A cold staining method for acid-fast bacilli

Bulletin of the World Health Organization, 64 (5): 741-743 (1986) ( World Health Organization 1986 A cold staining method for acid-fast bacilli R. VASANTHAKUMARI,l K. JAGANNATH,2 & S. RAJASEKARAN3 The Ziehl-Neelsen method is probably the best known and most frequently used procedure for staining tubercle bacilli. The method requires controlled heating for its success. However, in developing countries, such as India, where most laboratories rely mainly on spirit lamps as a source of heat, the Ziehl-Neelsen method often cannot be carried out because rectified spirit is difficult to obtain. The study describes a cold staining technique that uses the same staining solutions as the conventional Ziehl-Neelsen method. For direct smears, the correlation of results of the cold staining procedure with those of the Ziehl-Neelsen method was 97% and for concentrated smears was 99%. The method described is suitable for use in basically equipped laboratories. The Ziehl-Neelsen method is the most common laboratory technique for staining acid-fast tubercle bacilli. The surface of these bacteria contains a large amount of unsaponifilable waxy substance, and a number of staining methods have been developed to increase the permeability of the cell wall to the stain used. In the Ziehl-Neelsen method, the basic fuchsin- phenol dye is used hot, thus melting the waxy sub- stance and increasing the permeability of the cell wall. However, because of certain disadvantages of this technique, there have been various attempts to develop a cold method for staining tubercle bacilli. For example, Kinyoun used higher concentrations of basic fuchsin and phenol (1), while Tax Thiam Hok devised a method (2) by combining the staining tech- niques of Kinyoun and Gabbet (3), alleging that it was superior to and quicker than the Ziehl-Neelsen method. In contrast, Rao et al. used chloroform instead of ethanol as solvent, without increasing the concentration of the basic fuchsin-phenol staining solution, and reported that this variation was as effi- cient as the Ziehl-Neelsen method (4). We report here a variation of the Ziehl-Neelsen method that permits the cold staining of tubercle bacilli. The procedure uses the same reagents as the conventional Ziehl-Neelsen method, but avoids the l Assistant Professor of Microbiology, Institute of Tuberculosis and Chest Diseases, Madras, India. 2 Professor of Tuberculosis and Chest Diseases, Madras Medical College and Director, Institute of Tuberculosis and Chest Diseases, Chetpet, Madras-600 031, India. Requests for reprints should be sent to this author at the latter address. 3Assistant Professor, Government Tuberculosis Sanatorium, Tambaram, Madras, India. need to use heat by increasing the staining time. MATERIALS AND METHODS Sputum samples from patients with pulmonary tuberculosis who had been admitted to the Govern- ment Tuberculosis Sanatorium, Tambaram, India, were used in the study. Smears prepared from the sputa were stained using both the conventional Ziehl- Neelsen method and the cold staining method. Altogether 1100 samples of sputum were collected and divided into two groups, consisting of 700 and 400 samples, respectively. For the larger of these groups, direct smears were prepared from the thickest portion of each of the sputum samples. One set was then stained using the standard Ziehl-Neelsen method (5) and the other with the cold staining method. The 400 samples of sputum in the second group were concentrated using Petroff's method (5) and two smears were prepared from each of the concentrates. One set was then stained using the standard method and the other using the cold method described below. Solutions for the cold staining method Ziehl-Neelsen basic fuchsin-phenol solution, prepared as described by Allen & Baker (5). Gabbet's methylene blue (2) containing: Methylene blue 1 g Sulfuric acid (AR grade) 20 ml Absolute alcohol 30 ml Distilled water 50 ml 4718 -741- R. VASANTHAKUMARI ET AL. Staining procedure Smears were heat-fixed on microscope slides, flooded with basic fuchsin-phenol stain, and allowed to stand at room temperature for 10 minutes. The smears were then washed in running water, counter- stained with Gabbet's methylene blue for 2 minutes, and subsequently washed in tap water and air-dried. All smears were then examined microscopically by experienced technicians using an oil-immersion objective. Smears were graded as positive if a mini- mum of 3 bacilli were observed after scanning the slide for 15 minutes, while slides on which no acid- fast bacilli were found at the end of this time were rechecked. RESULTS Of the 700 direct smears examined (Table 1), 229 were positive by the cold method, in contrast to 225 by the conventional method; statistically, there was no difference between the results of the two methods (P>0.5). A common group of 216 smears was positive in both methods. Of the 229 smears that were positive in the cold method, 13 were negative in the Ziehl-Neelsen method, while 9 of the 225 smears that were positive in the Ziehl-Neelsen method were negative in the cold method. These discrepancies probably arose because single slides were used for each direct sputum smear and the distribution of tubercle bacilli in the sputum samples was not uniform. Similar results were obtained with the concentrated smears. Of the 400 concentrated sputum smears (Table 2), 170 were positive by the cold method and 171 by the standard Ziehl-Neelsen method. There was no difference statistically between the two methods (P>0.5). A group of 169 smears was positive in both the methods, and of the 170 smears that were positive Table 1. Comparison of the results of staining sputum (direct smears) from tuberculosis patients by the Ziehl- Neelsen method and the cold staining method Ziehi-Neelsen Cold staining Positive Negative Total Positive 216 13 229 Negative 9 462 471 Total 225 475 700 in the cold method only one was negative in the Ziehl- Neelsen method. Furthermore, of the 171 smears that were positive in the Ziehl-Neelsen method only 2 were negative in the cold method, indicating almost 100% correlation between the two methods. There was no difference in the intensity of staining in both methods. DISCUSSION The major difficulty in staining tubercle bacilli arises because their surface is coated with an unsaponifiable waxy substance. For such bacteria, the success of any staining technique depends on the ability of the dye to uniformly penetrate the cell wall through this waxy barrier, while leaving intact the acid-fast character of the bacilli. In the conventional Ziehl-Neelsen method, this is achieved by heating the microscope slide during the staining process. This operation requires fairly precise control of the temperature of the slide and experienced operators to carry it out successfully. In developing countries such as India, where smear microscopy is the main tool used to identify and follow up cases of tuberculosis, most laboratories use spirit lamps as the principal source of. heating. However, very often hot staining methods cannot be performed because either rectified spirit is not available or there is a shortage of suitably trained personnel. Numerous attempts have been made to develop a cold staining procedure for acid-fast bacteria (1, 2, 4). In the method described here longer exposure favours the uniform penetration of the dye through the cell wall. The concentration of staining solution is the same as that used in the classical Ziehl-Neelsen method, and hence no extra cost is involved. Use of a cold staining procedure has two distinct advantages: a high level of technical skill is not Table 2. Comparison of the results of staining sputum (concentrated smears) from tuberculosis patients by the Ziehl-Neelsen method and the cold staining method Ziehl-Neelsen Cold staining Positive Negative Total Positive 169 1 170 Negative 2 228 230 Total 171 229 400 742 COLD STAINING METHOD FOR ACID-FAST BACILLI 743 required, thus simplifying the training of personnel; and the problem of procuring rectified spirit for spirit lamps is removed, making it suitable for use even in remote areas. Heat fixation of smears on slides can be carried out using any available source of dry heat, such as a hot plate or even the closed lid of a boiling sterilizer. In addition, ready-made staining solutions are com- mercially available. Although the cold method described here is slightly longer to perform than the conventional Ziehl- Neelsen technique, its low cost, ease of use, and efficacy make it universally applicable, especially in those laboratories where facilities are poor. ACKNOWLEDGEMENTS We are thankful to the laboratory staff of the Government Tuberculosis Sanatorium, Tambaram, for their cooperation in conducting this study. RESUME METHODE DE COLORATION A FROID DES BACILLES ACIDO-RESISTANTS La methode de Ziehl-Neelsen est probablement la tech- nique la mieux connue et la plus usitee pour colorer les bacilles tuberculeux. Ces derniers se colorent mal en raison de la substance cireuse insaponifiable qui revet leur surface et la coloration n'est possible que si le colorant peut pen6trer librement dans les bacilles a travers cette barriere super- ficielle. Dans la methode de Ziehl-Neelsen, la perm6abilit6 de la paroi bacterienne est augment&e par le traitement a chaud par un colorant basique constitue de fuchsine pheniqu6e. Pour obtenir de bons r6sultats avec cette m6thode un controle assez precis de la temperature est necessaire et l'operateur doit etre un technicien qualifi6. Dans les pays en d6veloppement ou la bacilloscopie est le principal moyen de diagnostiquer la tuberculose, la plupart des laboratoires utilisent des lampes a alcool comme source de chaleur. Toutefois, il arrive souvent que la methode de Ziehl-Neelsen ne puisse etre appliqu6e faute de personnel qualifie et d'alcool rectifie. Le present article indique qu'une variante de la m6thode de Ziehl-Neelsen, pratiquee avec les memes reactifs, mais avec une periode de coloration plus longue, peut etre utilis&e pour colorer les bacilles tuberculeux a froid. Dans cette nouvelle methode, la fuchsine pheniquee basique est laissee en contact avec l'etalement pendant 10 minutes a froid, au lieu de 5 minutes avec chauffage intermittent comme dans la technique de Ziehl-Neelsen classique. Un groupe de 1100 etalements d'expectorations provenant de malades atteints de tuberculose pulmonaire ont e colores par la nouvelle methode et les resultats obtenus ont e compares a ceux que fournissait la m6thode classique: dans le cas d'etalements directs, la corr6lation 6tait d'environ 97%, alors que pour les etalements concentres la correlation depassait 99%. Aucun chauffage n'etant necessaire, la technique est d'exe- cution simple et peut etre confiee a des operateurs n'ayant requ qu'une breve formation. En raison de son faible cofit, de sa simplicite et de son efficacite, cette methode est aisement applicable, en particulier la oii les services de laboratoires sont reduits. REFERENCES 1. KINYOUN, J. J. American journal of public health, 5: 867 (1915). 2. TAx THIAM HOK. Simple and rapid cold staining method for acid-fast bacteria. American review of respiratory diseases, 85: 753-754 (1962). 3. GABBET, H. S. Lancet, 1: 757 (1887). 4. RAO, K. P. ET AL. A cold staining method for tubercle bacilli using chloroform. Indian journal of tubercu- losis, 14: 3-7 (1966). 5. ALLEN, B. W. & BAKER, F. J. Mycobacteria-isolation, identification, and sensitivity testing. London, Butter- worths, 1968, pp. 10 and 31-32.