A comparison of three media for isolation of Nocardia species from clinical specimens.S Ayyar, U Tendolkar, L Deodhar
Dept of Microbiology, LTMM College, Sion, Bombay.
The study was carried out to compare the efficacy of three media namely Modified Thayer Martin medium, McClung's carbon free broth with paraffin bait and paraffin agar in isolating Nocardia species from clinical specimens. Two hundred and seventy six clinical specimens from 245 cases were studied which included cases of bronchopulmonary and systemic infections and cases of mycetoma. Paraffin agar was found to be an inexpensive and selective medium for isolation of Nocardia species when compared with Modified Thayer Martin medium and paraffin bait techniques.
Keywords: Comparative Study, Culture Media, standards,Evaluation Studies, Human, India, epidemiology,Nocardia, growth &development,isolation &purification,Nocardia Infections, epidemiology,microbiology,Sensitivity and Specificity, Support, Non-U.S. Gov′t,
Nocardia organisms are gram positive, partially acid fast, non- spore forming bacilli found in nature in soil and aquatic environments. Nocardiosis is initiated by inhalation of the organisms. The organism grows well, but slowly on all laboratory media making their recovery difficult from clinical specimens. Moreover, the presence of Nocardia in such clinical specimens is often marred by the early growth of other flora. A variety of selective media for the recovery of Nocardia species have been suggested, which include Sabouraud's dextrose agar with chloramphenicol, which, however proved inhibitory to many Nocardia isolates, and the more recently described Modified Thayer Martin medium. Presently, the paraffin baiting technique devised by Gordon and Hagan is being used successfully in many laboratories. Shawar et al formulated three chemically defined media including paraffin agar medium for the selective recovery of Nocardia from clinical specimens.
The present study was designed to evaluate the comparative abilities of three media used for isolation of Nocardia species in our laboratory, viz. the conventional paraffin baiting technique using carbon free broth, Modified Thayer Martin medium and paraffin agar in recovering Nocardia species from clinical specimens.
Clinical specimens (sputa, bronchoscopic aspirates, gastric lavages, pus, CSIF) from patients of suspected tuberculosis/mycosis and biopsy material and aspirates from cases of mycetoma have been included in the study.
Sputum was homogenised by vigorous shaking with glass beads while biopsied tissues were homogenised using tissue grinders. Smear preparations were stained by the Grams and Ziehi Neelsen's techniques (using 1 % and 20% H2SO4 as decolouriser). All the specimens were processed on routine culture media (blood agar, Mcconkey's agar and Sabouraud's agar). Prior to the digestion procedure, the specimens were inoculated into carbon free broth for paraffin baiting, modified Thayer Martin medium and paraffin agar for recovery of Nocardia species.
I Paraffin baiting techniques:
About 2 ml of the specimen was inoculated into 5-7 ml of McClung's carbon free broth, which is essentially made up of salts.
NaNO3 - 29; K2HPO4 - 0.19; MgSO4.7H2O - 0.5g ;
FeC13 - 10mg ; MnC12.4H2O - 8mg; ZnSO4 -2mg
Distilled water - 1 litre ; pH 7.2
The carbon source was provided by means of a paraffin coated glass rod suspended in the medium by a cotton wool plug. Growth appearing after incubation at 370C was scraped off and plated on Sabouraud's dextrose agar for obtaining single colonies.
II Modified Thayer Martin medium (MTM):
Thayer Martin medium base (HiMedia) was used for the study. 5% haemolysed human blood and supplement containing 4 ug/mi of vancomycin, 7.5 ug/mi of colistin and 12.5 ug/m] of nystatin (VCN) was added to the sterilized basal medium, which was poured out in the form of slants. (No vitamin supplement/haemoglobin was added to the medium).
III Paraffin agar medium:
The paraffin agar medium used in our laboratory is a modified version of the medium suggested by Shawar et al. We have made use of paraffin wax (60-620C solid) as against paraffin oil (viscosity at 37.8?C - 345-355) used by Shawar et al. Carbon free agar was prepared by adding 2% agar and to Mclung's carbon free broth.
Liquefied paraffin wax was added in a ration of 9:11. While dispensing in petridishes, the flask was stirred continuously to ensure an even dispersion of paraffin in the medium. On solidifying, the paraffin set in globular form throughout the medium. Nocardia species was found to grow around the globules, on the surface of the medium in the form of a white chalky fringe [Figure - 1]
Two hundred and seventy-six clinical specimens from 245 cases were studied for recovery of Nocardia. Of these, 262 specimens were from 233 cases of bronchopulmonary and systemic infections and 14 from 12 cases of mycetoma.
Nocardiosis was detected in 6 of the 233 cases of bronchopulmonary and associated diseases investigated, giving a prevalence of 2.5%. Three isolates were recovered from the mycetoma cases studied.
All the 9 cases showed positivity for Nocardia in smear stained by the Ziehi-Neelsen technique using 1% H2SO4 as the decolouriser.
Growth patterns of Nocardia species on the three media used is as shown in [Table - 1].
[Table - 1] provides evidence of the equal sensitivity of paraffin baiting technique and paraffin agar in recovering Nocardia species. But paraffin agar could be the medium of choice considering the relatively lesser time required for isolation of the organism. The basis for the ability of Nocardia species to grow on such medium is its requirement of simple organic compounds as the source of carbon for growth. The fact that the paraffin baiting technique and paraffin agar were used successfully to grow Nocardia species, points towards the effective utilization of paraffin and its high suitability towards the growth of Nocardia species.
Modified Thayer Martin medium, in spite of being an enriched and highly nutritive medium, failed to give a good recovery rate of Nocardia owing to the growth of other bacteria, towards which VCN supplement proved non-inhibitory. However, MTM was found to be a good medium for subculture of Nocardia from paraffin agar and paraffin baits, for obtaining single colonies.
In conclusion, paraffin agar is an inexpensive, selective agar medium for Nocardia species. It is a modification of the paraffin baiting technique, with the added ability for quicker recovery of the organisms. Although the number of isolates in this study is relatively small, taking into consideration the low prevalence of Nocardia, the significance of our study is justified.
Since all the 9 culture positive cases were also found positive in smear, the efficacy of the three media in growing Nocardia species from smear negative cases warrants further investigation.
The authors wish to thank the R. D. Birla Smarak Kosh for their financial assistance in carrying out the above study.
[Figure - 1][Table - 1]