Surani, Kumar, Chauhan, Chawda, Ramanuj, and Trivedi: Comparison between ziehl-neelsen staining and fluorescent staining of sputum samples to detect acid fast bacilli in suspected case of pulmonary tuberculosis at tertiary care hospital, Amreli, Gujarat


Tuberculosis (TB) is one of the major community health problems, in India. Pulmonary tuberculosis (TB) is a lower respiratory tract infection, which is caused by Mycobacterium tuberculosis. It is the most common disease affecting the lower socio-economic class in developing countries, approximately one-third of the world’s population infected with TB as TB remain a major health problem.1 Tuberculosis can infect any system or organ of the body, but lung is the most common affected organ.2 Tuberculosis is one of the leading causes of death, when associated with HIV.3 There are various methods for diagnosis of tuberculosis eg. Microscopy, Culture, Molecular etc. Microscopy is the simple, quick and practical method for detection of TB bacilli.4 Specificity of AFB microscopy is high but sensitivity varies depends upon bacillary load.5 By using of concentration method will increase the sensitivity of the sample.6 Due to larger field of view and the better contrast, observation of acid fast bacilli by fluorescent staining have greater sensitivity than light microscopy.7 Culture methods are more sensitive than microscopy but takes 4 to 8 weeks as tubercle bacilli are slow growing organism so microscopic examination has the advantage of giving a result at once.8 So, the most hopeful diagnostic method to overcome this problem is Molecular techniques like PCR.9

Microscopic examination with staining methods like Ziehl-Neelson or Fluorescent, allows detection of acid fast bacilli in less than an hour. Ziehl-Neelson is most widely used procedure for the detection of Mycobacterium tuberculosis in smear.10 Fluorescent staining by Auramine stain is another method of staining. It is simpler and can be examined at lower magnification (40x) than ZN (100x), (40x Vs 100x). Fluorescent microscope (FM) may take upto 75% less time than ZN11. This are advantages of fluorescence staining.


Objective of the study was to compare the results of ZN stain with fluorescent stain by use of microscope.

Materials and Methods

The study was carried out in Microbiology Department, SMCGH, Amreli. All age groups of both the gender suspected to be a case of pulmonary tuberculosis as per RNTCP guidelines, both indoor patients and outdoor patients presenting with cough and expectoration for 2 or >2 weeks with or without symptoms like loss of appetite, loss of weight, chest pain and haemoptysis, included in this study. 350 sputum samples (Spot and early morning) collected from 175 suspected case of the pulmonary tuberculosis. Samples other than sputum and Samples macroscopically resembling saliva did not include in this study.

Sample collection method

Suspected case of pulmonary tuberculosis was asked to take two samples of sputum, one spot sample and another early morning sample into clean, sterile, heat proof, leak proof, wide mouth sputum container. Smear preparation from samples was carried out in a bio safety cabinet by trained RNTCP technician thereby ensuring the quality of smear. Each sample was processed by ZN stain and Fluorescent stain to detect acid fast bacilli. All stained smears were examined microscopically by using an oil-immersion microscope and fluorescent microscope and the results of the stained smears were given according to RNTCP guideline.

Ziehl-Neelsen stain

Reagent used: 0.1% Carbol fuchsin, 25% Sulfuric acid, 0.1% methylene blue.

Preparation and Staining of smear

A portion of the mucopurulent material is taken on a slide and a smear is made and dried. “Fixation of the smear”- fix the smear by gently passing the slide over the flame. Flood the slide with strong carbol fuchsin and heat until steam rises. Allow the preparation to stain for 5 minutes. Heat being applied at intervals until steam rises and stops it and continue in this way for 5 minutes (never heat too much to produce boiling and charring). The stain must not be allowed to evaporate and dry on the slide, if necessary, pour more carbol fuchsin on the slide. Wash in running tap water. Decolourise the smear with 25% sulphuric acid by dipping the slide in the acid solution in a wide mouth jar. Decolourisation should be continued till the film becomes faintly pink. Decolourisation must be done in stages and generally requires about 10 minutes. Wash in running tap water. Counter stain the smear with methylene blue for 1-2 minutes. Wash in running tap water. Blot dry and examine with the oil immersion objective lens of binocular microscope.

Fluorescent stain

Reagent used

Auramine-Phenol solution, 1% acid alcohol, 0.1% potassium permanganate solution.

Preparation and Staining of smear: A portion of the mucopurulent material is taken on a slide and a smear is made and dried. “Fixation of the smear”- fix the smear by gently passing the slide over the flame. Flood the slide with freshly filtered Auramine – Phenol. Let stand for 7 to 10 mins. Wash well with running tap water. Taking care to control the flow of water so as to prevent washing away the smear. Decolourise by covering completely with acid alcohol for 2 mins, twice. Wash well with running tap water as before to wash away the acid alcohol. Counter stain with 0.1% potassium permanganate for 30 secs. Wash well as before with running tap water and slop the slide to air dry and examine with 40x objective lens of fluorescent microscope.

Table 1

RNTCP guidelines for grading of slides in AFB microscopy

No. of AFB seen

OIF to be screened



No AFB in 100 OIF




1 to 9/100 OIF




10 to 99/100 OIF




1 to 10/ OIF




>10/ OIF




[i] (* - Record actual number of bacilli seen in 100 fields)

Table 2

Fluorescent microscopy grading system (40x)

No. of AFB seen

OIF to be screened



No AFB in 40 fields




1 to 19/ 40 fields




20 to 199 /40 fields




5 to 50 / field




>50/ field





Out of 350 sputum samples collected from 175 patients, 52 and 61 sputum samples were found to be positive for acid fast bacilli by Z-N and Fluorescence staining respectively. The Z-N smear positivity rate and fluorescence staining smear positivity rate in this study was 14.85% and 17.4% respectively.

Figure 1

ZN stain showing acid fast bacilli

Figure 2

Fluorescent stain showing tubercle bacilli

Graph 1

Comparison of ZN and Fluorescent staining report

Out of 175 suspected case of tuberculosis, 113 (64.57%) were males and 62 (35.43%) were females. Males were predominantly affected than females. Out of 61 positive sputum smear by fluorescent staining, 38 (62.29%) smear were of males and 23 (37.7%) smear were of female.

Graph 2

Gender wise distribution in of study participants

Graph 3

Gender wise distribution and positivity

We received sputum samples from different age groups: 15-30 year, 31-45 year, 46-60 year and above 60 year. Out of 61 positive sputum smear 6 (9.84%), 13 (21.31%), 15 (24.59%) and 27 (44.26%) were found positive from different age groups respectively. Majority positive samples were found in the age above 50 years.

Graph 4

Age wise distribution of study participants

Table 3

Comparison between spot sample and morning sample by ZN staining and Fluorescent staining

By ZN staining




Spot sample




Morning sample








By Fluorescent staining




Spot sample




Morning sample









Tuberculosis is the most serious infectious diseases and a major public health problem in India due to its high risk of transmission between person to person, morbidity and mortality. Even though we have many advance methods for early detection of tubercle bacilli and different modalities for treatment and management for tuberculosis still it is a major public health problem in all over the India with poor social and economic consequences. To prevent both morbidity and mortality, early diagnosis of disease followed by adequate treatment for the same is crucial. For the control of tuberculosis we can do early case detection, start treatment as early as possible, and by this prevent the transmission of the disease. Our main focus for its control should be quick and correct identification of the infected individuals by the detection of acid fast bacilli from the given sputum sample of suspected patient.11 This is considered as the indication of infective stage. The laboratory plays a crucial role in diagnosis of pulmonary tuberculosis by various alternative diagnostic methods like Microscopy, Culture, Molecular methods (PCR) etc. Culture methods are time consuming modality as Mycobacterium tuberculosis takes 4 to 6 weeks to grow on culture media. In developing countries, microscopy of the sputum is simplest and rapid method for detection of acid-fast bacilli. Also this is cheapest and more reliable method for diagnosis of pulmonary tuberculosis.

In this study shows, males are predominantly affected than female which is also seen in other studies.12, 13 The reason behind it is increased outdoor activity of men and therefore they are more prone to come in contact with active case of tuberculosis.

In this study, out of 350 sputum samples, 52 (14.85%) and 61 (17.4%) sputum samples were found to be positive for acid fast bacilli by Z-N and Fluorescence staining respectively. Rate of smear positivity depends upon the type (spot and early morning sample) and quality of sputum sample also presence of tubercle bacilli in the sputum sample. 104 bacilli/ml is required to give positive result.14 Fluorescent staining is a superior method of microscopy for demonstration of acid fast bacilli than ZN staining method. Many other studies have also revealed the increased efficacy of fluorescent staining.

Table 4

Comparisons of results of various studies with same objectives


Slide +ve rate by ZN stain

Slide +ve rate by Fluorescent stain

Sample size

Z. Khatun et al15




Suria et al16




Golia S et al




Ulukanligil et al17




Rahul achaeya et al 18




Our study




In this study we conclude early morning samples are more reliable than spot samples due to accumulation of the secretion get concentrate with bacilii over night.19

In this study it was observed that total number of 9 sputum smears which were negative by ZN staining and positive by Fluorescent staining method and So, we conclude that fluorescent staining with LED microscopy was more efficient over ZN staining.

Though Fluorescence staining has been added in Revised National Tuberculosis Control Programme (RNTCP) for elimination of TB because of its sensitivity and fast results.


Sputum smear examination for the acid fast bacilli is usually carried out for patients which are clinically and radio logically suspected for pulmonary tuberculosis. Though, the usual method of sputum examination, eg, ZN staining is not sensitive enough and same suspected cases are not confirmed. Also they remain undiagnosed and fail to get treatment. Therefore our study concludes that Fluorescent staining with LED is more efficient over ZN stain in detecting acid fast bacilli in sputum smear, and also Fluorescent Microscope LED (40x) has been found to be less time consuming as compared to Light Microscope (1000x) in the diagnosis of pulmonary tuberculosis. Nowadays, Molecular methods like PCR are even more sensitive and specific over microscopic methods. The only disadvantage with molecular method is high cost and time consuming and may not be affordable for developing countries.

Source of Funding


Conflict of Interest

The authors declare no conflict of interest.



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Received : 18-11-2021

Accepted : 24-11-2021

Available online : 15-12-2021

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