Another way? An investigation into an institution's use of the Wayson stain in re-evaluating "no organisms seen" on Gram stain smears from positive blood cultures

Abstract

Automated continuous monitoring blood culture instruments identify metabolism byproducts and flag blood culture bottles as "positive." A Gram stain is used to visualize and characterize the microbial growth in the broth and initiate additional testing. When no organisms are seen (NOS) on Gram stain, in our laboratory, bottles are reevaluated with a Wayson stain, a rapid one-step stain that provides contrast between organisms and the background, especially in Gram-negative organisms. In this study, we assess the performance of the Wayson stain on reevaluation of Gram stain NOS blood culture bottles. The study period, August 2022 to July 2023, includes blood cultures that were flagged as positive by the automated blood culture instrument (BACTEC) with accompanying Gram and Wayson stain interpretations and organism identification. 133,463 blood cultures were performed during the study period, and 20,129 cultures were flagged as positive by BACTEC. 601 NOS Gram stain cultures were identified of which 76 had a positive Wayson. The Wayson stain procedure increased the sensitivity of direct organism visualization from 99.32% to 99.71% by identifying 55% (76 of 137) of NOS Gram stain blood culture bottles. Of blood cultures that were positive by BACTEC with NOS Gram and Wayson stains (475), 414 were finalized as no growth and 61 had an isolate (false negatives) of which 49% (30) were yeasts. The Wayson stain aided in detecting gram-negative genera such as Capnocytophaga, Campylobacter, and Fusobacterium species and other rarely identified species.IMPORTANCEThe Wayson stain, a rapid one-step stain that provides contrast between microorganisms and the background, was historically used for the presumptive diagnosis of Yersinia pestis from bubo specimen. In our laboratory, the Wayson stain has long been used to reevaluate blood culture Gram-stained smears from bottles that were flagged as positive by the automated continuous monitoring instrument but where no organisms are seen. In this study, we show that the Wayson stain provides an easily implemented and interpreted technique, other than a repeat Gram stain or acridine orange, to increase the sensitivity of direct organism visualization from blood culture bottles, particularly for Gram-negative organisms.

Keywords: Gram-negative; bacteriology; blood cultures; clinical methods; wayson stain.

Fig 1

Photomicrographs comparing the Gram stains and Wayson stains of the following patient specimens: Capnocytophaga species Gram stain (1A) and Wayson stain (1B) and Campylobacter species Gram stain (2A) and Wayson stain (2B) initially not recognized on Gram stain with faint staining; Fusobacterium nucleatum Gram stain (3A) and Wayson stain (3B) initially interpreted as debris on Gram stain.