Mycobacteria from Sputum Specimens

Transcription

1 Microbiol. Immunol. Vol. 23 (9), , 1979 "T ween Egg Medium" for Isolating Mycobacteria from Sputum Specimens Michio TSUKAMURA, *Haruo TOYAMA, and Yuji FUKAYA *The National Chubu Hospital, Obu, Aichi (Received for publication, December 1, 1978) Abstract 'Tween egg medium,' in which Tween 80 was used as a carbon source, showed a higher rate of positive cultures than Ogawa egg medium in isolation of mycobacteria from sputum specimens. The Ogawa egg medium (9) is an egg medium widely used in Japan. It was shown to be a good medium comparable to the Lowenstein-Jensen medium (5) for isolating mycobacteria from sputum specimens (3, 6, 7). It is now desirable to find a better medium than these egg media for isolating dysgonic mycobacteria and for improving the rate of isolation of mycobacteria from clinical specimens. After discovery of the Dubos medium (2), Tween 80 (polyoxyethylene sorbitan monooleate) was shown to be utilized by tubercle bacilli as a good carbon source (1, 11, 17, 18). Because of this property, Tween 80 was supplemented in egg media without omitting glycerol, but this trial failed to improve the rate of positive cultures (4, 8), although the use of such media succeeded in obtaining a better growth of Mycobacterium tuberculosis and M. bovis. It has been suggested that this failure may be due to the fact that, in the presence of glycerol, Tween 80 can inhibit the growth of tubercle bacilli (10). On the other hand, it was shown that synthetic agar media, in which Tween 80 was used as the sole carbon source, supported good growth of M. tuberculosis (17) and of dysgonic strains of M. intracellulare (15). In the present study, a new egg medium, in which Tween 80 was used in the absence of glycerol, was shown to be somewhat superior to the Ogawa egg medium in obtaining a higher rate of positive cultures in the isolation of organisms from sputum specimens, even though the difference between these two media remained insignificant statistically. MATERIALS AND METHODS The composition of the Ogawa egg medium was as follows : basic solution ( 1 % (w/v) KH2PO4 and 1% (w/v) sodium glutamate), 100 ml; whole eggs, 200 ml ; glycerol, 6 ml ; and 2% (w/v) aqueous solution of malachite green, 6 ml. The composition of the 'Tween egg medium' was as follows : basic solution (1 % (w / v) KH2PO4, 1% (w/v) sodium glutamate, and 0.1 % (wily) MgSO4 E 7H2O), 100 ml ; whole eggs, 200 ml ; Tween 80 (Katayama Chemical Co., Osaka), 2 ml; and 2 % 833

2 834 M. TSUKAMURA ET AL (w/v) aqueous solution of malachite green, 6 ml. Therefore, the Tween egg medium is a modified Ogawa egg medium, in which Tween 80 has been substituted for glycerol. Both Ogawa egg medium (control) and Tween egg medium were poured in 7 ml amounts into tubes, 165 by 16.6 mm, and prepared in the form of slants by sterilization at 90 C for 60 min. As the materials examined, sputum specimens of patients hospitalized in the tuberculosis departments of this hospital were employed. A morning sputum specimen was added to an equal volume of a 4% (w/v) NaOH solution, shaken at room temperature for 15 min, and inoculated into an Ogawa egg medium slant and a Tween egg medium slant with a spiral loop that can deliver a 0.02 ml sample. The tubes inoculated were stoppered with a rubber cap with a pin hole and incubated at 37 C for 8 weeks. Readings of the results were carried out after 3, 4, and 8 weeks of incubation. Two methods of inoculation were employed : one with a one loopfulinoculation and another with a two loopful-inoculation in three experiments carried out independently. The method of inoculation with the spiral loop was shown to be superior to the inoculation with a 0.1 ml-pipette in obtaining more positive cultures (12, 13). All sputum specimens used in the experiments were taken from different patients. Growing colonies were examined by the Ziehl-Neelsen method and then screened for mycobacteria other than tubercle bacilli by testing growth on p-nitrobenzoic acid medium (16). The species of mycobacteria were identified by the methods described previously (14). RESULTS Contaminations occurred in 3, 5, and 1 samples in the three experiments, respectively. These occurred in both media. Hence, these samples were omitted in estimating the rate of positive cultures. The rate of positive cultures between the two media was compared in a total of 453, 473, and 431 samples, which were inoculated into the media with one loopful (Experiment I) and two loopfuls (Experiments II and III), respectively. The rate of positive cultures in the Tween egg medium was higher than that of the Ogawa egg medium (Tables 1 to 4) although difference between the two media was slight. The number of colonies grown in the Tween egg medium was greater than that in the Ogawa egg medium (Table 5). Furthermore, the growth on the Tween egg medium occurred faster than that on the Ogawa egg medium (Table 6). In the three experiments, 78, 69, and 71 samples, showed positive growth of mycobacteria (including Rhodococcus) in either or both media after incubation for 8 weeks. Out of these, a total of 21 and 29 cultures on the Ogawa egg medium and the Tween egg medium, respectively, belonged to mycobacteria other than tubercle bacilli. The 21 and 29 strains consisted of 18 strains of M. intracellulare, one strain of M. gordonae and two strains of Rhodococcus; and of 19 strains of M. intracellulare, four strains of M.gordonae,two strains of M.,fortuitum and four strains of Rhodococcus,

3 TWEEN EGG MEDIUM FOR ISOLATION OF MYCOBACTERIA 835 Table 1. Comparison of growth of mycobacteria after 8 weeks between Ogawa egg medium and Tween egg medium (Experiment I: Inoculation with one loopful of sputum samples) The numbers in the table show the number of sputum specimens obtained from different patients. Tablc 2. Comparison of grow th of mycobacteria after 8 weeks between Ogawa egg medium and Tween egg medium (Experiment II: Inoculation with two loopfuls of sputum samples) The numbers in the table show the number of sputum samples obtained from different patients. Table 3. Comparison of growth of mycobacteria after 8 weeks between Ogawa egg medium and Tween egg medium (Experiment III : Inoculation with two loopfuls of sputum samples) The numbers in the table show the number of sputum samples obtained from different patients.

4 836 M. TSUKAMURA ET AL Table 4. Comparison of the rate of positive cultures between Ogawa egg medium and Tween egg medium Table 5. Comparison of the amount of growth between Ogawa egg medium and Tween egg medium Table 6. Comparison of the rate of positive cultures after incubation for 3 or 4 weeks between Ogawa egg medium and Tween egg medium Table 7. Comparison of the rate of positive cultures in patients who showed positive smears between Ogawa egg medium and Tween egg medium

5 TWEEN EGG MEDIUM FOR ISOLATION OF MYCOBACTERIA 837 respectively. So-called 'smear-positive and culture-negative' samples occurred in the Ogawa egg medium for a total of 19 samples, and in the Tween egg medium for a total of 13 samples in the three experiments (Table 7). The number of 'smear-positive and culture-negative' samples decreased in the Tween egg medium. DISCUSSION The results of the present study demonstrated that the Tween egg medium was superior to the Ogawa egg medium in obtaining a higher rate of positive cultures, more colonies and a faster growth of mycobacteria. As a reason for this, it is suggested that, in the absence of glycerol, Tween 80 enhances the growth of some dysgonic mycobacteria in primary isolation, and that Tween 80 enhances growth as a good carbon source. The medium has been prepared based on the findings of Sattler and Youmans (10), who reported that the growth of tubercle bacilli utilizing Tween 80 as a carbon source may be inhibited by the presence of glycerol, and on the findings of several other authors (1, 11, 17, 18), who reported that Tween 80 is used by tubercle bacilli as a good carbon source. The new egg medium is recommended for isolating mycobacteria from sputum specimens. However, there is no advantage in using the medium for the purposes of subculturing mycobacteria or resistance tests, as M. tuberculosis tends to form S-type colonies in this medium and, therefore, differentiation among mycobacterial species by morphological characteristics of colonies becomes less clear-cut. The fact that M. tuberculosis tends to form S-type colonies in the Tween egg medium is not a great disadvantage since,according to our experience,recent isolates of M.tuberculosis from clinical specimens often tend to form S-type colonies even on the Ogawa egg medium and differentiation of M. tuberculosis from other mycobacteria always requires an exact indentification procedure. It is considered important that the use of the Tween egg medium permits the growth of some strains which do not form colonies on the Ogawa egg medium. In conclusion, it has been suggested that the Tween egg medium is more suitable than the Ogawa egg medium for isolating mycobacteria from sputum specimens. It has slightly improved the positive rate of isolation, and has provided more colonies at an earlier stage of incubation. REFERENCES 1) Dubos, R.J The effect of lipids and serum albumin on bacterial growth. J. Exp. Med. 85: ) Dubos, R.J., and Middlebrook, G Media for tubercle bacilli. Am. Rev. Tuberc. 56: ) Hayashi, O., Koyama, K., Moriwaki, F., and Ootake, A A comparative study on the two isolation culture methods (Ogawa and Lowenstein-Jensen) of tubercle bacilli in sputum. Japan. J. Bacteriol. 16: (in Japanese). 4) Hiramine, S., and Saito, H Cultivation of tubercle bacilli in a modified Ogawa egg medium to which Tween 80 was supplemented. Kekkaku 52: (abstract) (in Japanese). 5) Jensen, K.A Essai de standardisation des methodes de laboratoire. Bull. Union Int. Tuberc.

6 838 M. TSUKAMURA ET AL 24: ) Kalich, R., Bennert, G., Koedel, H., Ulber, H., Grunewald, R., Oqueka, F., Gerloff, W., und Knauf, B Vergleich der Leistungsfahigkeit eines modifizierten Ogawa-Nahrboden mit dem im DAB 7 zum kultuellen Nachweis von Mykobakterien vorgeschriebenen Nahrboden. Z. Erkr. Atmungsorgane. 145 : ) Krebs, A., und Kalich, R Ueber die Leistungsfahigkeit des asparagin-freien Einahrbodens nach Ogawa bei der Anzuchtung von Tuberkelbakterien. Z. Tuberk. 122: ) Nemoto, H., Oka, M., and Yokohori, H A study of the medium for bovine tubercle bacilli. Ann. Rep. Anim. Health Inst. Minist. Agri. 33: ) Ogawa, T., and Saba, K On the quantitative cultivation of tubercle bacilli. Kekkaku 24 (no. 2) : (in Japanese). 10) Sattler, T.H., and Youmans, G.P The effect of "Tween 80," bovine albumin, glycerol, and glucose on the growth of Mycobacterium tuberculosis var. hominis (H37Rv). J. Bacteriol. 56: ) Stinson, M. W., and Solotorovsky, M Interaction of Tween 80 detergent with mycobacteria in synthetic medium. I. Effect of Tween 80 on the growth and turbidimetric response of Mycobacterium avium cultures. Am. Rev. Respir. Dis. 104: ) Tsukamura, M Enumeration of viable numbers of Mycobacterium tuberculosis by utilizing spiral loop inoculation and egg medium slants. III. Kekkaku 35: (in Japanese). 13) Tsukamura, M A method of inoculation of samples with a spiral loop for isolation of tubercle bacilli from sputum specimens. Med. Biol. 91: (in Japanese). 14) Tsukamura, M Identification of mycobacteria, p In A publication of the Research Laboratory of the National Chubu Hospital, Obu, Aichi. 15) Tsukamura, M., Mizuno, S., and Toyama, H Enhancement of growth of slowly growing strains of Mycobacterium intracellulare by Tween 80. Kekkaku 45: (in Japanese). 16) Tsukamura, M., and Tsukamura, S Differentiation of Mycobacterium tuberculosis and Mycobacterium bovis by p-nitrobenzoic acid susceptibility. Tubercle 45: ) Yamane, I., Minami, K., and Yasui, T Tween 80 as a metabolite for tubercle bacilli. II. Culture medium containing Tween 80 as a sole source of carbon. Fukushima J. Med. Sci. 1: ) Youmans, A.D.,and Youmans,G.P.1954.Studies on the metabolism of Mycobacterium tuberculosis : IV. The effect of fatty acids on the growth of M. tuberculosis var. hominis. J. Bacteriol. 67: