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Original Article
Assessment of sputum staining techniques for the diagnosis of pulmonary tuberculosis
Niladri Sekhar Das*, DC Thamke**
*Department of Microbiology, College of Medicine and JNM Hospital, Kalyani, Nadia, West Bengal 741235, India.
**Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra-442102, India.
Place of study- Department of Microbiology, MGIMS, Sewagram, Maharashtra- 442102. India.
Source of funding- The present research work has received financial support from Department of Microbiology, MGIMS, Sewagram, Maharashtra- 442102. India.

Corresponding Author

Dr. Niladri Sekhar Das
(Assistant Professor of Microbiology), College of Medicine and JNM Hospital, Kalyani, Nadia,
West Bengal 741235, India.
E-mail: drniladrisekhar@gmail.com, Mob.: +91-9874383916

History : Received - 30-Aug-2013 Accepted - 19-May-2013 Published Online -  22-May-2013
DOI : http://dx.doi.org/10.7713/ijms.2013.0017


Introduction: Sputum smear examination plays a key role in routine diagnosis and treatment of pulmonary tuberculosis (PTB). The sensitivity of sputum microscopy is influenced by the number of mycobacterium present in the specimen, technique/s and quality of specimen. Ziehl-Neelsen (ZN) staining is commonly used methodology for the diagnosis of PTB but not very useful in paucibacillary cases. Although fluorescent microscopy is costly, it is considered more sensitive than ZN staining. The purpose of the study was to assess the efficiency of these two staining techniques for the detection of tuberculosis. 

Material and methods: We collected satisfactory sputum samples from 100 new symptomatics of PTB cases. This study was carried out from 2008-2011. For every sample, ZN and fluorescent (FL) staining was done and LJ (Lowenstein-Jensen) medium was used for isolation. Results were analysed using SPSS software.

Results: Positivity by ZN method was 33% and 34% with FL staining. The sensitivity of ZN microscopy was 82.9% and specificity was 93.8%, similar to that observed with FL microscopy and 94.3% correlation was found between ZN and LJ positive cases.

Conclusion: ZN staining alone is sufficient for the detection of Mycobacterium tuberculosis from a single sample, with no added advantage of FL staining.

Keywords : Fluorescence; Mycobacterium tuberculosis; sputum; staining; tuberculosis.


In developing countries, microbial diagnosis of pulmonary Tuberculosis (PTB) plays a key role in routine treatment and tuberculosis control programme. Only cost-effective, simple and rapid test for presumptive diagnosis of tuberculosis is sputum smear examination. It is estimated that between 60% to 70% of all PTB cases are diagnosed by means of sputum smear examination [1]. Ziehl Neelsen (ZN) stain is commonly used throughout the world [2,3] for smear examination but can detect bacilli when they are in the order of 105/mL of sputum, whereas more sensitive staining technique Fluorescent staining (FL) detects the bacilli when they are in the order of 104/mL of sputum though the disadvantage of the FL technique is that it requires costly microscope and may sometimes yield false positive results. Widely used ZN technique though rapid and simple to perform, has a low sensitivity, especially for single specimen for the demonstration of the acid fast mycobacteria. However the quality of the microscopy results depends on the quality of the collected sputum sample and the microscopic examination. For developing countries, with a large number of cases and financial constraints, evaluation of rapid and inexpensive diagnostic methods like demonstration of acid fast bacilli (AFB) in smears has great importance. The present study was conducted to reassess efficiency of the conventional ZN and the fluorescent auramine-rhodamine staining techniques of direct microscopy for detection of AFB from sputum sample in terms of correlation of positivity, sensitivity and specificity with respect to Lowenstein-Jensen (LJ) as a gold standard. 

Materials and Methods

The study was carried out at Department of Microbiology during 2008-2011. A total of one hundred suspected cases of pulmonary tuberculosis (according to RNTCP) who were supposed to provide sputum for acid-fast bacilli (AFB) staining were included in this study. Patients were given instructions and asked to collect satisfactory sputum sample in a wide mouth sterile container. A sample was considered satisfactory which was mucoid or mucopurulent with minimal oral or nasal material and having approximately 2 to 3 mL of sputum. Only satisfactory sputum samples were included in the study.

Two smears were made from the selected satisfactory sample, one for Ziehl–Neelsen (ZN) staining and the other for fluorescent staining (FL). From the mucoid or mucopurulent part of the sputum, smear of 2×3 cm size was made using a broomstick on each slide. ZN staining was done using standard procedures and grading was given according to the RNTCP guidelines [4] whereas Fluorescent staining [5] was also done by using 3% auramine phenol solution for the same sample and grading was given [5]. Both slides were blinded by the reader and were read by two independent readers. After staining rest of the sample was decontaminated by using NALC-NaOH method [6] and was inoculated on 2 Lowenstein-Jensen (LJ) slopes. Negative LJ culture was noted if there was no growth after incubation for 8 weeks. For the identification of the isolates as Mycobacterium tuberculosis, three tests- ‘Growth on para-nitrobenzonic acid incorporated in LJ medium’, ‘Niacin test’ and ‘Stability of catalase at pH 7.0/68°C’ were done.


Among 100 symptomatics microscopy (ZN + FL) detected 34% cases whereas individually ZN staining detected 33% as compared to 34% by FL microscopy (Table 1). This difference was found to be statistically significant (Fischer’s exact test, p < 0.05). We got 99% correlation between ZN and FL microscopy results, only one case which was negative on ZN staining was additionally detected by FL microscopy.

Isolation rate by LJ was 35% and all were Mycobacterium tuberculosis, using standard biochemical tests. Similar number of false positivity (n=4) was observed with ZN and FL staining techniques (Table 2). Only one case was additional false negative by ZN.

The specificity of both the staining were same (93.8%) and sensitivity of FL microscopy was slightly higher (85.7%) than ZN (82.9%) (Table 2). Among the six ZN negative but culture positive cases, five (83.3%) had low bacillary load and were also negative by fluorescence microscopy, but one case was detected additionally by fluorescence microscopy.

There was 97.14% correlation between microscopy (ZN+FL) and LJ positive cases (35) whereas individually it was 94.3% by ZN. Out of total 34 microscopy (ZN +FL) positive cases grading was similar in 27 cases (79.41%), whereas 5 cases showed discrepancy in grading (Table 3). One case of scanty was additionally detected by Fluorescence microscopy; whereas two scanty cases by ZN were detected as 1+ by Fluorescence microscopy.


Smear microscopy plays an important role in early diagnosis of mycobacterial infections as the method is highly specific, rapid and cheapest method used for detection of AFB in sputum. The only disadvantage of this method is low sensitivity (varying from 50%-80%) relative to culture [7-9]. The sensitivity of microscopy is influenced by the quality of specimen collection, the number of mycobacterium present in the specimen, the method of processing, the staining technique, and the quality of the examination [10]. To achieve maximum sensitivity of this diagnostic test, it is essential to have a good quality sputum sample, i.e. the sample should contain mucoid or mucopurulent material and the volume should be at least 3 mL [4]. The ZN method has commonly been used around the world, particularly in developing countries, because of its simplicity and low cost. Though the fluorescence microscopy was on average 10% more sensitive than ZN [11], but a real disadvantage of the fluorochrome method is that fluorescence fades with time along with the cost of the microscope. For this reason, the slides must be read within 24 hours. Fluorescence microscopy method can give “false positives” as compared to ZN staining, so it is recommended that the positive fluorescence microscopy smears should be confirmed by overstaining the smear by ZN method [10-12].

The sensitivity and specificity of ZN staining reported by various authors have been shown to range between 61% to 86.4% [12-15]; and 96.2% to 100% respectively [13-15]; whereas with respect to fluorescence microscopy, reports of sensitivity range between 59.7% to 83% [13-15] and that of specificity from 85.5% to 99% [13-15]. In the present study, there are slight differences in the sensitivity of ZN and fluorescence microscopy; however the specificity was exactly same for both (93.8%). The quality i.e. satisfactory sputum sample, in the present study, might have increased the sensitivity and specificity of ZN staining technique. However, we did not assess the effect of sputum quality on staining by using a control group. The correlation of positivity between the two microscopy techniques (ZN and FL) was 99%, and showed identical grading in 79.41% cases which is much higher than other studies [15-16]. Among total 34 microscopy positive cases, one case was missed by ZN microscopy and this case had low bacillary load as the growth obtained on LJ medium was scanty, moreover it took 42 days to grow on LJ. Among the microscopy negative but culture positive cases, 83.3% correlation was observed between the two staining techniques. Though the main advantage of fluorescence microscopy over ZN staining is in paucibacillary cases [16,18], our study did not reveal any significant advantage in samples having low bacterial counts. In our cases, we got same number of false positivity. Since none of our cases were on treatment (all new cases) presence of nonviable bacteria could not be the reason in our case, however sometimes sputum may contain acid fast particles resembling tubercle bacilli or precipitates of staining may hamper reading or mislead an inexperienced microscopist [13]. This could be a probable reason in our case. There were slightly higher false negative results with ZN as compared to FL, commonly due to deficiency in the preparation of the smear, such as too little material spread on the slide or some being too thin [13]. We performed three different techniques from a single sample; the less quantity or unequal distribution of the sample on the slide can be a reason of false negativity.

In our set-up, only ZN microscopy was able to detect 82.86% cases as Mycobacterium tuberculosis. In paucibacillary cases, ZN and FL staining appeared to detect similar grading in most cases (Table 3).

Key Points

• Performing both ZN and FL microscopy, on the single sample in our study had really no great advantage considering the cost of fluorescence microscope as no significant difference was observed with respect to sensitivity, specificity, grading and Mycobacterium tuberculosis detection.
• It is imperative to continue relying on simple ZN microscopy in satisfactorily-collected sputum samples.


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