by H. S. ANDLEIGH, M. D. (Path.), D. T. M., D. Bact., (Lond.)
Pro/essor o] Pathology and Bacteriology, S. M. S. Medical College, Jaipur, India. The isolation of pathogenic fungi from clinical material, such as the sputum, skin scrapings, and the discharges from the sinuses etc., is not a simple and straight-forward laboratory procedure. The contaminating microorganisms grow so profusely that they do not allow the proper growth of pathogenic fungi. Various techniques have been put forward from time to time with the object of suppressing the growth of saprophytic fungi and the contaminating bacteria. Several media have been described for tile isolation of fungi from clinical material such as sputum which is usually heavily contaminated with bacteria: In 1942 KURUHG used a dextrose-yeast extract medium of high acidity (pH 4.0) to inhibit bacterial growth. From this medium transfers were made to Sabouraud's agar for identification. In 1945, THOMPSON described the undesirable aspects of Sabourauds medium with and without crystal violet. He used successfully a hormone blood agar (pH 7.0) to which he added 2 units of penicillin and 10 units of streptomycin per ml of the medium. This supported the growth of several pathogenic fungi and proved useful ill isolating Coccidioides immitis from sputum in pure culture. A selective medium for Coccidioides immitis (1% ammonium chloride, 1 °/o Sodium acetate, 0.8 % tribasic potassium phosphate, 0.4 % cupric sulphate and 2 % agar) was described by SMIT~I in 1943. In 1947 and 1948 LITTMAN described an oxgaI1 streptomycin crystal violet medium for the cultivation of fungi from contaminated clinical material. The medium not only restricted bacterial growth, but restricted the size of fungus colonies. This latter is of advantage in that the fast growing fungus contaminants i.e. Aslbergillus, etc., do not overgrow most of the pathogenic fungi. H. caflsulatum grow only after heavy inoculation. In 194S HOWELL used a brain heart infusion blood agar with the addition of 20 units of penicillin and 40 units
of streptomycin per ml of medium for the successful cultivation of
H. capsulatum. These days Brain Heart Infusion liquid medium or Brain Heart Agar with 20 units of penicillin and 40 units of stremptomycin per ml of the medium, is supposed to be the best medium for the isolation of most of the pathogenic fungi. In the beginning of m y work in mycology in 1950, I started using media meant for growing pathogenic fungi, to which penicillin and streptomycin was added in the concentration of 20 units of penicillin and 40 units of streptomycin, respectively. This worked nicely for the isolation of some pathogenic fungi. Later on Actidione was discovered. Actidione is an antibiotic which is very active against m a n y yeasts and fungi but is tolerated in relatively high concentrations b y most bacteria. Actidione m a y be added to bacteriological media to facilitate the isolation of pathogenic fungi or counting of bacteria in the presence of yeast or molds. A medium containing 100 micrograms of Actidione per m[ has been reported to be useful in the isolation of pathogenic fungi from clinical lesions. Actidione proved of great help in the isolation of dermatophytes. The addition of 500 micrograms of actidione, 20 units of penicillin and 40 units of streptomycin per ml of Sabouraud's dextrose agar, that had previously been sterilised and cooled to 45 ° C, has been reported to make possible the isolation of T. [email protected], T. rubrum and Epidermophyton floccosum. However, several pathogenic bacteria can be cultured on nutrient dextrose agar medium containing upto 1000 micrograms of Actidione. per ml of the medium. A medium containing Actidione Polymixin, Bacitracin, Inculin and Crystal violet has been reported to be more effective in inhibiting the growth of fungal contaminants. During m y work on the investigations into the etiology of Maduromycosis in India, I found that it was very difficult to isolate the fungi in pure culture as a type of Gram's negative bacillus belonging to the Friedlander group always contaminated the growth and did not stop growing on either the liquid media or the solid media containing penicillin and streptomycin. I then tried to find out the sensitivity of these organisms against the different antibiotics and found that they were sensitive to achromycin even in the concentration of 100 micrograms per ml of the medium. They were found to be most sensitive to Achromycin and less sensitive to Chloromycetin and much less sensitive to streptomycin and Penicillin. Achromycin was then used in the medium along with another set of media containing Penicillin and Streptomycin to find out as to which was a more suitable antibiotic. All sorts of clinical material such as the sputum and the skin scrapings were tried. The medium used for these investigations was prepared in our laboratories as follows. 37 g of Difcos dehydrated brain heart infusion powder was added to 1000 ml of distilled water in a 2 litre flask. The material was
dissolved and it was sterilised at 15 lbs. pressure for 30 minutes. Beside the liqui~t medium, Brain Heart agar medium was also prepared. To one set of the medium cooled to 35 ° C, penicillin and streptomycin was added in the proportion of 20 units of penicillin and 40 units of streptomycin per ml of the medium and to another set of the medium achromycin in the proportion of 500 micrograms of pure achromycin powder per ml of the medium was added. Actidione in the proportion of 100 micrograms per ml of the medium was added to both types of media. Primary inoculation of the material from cases of mycetoma foot were put up on the two sets of each of the Brain Heart Infusion liquid medium, and brain heart infusion agar slopes containing peniciJ]in with streptomycin and achromycin respectively. Similarly sputum cultures of sinus discharges and scarpings were put up. It was found that while on the media containing penicillin and streptomycin some bacterial contaminants appeared, particularly the gram negative bacilli in cases of Mycetoma cultures, no such bacterial contaminants appeared on the media containing achromycin. Achromycin has another advantage in being much more stable than penicillin besides its being a broad spectrum antibiotic. Moreover, penicillin and streptomycin act in a neutral or slightly alkaline medium. The p H of Sabouraud's dextrose agar is fairly on the acid side. Achromycin on the other hand works best in a acid medium. Achromycin is therefore more suitable to be incorporated in the most widely used Sabouraud's dextrose agar medium than any other antibiotic. Achromycin is thus the antibiotic of choice to be incorporated in the media used for the isolation of pathogenic fungi from all sorts of clinical material such as sputum, skin scrapings and sulphur granules etc.
Summary The isolation of pathogenic fungi from heavily contaminated material has always been difficult uptil now. Brain Heart Infusion medium containing 30 units of penicillin and 40 units of streptomycin has been found to be the best medium for the isolation of pathogenic fungi. Better results have been obtained b y using Achromycin in place of penicillin and streptomycin.
Zusammenfassung Die Isolierung yon pathogenen Pflzen aus schwer kontaminiertem Material waz bis jetzt s c h w i e r i g . - H i r n - H e r z - Infusion Medium, das 30 I.E. Penicillin and 40 I.E. Streptomycin enth/ilt, ist als das beste Medium ffir die Isolierung pathogener Fungi gefunden worden. Bessere Resultate sind erzielt worden wenn Achromycin anstatt yon Penicillin und Streptomycin verwendet wurde.
H.S. ANDL]EIGH Acknowledgements
My t h a n k s are due to Messrs Lederle Laboratory (India) Private Ltd., for supplying me Achromycin. References 1. ANDLEIGH, H. S. (1951) Laboratory Diagnosis of Mycotic Lung Infections.
J. Ind. med. Assoc., 20: 360--371. 9,. ANDLEIGH, H. S. (1957) In vitro study of antifungal activity of Pentamidine and Stilbamidine. Mycopathol. Mycol. Appl. 8, 135--137. 3. ANDLEIGH, H. S. (1957) Etiology of Maduromycosis in India. Mycopathol. Mycol. Appl. 8, 138--153. 4. ANDLEIGH, H. S. (1958) Investigations into the role of fungi in P u l m o n a r y Diseases in India. Amer. Rev. Tuberculosis. 78, 644---646. 5. DEEVE, D. L., BUTT, E. ]Vi. & HAMMACK, R. W. (1952) Recent Advances ia clinical pathology 2nd Edition. Pp. 77 J. A. Churchill & Co. London.