Use of Acetic Acid instead of Sulphuric acid in

Zeihl-Neelsen staining

Article published in Northern Medical Journal, 2006 ;15(2)

                              Dr. Md. Mujibur Rahman


Shulphuric acid, used as decolorizing agent in acid fast staining (Zeihl-Neelsen.) is a mineral acid which is known to be a strong and so very much corrosive acid. On the other hand acetic acid is organic and weak acid and never been used as decolorizing agent in Zeihl-Neelsen (Z-N) staining. Incidentally when acetic acid is used in Z-N staining due to instant unavailability of shulphuric acid, it is found to be equally effective as shulphuric acid. Subsequently to find out the effectiveness of acetic acid as decolorizing agent in Z-N staining the study was undertaken. For the last seven years different specimens including sputum, ascetic fluid, CSF, pleural fluid, skin discharge and joint fluids were stained for Mycobacterium tuberculosis using both 20% shulphuric acid and 20% acetic acid simultaneously and 14 case were stained for Mycobacterium leprae using 5% strength of both acids. There were no differences of among the positive findings of using shulphuric acid and acetic acid as decolorizing agent in acid fast staining for detection of mycobacterium. So, acetic acid, which is less corrosive than shulphuric acid can be used for the purpose of Zeihl-Neelsen.


Mycobacterium tuberculosis can not be easily stained by common stains used in microbiological laboratories e.g. Gram's staining. So this organism is neither gram positive nor gram negative. In 1882 Ehrlich discovered that mycobacterium stained with fuchsin resist decolorization by mineral acids. In the same year, Zeihl changed the mordant to carbolic acid, and in 1883 Neelsen increased the concentration of carbolic acid and incorporated it with the dye to form carbol-fuchsin. Thus the standard stain for demonstrating acid-fastness was formulated -   the Zeihl-Neelsen stain. In this procedure 20% and 5% sulphuric acid is used for decolorization procedure Mycobacterium tuberculosis and Mycobacterium leprae respectively. Due to high lipid content of the mycobacterial cell wall (60% is lipid) these organism resist any access of stain inside the bacteria but if heated carbolfucshin is given the organism are easily stained. And subsequently when they are decolorized with sulphuric acid, all material including other bacteria and other cells are decolorized except the mycobacterium.

 Incidentally, one day in our laboratory sulphuric acid was not available during Zeihl-Neelsen (Z-N) staining of sputum and at that time an urgent Z-N staining of a sputum sample could not be done. Thinking for an alternative we find acetic acid in the laboratory and instantly tried with 25% acetic acid for decolorization purpose. Interestingly decolorization was complete but AFB was not found. After one hour sulphuric acid was available in the laboratory and staining was repeated with this acid and same result was found. After this time during every Z-N staining procedure I used both acetic acid and sulphuric acid separately during Z-N staining of each sample and found no differences between the two methods. Purpose of this study was to show that a weak organic acid can also serve the same purpose as strong acid can do in Z-N staining and to avoid limitation on use of only one type of acid.


Materials and Methods:

This study was done during the period from February, 1998 to December, 2004. A total 2050 different specimens including sputum, ascitic fluid, CSF, pleural fluid, joint fluids, discharges from skin ulcer were stained for Mycobacterium tuberculosis by Zeihl-Neelsen method. Two separate smears were prepared in clean sterile slides from each specimen. For sputum, Petroff's concentration method was adopted. Prepared smears were dried in air and then fixed by warming over flame and heated carbolfucshin was poured and kept for 5 minutes. Then excess stain was washed with water.  One smear was then decolorized using 20% sulphuric acid and another smear was decolorized by 20% acetic acid.

Similarly, for detection of Mycobacterium leprae slit smears from nasal mucosa and ear lobules were stained with heated carbolfucshin and subsequently decolorized by 5% strength of both sulphuric acid and acetic acid for separate smears of same sample respectively. After completing decolorization each smear was counterstained with methylene blue for 2 minutes. Then smear was washed in water and dried in air. Each smear was examined under oil immersion lens for at least 10 minutes or observed for at least 100 fields for mycobacterium.



A total of total 2050 different specimens including sputum, ascitic fluid, CSF, pleural fluid, joint fluids, and discharges from skin ulcers were stained for Mycobacterium tuberculosis. Among these the number of sputum specimens was 1520. Out the sputum specimens 162 cases (10.65%) were positive for AFB in both methods of staining. Cases of pleural fluid were 150; out of these 1(.66%) case was found to be positive in both methods. Specimens of ascitic fluid were 202 in number, out of these no cases were found to be positive for AFB. Discharges from chronic skin ulcer were 4 in number. Out of these 1 was found to be positive for AFB. A total of 560 cases of CSF were examined for AFB. No case was found to be positive for AFB. Specimens of synovial fluid were 45 in number. Among these no case was found to be positive for AFB.


(Broken - repairing)Table No. 1

Types of specimen

Total no of cases

Positive cases

(Z-N staining with Shulphuric acid

Positive cases

(Z-N staining with Acetic acid





Pleural fluid




Ascitic fluid








Discharges from skin ulcer




Slit smear for M. leprae




Slit smears from ear lobules, nostrils and skin, lesions were examined in 18 cases. Out of these 06 cases were found to be positive in both methods using 5% of shulphuric and acetic acid respectively.



During staining AFB from different materials, use of both sulphuric acid and acetic acid during decolorization shows similar sensitivity. The use of acetic acid in staining acid fast bacillus is not a modification of the classical method of Z-N staining; rather it can be an alternative to it. Thinking of the mildness of the acetic acid, the present method should be less hazardous than the original method and in this regard it should carry more advantageous position. We use many methods of staining in daily laboratory procedures including histo-pathological, cyto-pathological, and histochemical and other biological purposes. There may be many ways to simplify or improve these methods so that we can use these methods with ease, cost-effectively and more effectively. So, research should be undergone continuously to improve these staining methods and also other technical procedures.



1.  Joklik et al. Mycobacterium. Zinsser Microbiology, 20th edition, 1992 by Appleton & Lange. 497-525p.

2.      Watt B, et al. Mycobacterium.Mackie & McCartney Practical Medical Microbiology. Fourteenth edition, 1996. 329-341p.

3.      S. Frederick and M. Beverly. Mycobacterium. Manual of Clinical Mycobacteriology. Sixth edition, 1995. 400-437.



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