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Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 894-895
Evaluation of acridine Orange staining for rapid detection of mycobacteria in primary specimen

Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication27-Oct-2010

How to cite this article:
Sethi S, Chatterjee SS, Bedi C, Sharma M. Evaluation of acridine Orange staining for rapid detection of mycobacteria in primary specimen. Indian J Pathol Microbiol 2010;53:894-5

How to cite this URL:
Sethi S, Chatterjee SS, Bedi C, Sharma M. Evaluation of acridine Orange staining for rapid detection of mycobacteria in primary specimen. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Aug 5];53:894-5. Available from: https://www.ijpmonline.org/text.asp?2010/53/4/894/72048


Although isolation and identification of Mycobacterium tuberculosis in clinical specimens is the definitive proof of tuberculosis (TB), microscopy remains the most rapid technique for evaluation of a clinical specimen. Flourescent microscopy has long been used as a supplementary method for diagnosis of M. tuberculosis infections in developed countries. Recent recommendations encourage greater use of fluorescent staining methods along with traditional Ziehl-Neelson staining (ZNS) to reduce the smear turnaround time. [1] Experts in the western world opine that short time phenolic-Acridine Orange (P-AO) staining of mycobacteria may in fact be more specific and sensitive than other fluorescent methods of staining. [2] However, there have been no reports of the use of this particular fluorescent staining in routine clinical laboratories from developing countries like India, where tuberculosis is highly prevalent and thus quick screening of mycobacteria is essential. Therefore, we evaluated P-AO staining and compared it with Rhodamine-Auramine (RA) fluorescent staining, ZNS, and culture on Lowentstein-Jensen (LJ) medium to detect acid fast bacilli (AFB) in sputum samples obtained from suspected cases of TB.

In this study, the sputum samples were collected from 300 patients with clinically suspected TB, attending our tertiary care center. Decontamination of the sputum samples was carried out according to the N-acetyl-l-cysteine-NaOH method. [3] Each sputum sample was stained using three different staining techniques, viz. P-AO fluorescent staining, [4] RA fluorescent staining at 37°C [4] and ZN stain. The Acridine Orange (AO) reagent was prepared by dissolving 5 g of phenol crystals in a solution containing 50 ml of deionized water, 25 ml of glycerol (Sigma, Mumbai, India) and 25 ml of 95% ethanol. Then, 1 g of AO (Sigma) was added and the mixture was stirred briefly. This mixture was stirred and allowed to settle overnight to allow the AO to dissolve completely. Acid alcohol destaining-counterstaining solution was prepared by mixing 74 ml of 95% ethanol, 26 ml of deionized water, 0.5 ml of concentrated HCl and 0.2 g of methylene blue. For staining, smears were heat fixed on a clean glass slide and were covered with AO reagent for 15 minutes. These were then rinsed with deionized water and drained. Smears were then covered with the destaining-counter staining solution for 2 minutes, rinsed with water, drained and dried. RA staining was done according to the standard techniques. [3] AFB positive control smears were used with each day's staining. The same staining reagent solutions were used throughout the study. The smears were screened for the presence of AFB, with a fluorescent microscope (Nikon, Tokyo, Japan) by using a 20× objective. The presence of bright orange red fluorescing rods against a dull green background was confirmed with a 40× objective [Figure 1]. Each sample was inoculated in two slants of LJ medium and incubated at 37°C for 7 weeks and examined at regular intervals for growth. Organisms were then identified by standard biochemical reactions. [4] Culture results were taken as gold standard. Results of the study [Table 1] revealed that P-AO (79.7%) was nearly as sensitive as ZNS (83.5%) and fared better than RA (73.4%).
Figure 1: AO fluorescent staining showing bright orange fluorescing bacilli in a pale green background (AO stain, ×400)

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Table 1: Comparison of different methods of diagnosis in 300 sputum samples

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Smithwick et al.[4] and Katila et al.[5] previously showed that results of P-AO were comparable to those of the RA method in four laboratories. A study conducted across six laboratories in Europe [2] also found that P-AO outperformed RA staining. The differentiation of the bacilli was better in P-AO staining as the dull green background enables easy visualization. It was more difficult in the case of RA staining because the bacilli fluoresce as yellowish green and the background is also pale green. Moreover, methylene blue, used in P-AO staining method, partially blocked the fluorescence of the background debris, but there was a little yellow green fluorescing background to search for the bright red fluorescing bacilli [Figure 1]. On the other hand, in case of RA staining, fluorescent debris can be mistaken for AFB and potassium permanganate quenching of the background fluorescence frequently leaves little visible background material, in which to search for AFB. In addition, the shelf life of P-AO stain is 6 months. [4]

   Acknowledgments Top

We thank Dr. Shiv Kumar Sharma for technical assistance.

   References Top

1.Tenover FC, Crawford JT, Huebner RE, Geiter LJ, Horsburgh CR Jr, Good RC. The resurgence of tuberculosis: Is your laboratory ready? J Clin Microbiol 1993;31:767-70.  Back to cited text no. 1
2.Kalich R, Bennert G, Fiedler T, Kubin M, Mezensky L, Turzova M, et al. Comparison of auramine-rhodamine B and acridine orange for staining of acid-fast bacteria. J Hyg Epidemiol Microbiol Immunol 1979;23:307-17.  Back to cited text no. 2
3.Kent PT, Kubica GP, editors. Public health mycobacteriology: A guide for the level III laboratory. 1 st ed. Atlanta: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control; 1985.  Back to cited text no. 3
4.Smithwick RW, Bigbie MR Jr, Ferguson RB, Karlix MA, Wallis CK. Phenolic Acridine Orange fluorescent stain for mycobacteria. J Clin Microbiol 1995;33:2763-4.  Back to cited text no. 4
5.Katila ML, Mäntyjärvi RA. Acridine Orange staining of smears for demonstration of Mycobacterium tuberculosis. Eur J Clin Microbiol 1982;1:351-3.  Back to cited text no. 5

Correspondence Address:
Sunil Sethi
Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.72048

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