INDIA

Mycobacterium tuberculosis strains can be detected directly in the sputum specimen within 2 or
3 hours, but in practice, this method has not become a routine laboratory technique, particularly
due to lack of sufficient specificity and sensitivity. Serological tests are currently not reliable
enough for the diagnosis of tuberculosis [2]. Microscopic examination and culture are still
essential elements of the bacteriological diagnosis of tuberculosis in microscopic examination;
the diagnosis of tuberculosis is confirmed on the basis of demonstration of tubercle bacilli in the
sputum or any other pathological material [1, 2, 4, 6].

References

[1]. Ayahs Gupta, S. K. sharma and J.N Pande(1993) Diagnostic methods for tuberculosis,
the India Journal of Chest Diseases and Allied Services.
[2]. Ba F and Rieder HL (1999) A comparision of flurescence microscopy with the Zeihl-
Neelson technique in the examination of sputum for acid-fast bacilli, Int J Tercle Lung Disease.;
3 (12): 1101-5
[3]. Clancey JK, Allen BW, Rogers DT, Smith LS, Aber V and Mitchison DA (1976)
Comparison of machine and manual staining microscopy, J Clin Pathol.; 29 (10); 931-3
[4]. Claxton PD, Eamens GT and Mylrea PJ (1979), Laboratory diagnosis of bovine
tuberculosis, Auet 1Vet J.; 55 (11):514-20
[5]. Desgmukh SR, Mantri SB, Kendre PB and Nagoba BS (1996) A comparison of sputum
examination for acid fast bacilli by modified Schaeffer and fulton stain, Zeihl-Neelson stain and
cold stain, Indian J Med Res.;103; 294-5.
[6]. 6. Dandapat MC, Mishra BM, Dash SP, Kar PK. Peripheral lymph node tuberculosis.A
review of 80 cases. Br J Surg 1990;77:911-2.

AFB can be more sensitive if fluorescent stain is used over ZN stain in detecting Tubercle bacilli
in sputum (Goyal R and Kumar A, 2013).

A Comparison of Ziehl-Neelsen Staining and Fluorescent Microscopy for Diagnosis of

Pulmonary Tuberculosis

Roma Goyal1, Anil Kumar2

1. Demonstrator, Dept. of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar.

2. Demonstrator, Dept. of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar.

AFRICA

The minimum number of AFB necessary to produce a positive smear result has been estimated to
be 5000-10,000/ml of sputum. If AFB concentration is below 1000 bacilli per ml of sputum, the
chance of observing the bacilli in a smear is less than 10% [4].
4. Maria Luisa Moro, Simona Nascetti, Filomena Morsillo, Matteo Morandi et al. Laboratory
procedures for the diagnosis of tuberculosis. Ann Ist super Sanita. 2010; 46(2): 178-184.
PubMed | Google Scholar

IUATLD

In low income and high tuberculosis prevalence countries, sputum smear microscopy is, and is likely to
remain for the foreseeable future, the only cost-effective tool for diagnosing patients with infectious
tuberculosis and to monitor their progress in treatment. Sputum smear microscopy is a simple,
inexpensive, appropriate technology method which is relatively easy to perform and to read. It yields
timely results with a very high sensitivity of detection of tubercle bacilli transmitters, and provides most
of the essential laboratory-epidemiological indicators needed for the evaluation of the National
Tuberculosis Programme (NTP).

The IUATLD recommends: – The examination of three sputum specimens – “SPOT” + “MORNING” +
“SPOT” – for the diagnosis of tuberculosis cases. – The examination of single “MORNING” sputum
specimens on three occasions for follow-up of treatment: one at the end of the intensive phase, one
during the continuation phase, and one at the end of treatment. Smear positive pulmonary TB
(grade 2–3+ by IUATLD criteria on Ziehl-Neelsen staining [14]) were recruited in this study.

The staining method

The method of choice for sputum smear microscopy is the Ziehl-Neelsen (ZN) staining technique
because it is the only one that provides consistently good results without the need for special
equipment. Preparing the necessary reagents requires a weighing scale that is not always available in a
peripheral laboratory, and preparing the reagents in the National Reference Laboratory or in the nearest
intermediate laboratory is therefore a frequently used option. The advantages of this option, i.e., better
standardisation and quality assurance, outweigh the disadvantages of long term storage. Cold staining
procedures such as the Kinyoun and Tan Thiam Hok methods are not recommended, as evidence shows
that they have difficulty detecting acidfast bacilli (AFB) in paucibacillary samples and the staining fades
rapidly. Fluorescence microscopy, which is recommended when the daily workload exceeds 50
specimens, has no place in most peripheral laboratories of low-income countries (ref below)

Disease I.U.A.T.a.L. IUATLD; Paris: 2000. Sputum examination for tuberculosis by direct
microscopy in low income countries. Technical Guide

The initiation of infection with Mycobacterium tuberculosis occurs when organisms in small
droplet nuclei are inhaled into the alveoli (13, 14, 36). Subsequent passage of these bacilli
through the alveolar epithelium is required for the establishment of infection and disease
progression (2, 13, 14, 36). Later in disease, destruction of the alveolar epithelium occurs
during caseation and liquefaction of the granuloma, leading to transmission of the bacilli
(13, 14, 36).