p. 1891-1892

Vol. 30, No. 7

0095-1137/92/071891-02$02.00/0 Copyright © 1992, American Society for Microbiology

Selective Buffered Charcoal-Yeast Extract Medium for Isolation of Nocardiae from Mixed Cultures MELISSA A. GARRETT, HARVEY T. HOLMES,t AND FREDERICK S. NOLTE* Department of Pathology and Laboratory Medicine, Emory University School of Medicine and Emory University Hospital, Atlanta, Georgia 30322 Received 10 January 1992/Accepted 16 April 1992

The recovery of Nocardia species from mixed cultures is facilitated by use of a selective medium. We show that buffered charcoal-yeast extract medium with polymyxin, anisomycin, and vancomycin can be used for the selective isolation of nocardiae from contaminated specimens.

Some Nocardia species cause a wide spectrum of diseases including pulmonary infection; cutaneous and subcutaneous abscesses; bloodstream invasion with secondary involvement of the brain, kidneys, and other organs; and actinomycotic mycetoma. Pulmonary nocardiosis has been increasingly recognized as a serious infection in patients with underlying neoplastic disease and in organ transplant recipients (5, 8). Nocardiae are aerobic, filamentous, partially acid-fast bacilli that grow slowly on most culture media. They may be obscured by rapidly growing contaminants present in specimens such as sputa. Several approaches to the selective isolation of nocardial species have been described. These include the use of selective media (1, 4, 7), specimen decontamination (3), and paraffin baiting (9). Decontamination of respiratory specimens with N-acetyl-L-cysteine, NaOH, or benzalkonium chloride in trisodium phosphate is toxic for nocardiae, and paraffin baiting, although it results in higher efficiency of isolation, may take weeks to yield positive results. Sabouraud dextrose agar with chloramphenicol was suggested for recovery of Nocardia species from sputum, but many isolates are susceptible to this antibiotic (1). Murray et al. demonstrated that modified Thayer-Martin medium supports the growth of nocardiae and suppresses the normal oropharyngeal flora (4). Media containing paraffin as the sole source of carbon have also been shown to be effective for the selective isolation of nocardiae from seeded sputum specimens (7). We recently recovered Nocardia asteroides from two bronchoalveolar lavage fluids on selective buffered charcoalyeast extract medium (sBCYE), a medium formulated for the isolation of Legionella species from contaminated specimens. In both cases the Nocardia species was completely obscured on the nonselective media by the normal upper respiratory tract flora. This observation prompted us to investigate whether sBCYE could serve as a selective medium for nocardiae as well as for Legionella species. (This work was presented in part at the 90th Annual Meeting of the American Society for Microbiology, Anaheim, Calif., 13 to 17 May 1990 [la].) Eleven recent clinical isolates of N. asteroides and one isolate of Nocardia brasiliensis were obtained for the study from the culture collections of the Centers for Disease


Corresponding author.

t Present address: St. Joseph Mercy Hospital, Pontiac, MI 48341.

Control (June Brown) and the Clinical Microbiology Laboratory, Emory University Hospital. The cultures were identified by accepted methods (2) and stored on Sabouraud dextrose agar at room temperature until use. Growth from Sabouraud dextrose agar was suspended in saline and dispersed by vortex mixing with glass beads. The large aggregates were allowed to settle, and the resulting clarified suspension was adjusted to a turbidity of a 0.5 McFarland standard. Serial 10-fold dilutions were made in saline, and 0.1 ml was inoculated onto sBCYE agar containing polymyxin (40,000 U/liter), anisomycin (80 mg/liter), and vancomycin (0.5 mg/liter) (Remel, Lenexa, Kans.) and chocolate agar (CA) (GC agar base with Iso VitaleX enrichment; BBL, Cockeysville, Md.). The average colony size of 10 well-isolated colonies on each medium and the plating efficiency of sBCYE were determined after incubation for 5 days at 35°C. The colony diameters were measured with a micrometer and a stereomicroscope. The plating efficiency was determined by dividing the colony counts on sBCYE by the colony counts on CA. Colony counts were determined for all strains at the same dilution (10-4) by counting from 8 to 291 colonies per plate. Tests were performed in duplicate. In a second set of experiments, serial 10-fold dilutions of standardized suspensions of five strains of N. asteroides were prepared as described above. Cotton-tipped applicators were sequentially dipped in pooled expectorated sputum and then in a 10-3, 10-4, or 10-5 dilution of the nocardial suspension. The same applicator was used to inoculate both media, and in each case the CA was inoculated first. Plates were streaked for isolated colonies by using a quadrant pattern, and the growth of nocardiae was assessed semiquantitatively and scored from 1+ to 4+. The comparison of the growth of 12 strains of Nocardia on sBCYE and CA is shown in Table 1. All strains grew on sBCYE, and the plating efficiency ranged from 64 to 132% (mean, 100%). The mean colony diameter ranged from 0.42 to 1.69 mm (mean, 1.00 mm) on CA, compared with 1.22 to 2.51 mm (mean, 1.76 mm) on sBCYE (P < 0.01). The use of sI3CYE substantially enhanced the detection of five strains of N. asteroides in seeded sputum (Table 2). There was a 1- to 2-log increase in detection when results of cultures on sBCYE and CA were compared. In addition, growth of nocardiae was evident 1 to 2 days earlier on sBCYE. Our study demonstrated that medium developed for the selective isolation of Legionella strains can also support the growth of Nocardia strains, and its use may enhance the 1891




TABLE 1. Comparison of the growth of 12 Nocardia strains on sBCYE and CA Avg colony diam

% Plating efficiencya

(mm) on:


N. asteroides 308 309 310 311 313 314 315 316 319 475 3318 N. brasiliensis



1.80 1.77 2.05 1.91 1.26

1.22 1.69

80 122





2.11 1.40 1.43

0.49 1.18 0.84 0.56

2.09 1.22 1.55

0.98 0.63 1.17

67 101 66 132 64



The sBCYE used in the study was a gift from Remel.

64 106 118

a Percent plating efficiency = (number of colonies on sBCYE/number colonies on CA) x 100.

when techniques and selective media for Legionella strains were used (10). A similar selective medium, sBCYE agar, with polymyxin, anisomycin, and cefamandole, supports the growth of Brucella species and, potentially, can also be used for the selective isolation of these organisms from mixed cultures (6). In summary, the medium used for the selective isolation of Legionella species can also be used for the recovery of nocardiae from mixed cultures. Inclusion of sBCYE routinely in sputum cultures from immunocompromised patients may be justified, considering that this medium will facilitate the recovery of two important opportunistic pulmonary pathogens.


recovery of these organisms from contaminated specimens such as sputum. The growth of nocardiae on sBCYE was equivalent or superior to that on nonselective, nutritionally rich CA. The clinical utility of these observations is supported by our own experience and the recent report of Vickers et al. describing three cases of pulmonary nocardiosis in which N. asteroides was isolated from sputum only TABLE 2. Comparison of the recovery of five N. asteroides strains from seeded sputum on sBCYE and CA Growth of Nocardia strains at indicated dilution' 10-4 10-5 10-3





2+ 2+

2+ 1+

1+ NG



1+ NG

1+ NG




3+ 2+

2+ NG

1+ NG



1+ 1+

1+ NG




1+ NG

1+ NG


aGrowth was assessed semiquantitatively and scored from 1 + (least growth) to 4+ (most growth); NG, no growth.

REFERENCES 1. Ajello, L., and G. D. Roberts. 1983. Mycetomas, p. 1033. In W. J. Hausler (ed.), Diagnostic procedures for bacterial, mycotic, and parasitic infections. American Public Health Association, Washington, D.C. la.Malone, M. A., H. T. Holmes, and F. S. Nolte. 1990. Selective medium for Legionella also supports the growth of Nocardia, abstr. C-48, p. 352. Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990. American Society for Microbiology, Washington, D.C. 2. Mishra, S. K., R. E. Gordon, and D. A. Barnett. 1980. Identification of nocardiae and streptomycetes of medical importance. J. Clin. Microbiol. 11:728-736. 3. Murray, P. R., R. L. Heeren, and A. C. Niles. 1987. Effect of decontamination procedures on recovery of Nocardia spp. J. Clin. Microbiol. 25:2010-2011. 4. Murray, P. R., A. C. Niles, and R. L. Heeren. 1988. Modified Thayer-Martin medium for recovery of Nocardia species from contaminated specimens. J. Clin. Microbiol. 26:1219-1220. 5. Palmer, D. L., R. L. Harvey, and J. K. Wheeler. 1974. Diagnostic and therapeutic considerations in Nocardia asteroides infection. Medicine (Baltimore) 53:391-401. 6. Raad, I., K. Rand, and D. Gaskins. 1990. Buffered charcoalyeast extract medium for the isolation of brucellae. J. Clin. Microbiol. 28:1671-1672. 7. Shawar, R. M., D. G. Moore, and M. T. LaRocco. 1990. Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens. J. Clin. Microbiol. 28:508-512. 8. Simpson, G. L., E. B. Stinson, M. J. Egger, and J. S. Remmington. 1981. Nocardial infections in the immunocompromised host: a detailed study in a defined population. Rev. Infect. Dis. 3:492-507. 9. Singh, M., R. S. Sandhu, and H. S. Randhawa. 1987. Comparison of paraffin baiting and conventional culture techniques for isolation of Nocardia asteroides from sputum. J. Clin. Microbiol. 25:176-177. 10. Vickers, R. M., J. D. Rihs, and V. L. Yu. 1992. Clinical demonstration of isolation of Nocardia asteroides on buffered charcoal-yeast extract media. J. Clin. Microbiol. 30:227-228.

Selective buffered charcoal-yeast extract medium for isolation of nocardiae from mixed cultures.

The recovery of Nocardia species from mixed cultures is facilitated by use of a selective medium. We show that buffered charcoal-yeast extract medium ...
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