Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species

Ali M. Bazzi1, *, Jaffar A. Al-Tawfiq3, 4, Ali A. Rabaan2
1 Microbiology Lab, Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia
2 Molecular Diagnostic Lab, Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia
3 Specialty Internal Medicine, Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia
4 Department of Medicine Indiana University School of Medicine, Indianapolis, IN, USA

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© 2017 Bazzi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the John Hopkins Aramco Health care Saudi Aramco -Dhahran, zip code 31311, Dhahran health center, bdg 62, room 273, Saudi Arabia; Tel: +966138776636; Fax: 966138776629; E-mails: bazziamh@gmail.com; ali.bazzi@JHAH.com



Acinetobacter baumannii and Brucella species are Gram-negative organisms that are vulnerable to misinterpretation as Gram-positive or Gram-variable in blood cultures.


We assess the random errors in gram stain interpretation to reduce the likelihood of such errors and therefore patient harm.


Aerobic and anaerobic blood cultures from two patients in an acute care facility in Saudi Arabia were subjected to preliminary Gram-staining. In case 1, VITEK-2 Anaerobe Identification, repeat Gram staining from a blood agar plate, Remel BactiDrop™ Oxidase test, Urea Agar urease test and real-time PCR were used to confirm presence of Brucella and absence of Coryneform species. In case 2, repeat Gram- staining from the plate and the vials, VITEK-2 Gram-Negative Identification, real-time PCR and subculture on to Columbia agar, blood agar, and MacConkey agar were carried out to identify A. baumannii.


In case 1, initially pleomorphic Gram-positive bacteria were identified. Coryneform species were suspected. Tiny growth was observed after 24 h on blood agar plates, and good growth by 48 h. Presence of Brucella species was ultimately confirmed. In case 2, preliminary Gram-stain results suggested giant Gram-positive oval cocci. Further testing over 18-24 h identified A. baumannii.


Oxidase test from the plate and urease test from the culture vial is recommended after apparent identification of pleomorphic Gram-positive bacilli from blood culture, once tiny growth is observed, to distinguish Brucella from Corynebacterium species. If giant Gram-positive oval cocci are indicated by preliminary Gram-staining, it is recommended that the Gram stain be repeated from the plate after 4-6 h, or culture should be tested in Triple Sugar Iron (TSI) medium and the Gram stain repeated after 2-4 h incubation.

Keywords: Gram-stain, Blood culture, Brucella, Acinetobacter, Coryneform.