.1128/JCM.02523-06
12. Tille P. Bailey & Scott’s Diagnostic Microbiology. 13rd ed. Elsevier Health; St. Louis Missouri, USA; 2013.
13. Uehara Y, Yagoshi M, Tanimichi Y, et al. Impact of reporting Gram stain results from blood culture bottles on the selection of antimicrobial agents. Am J Clin Pathol 2009;132:18-25. DOI: https://doi.org/10.1309/AJCP0H2DAMBXZUSS
14. Winn WC, Koneman EW. Koneman’sColor Atlas and Textbook of Diagnostic Microbiology. 6th ed. Lippincott Williams & Wilkins; Philadelphia, USA; 2006.
How to Cite
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
PAGEPress has chosen to apply the Creative Commons Attribution NonCommercial 4.0 International License (CC BY-NC 4.0) to all manuscripts to be published.