Comparison of strip and Ziehl—Neelsen methods for staining acid-fast bacteria*

Bull. Org. mond. Sante 11974, 51, 83-91Bull. Wid Hith Org. Comparison of strip and Ziehl-Neelsen methods for staining acid-fast bacteria* P. VARUGHESE, D. M. HELBECQUE, K. B. McRAE, & L. EIDUS The efficiency of the Ziehl-Neelsen method for staining acid-fast bacteria was compared with that ofthe strip-staining procedure in which one kind ofimpregnated strip is used to stain the bacteria and another kind for simultaneous decolorization and counter- staining of the smear. The methods were evaluated in 1 136 duplicate smears preparedfrom digested sputum and 307 pairs of direct smears. The efficiency of the strip method was comparable to that of the Ziehl-Neelsen method with digested sputum; with direct smears, however, it generally depended on the quality of the smear. With thick, uneven smears, lower bacterial counts were obtained by strip staining. On the basis ofthis trial, the authors suggest improvement of the strip method. Nearly 90 years have elapsed since the introduc- tion of the Ziehl-Neelsen staining method for the microscopic detection of acid-fast bacteria. In the years following the introduction of this procedure, several modifications have been suggested for the preparation of smears and for the staining process itself. Simplification of the somewhat cumbersome procedure would result in a saving of time and enable unskilled personnel to perform the staining with acceptable accuracy, even under poor working condi- tions. This paper presents an early effort to break away from the classical concept of staining and from cold- staining methods involving highly concentrated solu- tions and the time-consuming preparation of suitable microscopic slides. The strip method investigated requires ready-made impregnated strips for staining bacteria and for the simultaneous decolorization and counterstaining of smears. In the present study, consisting of two experiments, three types of smear were used for the comparison of the strip and Ziehl-Neelsen staining methods a-i.e., smears prepared from digested and washed sputum concentrates; uniformly thin films made from spu- tum diluted in a 1 : 1 proportion with Sauton's medium; and thick smears prepared from sputum diluted 1 : 1 with water. * From the National Reference Centre for Tuberculosis (Laboratory Centre for Disease Control), Health Protection Branch, Health and Welfare, Ottawa, Canada. a For convenience, the abbreviations S (for the strip method) and Z (for the Ziehl-Neelsen method) are used in the tables, formulae, and figure. MATERIALS AND METHODS Experiment 1 Sputum specimens were obtained from tuberculo- sis pretreatment cases or patients treated with rifam- picin and isoniazid, with or without streptomycin, or the standard triple regimen-isoniazid, para-amino- salicylic acid (PAS), and streptomycin. Five millilitres of each sputum sample were di- gested with 20 ml of 4% sodium hydroxide and shaken at intervals for 15 min. The digested sputum specimens were concentrated by centrifugation (700g for 15 min) and the supernatant drained by suction. The sediment was neutralized by IN HC1, using phenol red as an indicator, and diluted with 5 ml of distilled water. The bacterial suspension was auto- claved at a pressure of I x 106 Pab for 15 min and centrifuged as before. After decantation of the super- natant, the sediment was used for the preparation of smears. Two smears were prepared from each of the sputum concentrates. The slides were cleaned with 95% ethanol and one loopful (3 mm in diameter) of 1: 10 diluted sterile bovine serum was placed on each slide. Three loopfuls of the sediment were then added to each slide, mixed thoroughly with the serum, and spread uniformly over a circular area 1 cm in diameter. The slides were dried at 37'C and fixed by flame. One of the smears was stained by the Ziehl- Neelsen method and the other by the strip method. b Equivalent to 15 lbf/in'. 3240 -83 P. VARUGHESE ET AL. Experiment 2 Sputum specimens were collected from six patients hospitalized for tuberculosis. Four of the specimens (Nos. 2, 3, 4, and 5), selected at random, were diluted with Sauton's medium, and the two others (Nos. 6 and 9) with distilled water, all in the proportion 1: 1. Both diluents contained thiomersal in a final concen- tration of 1 in 1 000 w/v. The diluted sputum specimens were then homogenized for 15 min in 50- ml thick-walled tubes containing 30 glass beads, by means of a Junior Vortex mixer. Sample No. 6 was divided into 3 aliquots. One of these contained the amount of bacteria originally present in the sputum (No. 6); to the second and third aliquots (referred to as samples Nos. 7 and 8) bacterial suspension derived from other positive sputum specimens was added in order to increase the bacterial content of the original sputum 15-fold (No. 7) and 4-fold (No. 8). Two millilitres of the 8 homogenized mixtures were trans- ferred into separate Bijou bottles, each containing 20 glass beads, and stored at 4°C. The bottles were shaken vigorously before preparation of the slides. Two loopfuls of each of the above-mentioned homogenized sputum specimens were transferred to each of 2 clean slides and spread uniformly over a circular area 1 cm in diameter. The slides were then dried, fixed by flame as before, and placed overnight on a hot plate at 75°C to kill the bacteria. From each sample, 27-50 replicate pairs of slides were prepared over a period of 20 days; half of the duplicate slides, selected at random, were stained by the Ziehl-Neel- sen method and the other by the strip method, each slide receiving a separate code number. Microscopic reading The slides were read under oil immersion with an Orthoplan research microscope. Twenty-five fields were examined longitudinally at the diameter, ex- cluding the marginal regions of the smear. The slides of Experiment 1 were read by one reader and those of Experiment 2 by another reader. Both were skilled bacteriologists. The reader of Experiment 2 (in addi- tion to counting the acid-fast rods) also assigned a rating to each slide for the quality of the bacterial staining: rating 3 was given to slides in which most of the bacterial population was well stained, rating 2 to mediocre smears, and rating 1 to those containing poorly-stained organisms. RESULTS Experiment I Of the 1 136 digested sputum specimens stained by both methods, 655 were negative and 481 were posi- tive-i.e., acid-fast organisms were detected in them -by at least one of the methods. Ziehl-Neelsen- positive and strip-negative results were obtained in 52 cases, whereas 26 specimens were positive only by the strip procedure. The results obtained by the two methods were in agreement for 93.1 % of the specimens examined. With the Ziehl-Neelsen method, 94.6% of the 481 positive specimens were detected, whereas the efficiency of the strip method was only 89.2%. The grouping of the 1 136 pairs of slides into 9 classes, each with a different range of bacterial counts per slide, is demonstrated in Table 1. The strip method gave results very similar to those of the Ziehl-Neelsen method in all classes, including specimens containing only a small number of acid- fast bacteria. This similarity is remarkable in view of the somewhat uneven distribution of bacteria despite careful preparation of the smears. The distribution of bacteria on three pairs of slides (one of each pair stained by the Ziehl-Neelsen method and the other by the strip method) is illustrated in Fig. 1. The duplicates were selected at random from three classes, each with a different bacterial range. Demonstration of the bacterial dis- tribution based on the average of 10 slides, as proposed by Engbaek et al. (1), was avoided, since it would have simulated a more uniform dispersion of acid-fast rods. Statistical comparisons of the methods were made in the categories containing acid-fast organisms. The analysis was carried out by means of logarithms of the positive counts (Table 2). The average logarith- mic bacterial counts per field of the 403 pairs of positive slides stained by the Ziehl-Neelsen and strip methods were 1.9901 and 1.9490, respectively. The correlation between the two methods is very high (0.9571). However, the paired t test shows that the mean of the Ziehl-Neelsen method is significantly higher (P = 0.001) than that of the strip method. Therefore, it may be concluded that the latter yields lower counts on the whole, and the relative efficiency may be estimated by 10-0.0411 = 91 % Moreover, the variance of the strip method is significantly greater (P < 0.01) than that of the Ziehl-Neelsen method when calculated by the F test for correlated variances (see Snedecor & Cochran (2), pp. 195-197). In the categories having negative counts (Z > 0 when S = 0, and Z = 0 when S > 0), the Ziehl-Neelsen method yielded twice as many positive slides as the strip method, the difference being significantly greater (P < 0.005) by the X' test for paired samples (see Snedecor & Cochran (2), p. 213). 84 STAINING METHODS FOR ACID-FAST BACTERIA 85 Table 1. Comparison of 1 136 pairs of slides stained by the strip method and by the Ziehl-Neelsen method, respec- tively, according to bacterial count Strip method Range 0 1-5 6-10 11-50 51-100 101-500 501-1000 1001-5000 5000+ 0 655 9 11 6 - - - - 1-5 23 24 15 5 - - - - - 6-10 16 11 10 7 1 - - - - 11-50 11 13 6 52 11 2 - - - Ziehi- Neelsen 51-100 2 2 1 20 23 10 - method 101-500 - - - 4 7 63 9 - - 501-1000 - - - - - 5 23 10 - 1001-5000 - - - - - 2 8 56 1 5000+ - - - - - - - 1 1 Range: 2515-00 S 1 5 10 15 20 25 1 5 10 15 20 25 a SIGHT FIELDS - > Pange: S01-1000 1 5 10 15 20 25 Fig. 1. Distribution of acid-fast bacteria in 25 microscopic fields of 3 pairs of slides (Z = Ziehl-Neelsen, S = strip method). Range: 10 -25 Z 60 40 2 - 0 2020 0 c 9 Y1 U0 .- I. s a I a a a I s P. VARUGHESE ET AL. Table 2. Statistical analysis (carried out on the logio scale) of duplicate smears prepared from 481 digested positive sputum specimens stained by the Ziehl-Neelsen (Z) and strip (S) methods Method Sample log Z log S log S - log Z Coefficient category size mean s.d. mean s.d. mean s.d. of correlation Z-0, S-0 655 Z>0, S-0 26 0.8959 0.3205 Z-0, S>0 52 . 0.8560 0.3607 Z>0, S>0 403 1.9901 0.8740 1.9490 0.9180 -0.0411 0.2659 0.9571 To estimate the overall difference in detection rates, we used the value 52/1 136 - 26/1 136 = 2.3% with an estimated standard error of 0.77 %. In the 52 smears that were positive only with Ziehl-Neelsen staining, the average logarithmic bacterial count per slide was 0.8959, whereas it was 0.8560 in the 26 strip-positive slides. The slides positive by only one of the staining procedures generally had a very low bacterial count. Experiment 2 The results of the bacterial count and the quality of stain uptake in 307 pairs of slides are shown in Table 3. With the sputum specimens diluted in Sauton's medium (Nos. 2, 3, 4, and 5), the strip method yielded average bacterial counts ranging from 71.9% to 97.6% of the Ziehl-Neelsen readings. The bacterial staining quality of the two methods was similar. With both, the rating was high for samples Nos. 3 and 5, somewhat lower for No. 2, and lowest for No. 4. For the water-diluted sample No. 6 and its bacteria-enriched modifications (Nos. 7 and 8), the strip method yielded considerably lower bacterial counts than the Ziehl-Neelsen method. As lower counts were repeatedly obtained in most of the strip-stained replicates of sample No. 6, it is clear that the reduced harvest of acid-fast organisms was not a chance occurrence resulting from the unsatis- factory preparation of a few slides. With the strip method, 26 (65.0%) of 40 replicate smears gave Table 3. Bacterial counts and quality of staining with the strip and Ziehl-Neelsen methods in direct sputum smears No. of Mean of the bacterial Rating of the Bacterial counts of S Sputum pairs of counts per slide bacterial staining staining expressd sample slidespZiehl- strip Ziehl- strip as a percentage of Zprepared Neelsen staining Neelsen staining couInts 2 45 110.2 79.2 77.8 71.1 71.9 3 35 4.2 a 4.1 b 85.4 88.5 97.6 4 50 124.7 100.0 52.7 62.0 80.2 5 46 1195.0 1 016.0 100.0 92.8 85.0 6 40 69.5 18.2 C 94.2 65.0 26.2 7 33 1 095.9 572.2 87.9 57.6 52.2 8 27 272.3 154.6 90.1 59.3 56.8 9 31 1.2 d 1.4 d 76.9 100.0 116.7 a b d 3 slides did not exhibit bacteria. 6 slides did not exhibit bacteria. 1 slide did not exhibit bacteria. 18 slides did not exhibit bacteria. 86 STAINING METHODS FOR ACID-FAST BACTERIA bacterial counts below 30% of those obtained with the Ziehl-Neelsen method; 10 (25.0%) produced counts of between 31 % and 50%; and only 4 (10.0%) achieved a comparable reading, ranging from 51 % to 100% of the acid-fast bacilli detected by the Ziehl-Neelsen method. The decrease in the bacte- rial count obtained with the strip method is accom- panied by a lower rating of the bacterial staining. The same applies to the enriched aliquots of sputum specimen No. 6 (Nos. 7 and 8) in which the bacterial counts with the strip method, although higher than in the parent sputum, were still considerably dimin- ished. The increase in the count could be attributed either to further dilution of the specimen by the addition of bacterial suspension or to the better staining capacity of the supplementary strain. It is noteworthy that, with samples Nos. 7 and 8, similar bacterial yields (52.2% and 56.8% of that with the Ziehl-Neelsen method) and stain ratings were pro- cured although No. 7 was enriched with nearly four times as many bacteria as No. 8. The results for these slides imply that the poor staining quality of the organisms was not responsible for the decreased bacterial count with the strip method. The relationship between the two facts is indirect, arising from a common factor-probably the physical properties of the smear. Sputum speci- mens yielding low counts by the strip procedure frequently evinced unsatisfactory staining intensity of the bacteria in several replicates. It appears that consistency, thickness, and penetrability of the smear have a greater bearing on the strip method than on the Ziehl-Neelsen method. This is in conformity with the satisfactory performance of the strip method in digested sputum specimens spread evenly in a thin layer, as well as the fairly acceptable results achieved in direct sputum specimens diluted with Santon's medium, which yielded uniform films. The sputum specimens diluted with water varied in consistency and dried unevenly, with an impermeable thicker core. Consequently the strip staining method gave less satisfactory results. For statistical comparison of the bacterial counts of 307 pairs of slides, the transformation loglo (bacterial count+ 1) was effected. The paired t test was used to compare the means within each sputum sample, the difference being log (S+1)-log (Z+ 1). The results are shown in Table 4. It may be noted from the mean differences that the counts obtained with the strip method were significantly lower, on the logarithmic scale, than those with the Ziehl-Neelsen method, except in the paucibacillary samples (Nos. 3 and 9). The difference was more pronounced in the samples diluted with water. In order to evaluate the day-to-day variation of the staining methods with each diluent, a nested analysis was carried out for each method and for the paired differences of the corresponding smears. Table 5 gives the results for the samples diluted with Sauton's medium (Nos. 2, 3, 4, and 5). It may be inferred from the analysis that both staining procedures differen- tiate between the above-mentioned sputum samples Table 4. Summary of the statistical analysis of the two staining methods carried out on samples diluted with Sauton's medium and with water, by means of the paired t test Sputum Sample Mean ~~~~~StandardSputum Sampl Diluent difference a deviation t Probabilitysample No.size difference G ~~of difference 2 45 Sauton -0.205 0.28 -4.98 <0.001 3 35 Sauton -0.077 0.47 -0.97 n.s. 4 50 Sauton -0.159 0.35 -3.24 0.01 5 46 Sauton -0.071 0.21 -2.29 0.03 6 40 water -0.675 0.42 -10.13 <0.001 7 33 water b -0.291 0.21 -7.76 <0.001 8 27 water b -0.249 0.19 -6.65 <0.001 9 31 water 0.012 0.44 0.15 n.s. a Mean of logio (S + 1) - mean of logio (Z + 1). b Aliquot of sample No. 6 enriched with bacterial suspension. 87 P. VARUGHESE ET AL. Table 5. Nested analyses of variance for the Ziehl-Neelsen and strip methods carried out on sputum samples (Nos. 2, 3, 4, and 5) diluted with Sauton's medium d.f. S.S. M.S. Variance F P LOG (Z+1) Between sputa 3 117.6492 39.2164 0.8950 880.82 (<0.001) Between days/sputa 93 3.4535 0.0437 0.0044 1.27 n.s. Replicates/days/sputa 79 3.2079 0.0345 0.0345 LoG (S+1) Between sputa 3 118.4226 39.4742 0.8990 315.77 (<0.001) Between days/sputa 93 9.3970 0.1189 0.0321 2.323 (0.001) Replicates/days/sputa 79 4.7615 0.0512 0.0512 Lo (S+1) - LO (Z+1) Between sputa 3 0.5476 0.1815 0.0007 1.208 n.s. Between days/sputa 93 11.4681 0.1452 0.0311 1.826 (0.01) Replicates/days/sputa 79 7.3901 0.0795 0.0795 and that the difference is relatively constant. Whereas the Ziehl-Neelsen method is not significantly affected by day-to-day differences in smear preparation and staining, the strip procedure shows significant varia- tion between days. The correlation coefficient be- tween log (Z+ 1) and log (S+ 1) was 0.925 for specimens Nos. 2, 3, 4, and 5. Nested analyses of the samples diluted with water were undertaken separately on those containing only the bacteria originally present (Nos. 6 and 9) and those enriched with bacterial suspension (Nos. 7 and 8). The results of the nested analyses of variances for log (Z+1) and log (S+1) as well as for the difference of the two values are shown in Tables 6 and 7. It may be inferred from these tables that both staining methods differentiate between sputa. For the samples diluted with water (Nos. 6 and 9) the difference between the methods is not constant over Table 6. Nested analyses of variance for the Ziehl-Neelsen and strip methods carried out on sputum samples (Nos. 6 and 9) diluted with water d.f. S.S. M.S. components F P LOW (Z+1) Between sputa 1 43.6748 43.6748 1.2482 589.51 (0.001) Between days/sputa 37 2.5670 0.0696 0.0198 2.06 (0.05) Replicates/days/sputa 32 1.0816 0.0338 0.0338 Lo (S+1) Between sputa 1 14.000 14.000 0.396 84.20 (0.001) Between days/sputa 37 5.861 0.158 0.035 1.66 (0.10) Replicates/days/sputa 32 3.059 0.096 0.096 LOO(S+1) - LOG (Z+1) Between sputa 1 8.250 8.250 0.229 31.68 (0.001) Between days/sputa 37 9.054 0.245 0.069 2.05 (0.05) Replicates/days/sputa 32 3.820 0.119 0.119 88 STAINING METHODS FOR ACID-FAST BACTERIA Table 7. Nested analyses of variance for the Ziehl-Neelsen and strip methods carried out on sputum samples (Nos. 7 and 8) diluted with water and enriched with bacterial suspension d.f. S.S. M.S. Variance F Pcomponents LOG (Z+1) Between sputa 1 5.2118 5.2118 0.1809 159.04 (<0.001) Between days 38 1.3224 0.0348 0.0109 1.76 (0.1) Replicates/days 20 0.3947 0.0197 0.0197 LOG (S+1) Between sputa 1 4.5136 4.5136 0.1565 134.36 (<0.001) Between days/sputa 38 1.3436 0.0354 0.0095 1.59 (0.15) Replicates/days/sputa 20 0.4457 0.0223 0.022 LOG (S+1) - LOG (Z+1) Between sputa 1 0.0252 0.0253 0 0.612 n.s. Between days/sputa 38 1.5456 0.0406 0 0.88 n.s. Replicates/days/sputa 20 0.9220 0.0461 0.0461 samples and is significantly variable over days, whereas, for the samples enriched with bacterial suspension (Nos. 7 and 8) the difference between methods is relatively constant over both samples and days. Moreover, for the samples diluted with water (Nos. 6 and 9), both methods showed significant day-to-day variations, whereas, for Nos. 7 and 8, only slight day-to-day variation occurred within each method. It appears that, with those samples, the difference in the physical properties of the sputum produced significant day-to-day variations with both methods. However, the strip method may have been affected to a greater extent, as a result of which it gave lower bacterial counts. The correlation co- efficient between log (Z+ 1) and log (S+ 1) was 0.739 for samples Nos. 6 and 9 and slightly higher (0.812) for Nos. 7 and 8. DISCUSSION Considerable efforts have been made to improve the Ziehl-Neelsen method and to develop simple cold-staining procedures. The heating of slides is cumbersome, particularly under field conditions, and accidental overheating of the stain renders decoloriza- tion difficult. With insufficiently trained staff, the disadvantage of the Ziehl-Neelsen method is aggravated. With cold-staining techniques, the concentrated carbolfuchsin solution may precipitate prior to uti- lization or on the smear. Removal of the stain film by decolorizing agents is difficult and can affect the colour intensity of the acid-fast organisms. Kinyoun's method (3), involving 1-3 min of staining time, was designed originally for sputum concentrates yielding thin smears. The application of concentrated carbol- fuchsin solutions to ordinary sputum usually required an extended staining period, according to Smith (4) and Lynch et al. (5), in order to achieve the necessary efficiency. Muller & Chermock (6); Aubert (7); Desbordes et al. (8); and Gross (9) reported that the addition of detergents to the dye resulted in better stain penetration in a shorter time. The use of dimethylsulfoxide for cold staining, as recommended by Pottz et al. (10) and Muftic & Redmann (11), was rather disappointing in diagnostic microscopy (un- published data). It produced a granular staining of the mycobacteria and, despite careful decolorization, bacterial stain was often removed. The introduction of impregnated strips for the staining of mycobacte- ria is a new approach towards simplification of the Ziehl-Neelsen technique. Comparison of the results of the two methods on 403 positive slides in Experiment 1 showed a high correlation. On the other hand, the strip method had a greater variance (P < 0.01) than the Ziehl-Neelsen method, and the mean of the latter-calculated by the paired t test-was significantly greater (P = 0.001). Although the greater variance of a procedure may reflect on the accuracy and reproducibility of 89 P. VARUGHESE ET AL. the method, from a practical point of view it appears that both procedures can be employed with similar efficiency in the staining of digested sputum speci- mens. The strip method is convenient, particularly in laboratories where specimens are generally processed for seeding on culture medium and the digested concentrate can be utilized for smear preparation. Under these circumstances, the simplicity of the method offers a definite advantage over the rather cumbersome Ziehl-Neelsen technique. In experiment 2, the smears prepared from sputum mixed with Sauton's medium yielded lower counts by the strip method than by the Ziehl-Neelsen method; however, the staining quality of the organisms was not affected. In the case of specimens diluted with water, the strip method generally produced not only a lower bacillary count but also a poorer stain. The efficiency of the procedure appears to depend to a large extent on the preparation and consistency of the smears. In its present form, the method lacks flexibility, whereas the Ziehl-Neelsen method is bet- ter able to adjust to differences in the quality of smears. The 4 minutes allocated to the staining with carbolfuchsin-impregnated strips may not be suffi- cient for the complete penetration of thick smears. A prolonged staining time would produce a strong red background that could not be decolorized by the simultaneous decolorization-counterstain strip in one minute. An extension of the latter process may accentuate the counterstain and interfere with the detection of the acid-fast organisms. To standardize this type of " matched " procedure, the quality of the smear must be adjusted to the method. A selective preparation of the smears, as advised by Koch (12), would promote the use of strips in routine work. By careful removal of caseous debris or purulent mate- rial from the sputum, as described by Spendlove (13), a thin smear can readily be prepared. The authors are well aware of the limitations of procedures involving simultaneous decolorization and counterstaining. However, the new method employing carbolfuchsin-impregnated strips for staining is not bound to the use of simultaneous decolorizing and counterstaining. The preparation of carbolfuchsin-impregnated strips is extremely simple (see Annex 1). The strips may be employed indepen- dently of the simultaneous decolorization-counter- staining strips, which are an impediment rather than an asset to the method. In a current study based on the experience of the present investigation, the carbolfuchsin strip is being used on undiluted sputum with a well-matched decolorizing procedure, independent of counterstain- ing. This modification is being compared with the present strip method and with the Ziehl-Neelsen method. ACKNOWLEDGEMENTS This study was assisted by a grant from the World Health Organization and the authors express their gratitude for the support. They also thank Mrs Vera Handzel for technical help and Mrs Vasantha Narasimhan for assisting in the pre- paration of the manuscript. RtSUMIt COMPARAISON DE LA METHODE DES BANDES ET DE LA METHODE DE ZIEHL-NEELSEN POUR LA COLORATION DES BACTERlES ACIDO-RPSISTANTES On a entrepris de comparer la m6thode de Ziehl- Neelsen et la m6thode des bandes impr6gn6es. Cette derniere utilise deux types de bandes: l'une pour la coloration des bacteries et l'autre pour la d6coloration et la contre-coloration simultan6es des frottis. Les deux methodes ont 6te 6valuees sur 1136 frottis obtenus a partir de crachats trait6s par l'hydroxyde de sodium et sur 307 paires de frottis de crachats non trait6s. Le taux d'efficacit6 de la m6thode des bandes s'est revel6 comparable a celui de la m6thode de Ziehl- Neelsen pour la coloration des 6chantillons trait6s, mais avec le mat6riel non trait6 les r6sultats ont vari6 en fonction de la qualit6 des pr6parations. Avec des frottis d'epaisseur in6gale, la m6thode des bandes a fourni des num6rations bact6riennes inferieures a celles obtenues par la m6thode de Ziehl-Neelsen. Les auteurs exposent un certain nombre de suggestions destin6es a am6liorer la technique des bandes impr6gn6es. 90 STAINING METHODS FOR ACID-FAST BACTERIA 91 Annex I PROCEDURE FOR ACID-FAST STAINING AND PREPARATION OF CARBOLFUCHSIN STRIPS Ziehl-Neelsen method Filter paper cut to the size of a slide is placed over the smear and flooded with carbolfuchsin solution. The slide is heated 3 times until vapour appears. Staining time is approximately 5 min. The slide is washed with water, decolorized briefly with 15% sulfuric acid, and rinsed with water. After treatment with 95% ethanol for a few seconds, the slide is washed in running water, counterstained with a 0.2% aqueous solution of malachite green for 60 s, rinsed again with water, and dried in a vertical position. Strip method An acid-fast red strip a is dipped quickly into a 20% aqueous solution of methyl alcohol and placed on the smear for exactly 4 min. The strip is removed and excess stain is washed off in running water. A decolorizing-counterstaining strip immersed in methyl alcohol is placed over the smear for 1 min precisely. The slide is washed and dried in a vertical position. Modification of the strip method This tnodification calls for strips impregnated with carbolfuchsin-which may be prepared on the spot as shown below-and decolorizing solution. Preparation of carbolfuchsin-impregnated strips: 10 g of acid fuchsin b are dissolved in 100 ml of absolute ethyl alcohol. Pure, phenol crystals are melted at 65°C and 20 ml are added to the acid fuchsin solution. Large sheets of filter paper are cut into ribbons of the approximate width of a slide (2.5 cm). These strips are drawn through the staining solution and allowed to dry on a line in a vertical position. The alcoholic staining solution dries fast and the ribbons must be cut into strips without delay. The strips should be approximately 3/4 of the length of a slide (7.5 cm). They are packed in lots of 10-20 in small, airtight polyethylene bags and stored in a cool, dark place. Use of carbolfuchsin-impregnated strips: A strip is dipped quickly into a 20% aqueous solution of methyl alcohol and placed on the smear for 15 min. After the strip has been removed, the slide is washed under running tap-water to remove the excess stain. Decolorization is performed with a freshly prepared 5% aqueous solution of sodium sulfite. If necessary, it may be repeated in order to obtain colour-free smears. (A measuring spoon and measuring cylinder can be used for preparing the 5% sodium sulfite solution, so as to avoid weighing the chemical.) The slide is counterstained with an aqueous solution of 0.1 % malachite green for 30 s. a Supplied by ICN Winley-Morris Ltd, Montreal, Canada. b As acid fuchsin is more readily soluble in alcohol than basic fuchsin is, its concentration in the staining solution may be doubled, yielding strips with a higher fuchsin content. 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