Tubercle 65 (19841 123-125 0 Longman Group Ltd.

BULK STAINING OF SPUTUM BY THE ZIEHL-NEELSEN METHOD

Tamas Fodor

Koranyi National institute of Tuberculosis and Pulmonology, Budapest, Hungary

Summary A trial of the Ziehl-Neelsen staining method employing bulk decolorization, rinsing and counter staining yielded reliable results. Of 110 patients who proved to be positive by direct microscopy, cross-contamination could not be proved in any. The advantage of the mass method is its economy.

En procedant avec une grande quantite de frottis a la fois pour les etapes de d&coloration, rincage et contre-coloration, on a obtenu des resultats fiables. Parmi les 110 malades trouves positifs a la microscopic directe, aucune contamination croisee n’a pu Qtre mise en evidence. L’avantage de la coloration en grande quantite est l’economie qu’elle represente.

Resumen La utilization de la tincion de Ziehl-Neelsen con un metodo masivo de decoloracion, enjuague y contratincion ha producido resultados fiables. En ninguno de 10s 110 pacientes estudiados, con positividad probada por microscopia directa, se pudo comprobar una contamination cruzada. La ventaja del metodo masivo es su economia.

Introduction The technical guide of the International Union against Tuberculosis-on sputum examina- tion for tuberculosis by direct microscopy states that bulk staining, rinsing and acid decolorizing and counter-staining should be avoided because of the danger of cross contamination from one slide to another [Il. Similarly, Kubica and Vestal recommend that slides should be stained individually, because mass staining in a common container may allow cross transfer of acid-fast organisms [2].

In our experience, the use of a common container has not resulted in cross contamination. The present paper is a summary of our results.

Methods To one volume of sputum 4 volumes of a solution of 0.125 % chlorhexidine gluconate were added. After mechanical mixing the mixture was incubated for 4 hours at 37°C and for 12 hours at room temperature. The resulting sediment was inoculated with a Pasteur-pipette on to each of 3 slopes of Lowenstein-Jensen medium and onto defatted slides to make smears. These were fixed in a hot air sterilizer for IO minutes at 120°C [31. The slides were

124 Fodor

then placed on a slide-rack and covered with a piece of filter paper. The filter paper was covered with carbol fuchsin and heated. After removal of the paper strips, the slides were placed perpendicularly, side by side, but separated, in a rack. The rack contained slides in batches of up to 24. Rinsing, decolorizing and counter staining took place in common containers.

The reagents were: Ziehl’s carbol fuchsin, 95 % ethyl alcohol containing 3 % by volume of concentrated hydrochloric acid, and methylene blue solution. For decolorizing each batch, two containers were used successively. Tap-water served for rinsing. For each rack, the counter stain was changed monthly, the acid alcohol weekly or fortnightly depending on the grade of utilization.

At least 100 microscopic fields were examined. If necessary a more thorough search was made, sometimes of several hundred fields. All positive smears were double-checked by a second microscopist. The presence of only l-3 acid-fast bacilli on a slide was reported positive if it was confirmed by another smear. Four or more organisms per slide were reported as positive.

All cultures were kept for up to 10 weeks before being discarded as negative. A positive smear report was considered to be verified if a positive culture was obtained

from the same material. In some cases the confirmatory culture was not inoculated from the same material, in which case only a few days elapsed between microscopy and the second inoculation.

Results In the course of 118 stainings of 6330 slides, 408 were positive. The positive smears were obtained from 110 patients (Table I). Forty-eight were examined simultaneously. The number of positive smears never exceeded 8. Obtaining positive slides several times from the same patient made the possibility of cross contamination highly unlikely. There were 107 cases verified by culture (97 %I, which ruled out errors in the staining procedure.

There were 3 patients whose cultures were negative despite positive smears, although in 2 positive cultures had been obtained previously. One of the 2 died shortly after the positive microscopic examination and active tuberculosis was found at autopsy. The second patient showed newly developed cavitation on the chest X-ray, and the third patient, who had 2 positive smears, died of lung cancer: at autopsy he was found to have pulmonary tuberculosis as well.

Out of the 118 stainings, in 96 one or more slides were strongly positive. Despite the risk of cross contamination, transfer of bacilli from positive to negative slides could not be proved in any of these cases.

Discussion Clancy et a/ stained direct smears of sputum in an automatic machine which permitted 48

Table 1. Incidence of positive slides per patient in relation to positive cultures

Positive results Number of positive slides per patient

1 2 3 or more total

Number of patients 26 20 64 110 Positive by culture 24 19 64 107

Bulk staining of sputum 125

slides to be stained in each cycle. They obtained no evidence of carry-over of acid-fast bacilli from positive to negative smears.141

Aber and colleagues have studied the sources of laboratory error in examining cultures and smears of sputum [5]. They found that elimination of the more obvious hazards, such as the use of common reagent containers for several specimens, failed to reduce the errors. False positive reports seemed to be related more to the quality of the technician, especially in developing countries, than to the detail of the technical methods.

Our findings show that the use of mass decolorising, rinsing and counter-staining can yield reliable results. The advantage of the bulk method lies in its economy-it spares work, time and material. Our experience is but limited. However, considering that the Ziehl- Neelsen staining technique is still used in many laboratories, it seems worth-while to test this method of bulk staining under different conditions.

References 1 Technical guide for sputum examination for tuberculosis by direct microscopy. (1978). Bulletin of fhe

lntemafional Union against Tuberculosis, Supplement No. 2. 2 Kubica, G. P. & Vestal, A. L. (1960). Tuberculosis Laboratory Methods in Diagnosis, DHEW Publication No. 770,

Communicable Disease Center, Atlanta, Georgia. 3 Bartmann, K. (1980). Mikrobiologische Grundlagen in Handbuch der inneren Medizin /V/3 Atmungsorgane,

edited by Jentgens, H. Springer-Verlag Berlin Heidelberg, New York. 4 Clancey, J. K., Allen, B. W., Rogers, D. T., Smith, L. S., Aber, V. & Mitchison, D.A. (1976). Comparison of machine

and manual staining of direct smears for acid-fast bacilli by fluorescence microscopy. Journal of clinical pathology, 29, 931.

5 Aber, V. R., Allen, B. W., Mitchison, D. A., Ayuma, P., Edwards, E. A. & Keyes, A. B. (1980). Quality control in tuberculosis bacteriology. 1. Laboratory studies on isolated positive cultures and efficiency of direct smear examination. Tubercle, 61, 123.