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  Table of Contents  
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 561-562

Stamp's modified Ziehl–Neelsen staining for Brucella: Beware of the first impressions

1 Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India
2 Directorate of Research, Manipal University, Manipal, Karnataka, India

Date of Submission16-Nov-2015
Date of Acceptance07-Sep-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
V K Eshwara
Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.195363

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How to cite this article:
Tilak K, Eshwara V K, Tellapragada C, Mukhopadhyay C. Stamp's modified Ziehl–Neelsen staining for Brucella: Beware of the first impressions. Indian J Med Microbiol 2016;34:561-2

How to cite this URL:
Tilak K, Eshwara V K, Tellapragada C, Mukhopadhyay C. Stamp's modified Ziehl–Neelsen staining for Brucella: Beware of the first impressions. Indian J Med Microbiol [serial online] 2016 [cited 2021 Jan 16];34:561-2. Available from:

Dear Editor,

Human brucellosis is an important zoonotic infection and public health problem of global importance. Due to protean clinical manifestations, correct diagnosis of disease relies largely on laboratory evidence such as antibody detection or culture.[1] Inadvertent handling of cultures might put the laboratory worker to a risk of accidental exposure to the highly aerosolised brucellae.[2] Stamp's modified Ziehl–Neelsen (S-ZN) staining is a simple and inexpensive method for presumptive identification of Brucella, and when applied on positive blood culture broths, it minimises dangers of laboratory accidents along with rapid reporting of results.[3] Herein, we report for the first time false-positive result in S-ZN stain on blood cultures growing bacteria other than Brucella leading to misidentification.

We analysed fifty blood cultures with a high index of suspicion of brucellosis based on Gram staining that showed small Gram-negative coccobacilli in BacT/ALERT FA Plus blood culture bottles. Stamp's modified cold Ziehl–Neelsen staining was performed on these blood culture broths using 0.5% acetic acid as decolouriser. Broth was subcultured on 5% sheep blood agar and MacConkey agar. Bacterial colonies were screened by Gram's staining, oxidase test and Christensen's urease test. Isolates were further confirmed by Vitek 2 Compact system and multiplex polymerase chain reaction (PCR) targeting bcsp31 gene of Brucella for the simultaneous identification of genus Brucella (208 bp), Brucella abortus (498 bp) and Brucella melitensis (731 bp).[4]

All the analysed blood culture fluids showed pink coccobacilli in S-ZN stain which were identified pre-emptively as Brucella. All the 50 isolates were oxidase positive, but only 45 did hydrolyse urea majorly within 15 min. Isolates showing both oxidase and urease positivity were confirmed as B. melitensis by Vitek 2 Compact system and PCR while five oxidase-positive and urease-negative coccobacilli were identified as Brevundimonas diminuta by Vitek 2 Compact system, and these isolates were found to be negative by PCR for Brucella. Colonies of Brucella were faintly visible after 18 h of incubation while B. diminuta showed larger colonies at the same time. However, all the isolates of Brucella and Brevundimonas were morphologically indistinguishable both in Gram's stain and S-ZN stain.

While an early and rapid detection of Brucella in cultures is important to initiate appropriate antimicrobial therapy, culture manipulations increase the chance of laboratory-acquired brucellosis in under-equipped laboratories of developing nations. S-ZN staining method serves as a safe and economical method for presumptive identification of Brucella from blood cultures in resource-poor settings. Brucella is not truly acid fast, but the organisms retain certain dyes, including basic fuchsin, in the presence of dilute acids or alkalies, and this has been used as a basis of differential staining methods. Brucella organisms are stained pink against a blue background. Available reports demonstrated that organisms of enzootic abortion (Chlamydia) and Q fever (Coxiella burnetii) are also stained pink within tissue cells and are very difficult to differentiate from Brucella organisms.[5] However, due to their uncommon occurrence in humans, false-positive results are not worrisome in blood cultures for human brucellosis.

We report for the first time that B. diminuta, a Gram-negative, glucose non-fermenting coccobacilli, also closely resembles Brucella in being oxidase positive and an inability to grow on MacConkey's agar. Adding to the complexity that like Brucella, it appears as small pink-coloured coccobacilli against a blue background when stained by S-ZN staining method. This can result in B. diminuta being incorrectly identified as Brucella spp. and thereby leading to administration of anti-Brucella therapy for a prolonged duration if further confirmatory tests are not carried out. These findings alert microbiologists to use additional tests before declaring pink coccobacilli in S-ZN stained smear as Brucella.

Financial support and sponsorship

This study was financially supported by MD/MS/DM/MCH thesis financial assistance programme of ICMR.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Corbel MJ. Brucellosis in Humans and Animals. World Health Organization in Collaboration with the Food and Agriculture Organization of the United Nations and World Organization for Animal Health; 2006. Available from: [Last accessed on 2015 Oct 10].  Back to cited text no. 1
Fiori PL, Mastrandrea S, Rappelli P, Cappuccinelli P. Brucella abortus infection acquired in microbiology laboratories. J Clin Microbiol 2000;38:2005-6.  Back to cited text no. 2
Joshi PA, Kulkarni RD, Powar RM. Modified cold Z-N staining for presumptive identification of Brucella. Indian J Med Res 2005;121:108-10.  Back to cited text no. 3
Kumar S, Tuteja U, Sarika K, Singh D, Kumar A, Kumar O. Rapid multiplex PCR assay for the simultaneous detection of the Brucella genus, B. abortus, B. melitensis, and B. suis. J Microbiol Biotechnol 2011;21:89-92.  Back to cited text no. 4
Poiester FP, Nielsen K, Samartino LE, Yu WL. Diagnosis of brucellosis. Open Vet Sci J 2010;4:46.  Back to cited text no. 5


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