INSTRUCTIONS FOR USE – READY-TO-USE PLATED MEDIA PA-254039.08 Rev.: October 2019 BD Sabouraud Glucose Agar • BD Sabouraud Agar with Chloramphenicol • BD Sabouraud Agar with Gentamicin and Chloramphenicol • BD Sabouraud Agar with Penicillin and Streptomycin • INTENDED USE BD Sabouraud Glucose Agar is used for the isolation and cultivation of fungi (yeasts, molds, and dermatophytes) from clinical specimens. BD Sabouraud Agar with Chloramphenicol, BD Sabouraud Agar with Gentamicin and Chloramphenicol, and BD Sabouraud Agar with Penicillin and Streptomycin are selective media for the isolation of fungi from clinical specimens. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Sabouraud Glucose Agar is a general purpose medium originally devised for the cultivation of dermatophytes.1,2 Today it is used for the isolation and cultivation of all fungi.3-5 The peptones in Sabouraud Glucose Agar are sources of nitrogenous growth factors. Glucose provides an energy source for the growth of microorganisms. The high glucose concentration provides an advantage for the growth of the (osmotically stable) fungi while most bacteria do not tolerate the high sugar concentration. In addition, the low pH is optimal for fungi, but not for many bacteria.3 Sabouraud Glucose Agar is only slightly selective against bacteria. BD Sabouraud Agar with Chloramphenicol, BD Sabouraud Agar with Gentamicin and Chloramphenicol, and BD Sabouraud Agar with Penicillin and Streptomycin are selective media based on Sabouraud Glucose Agar. Selective agents to inhibit bacteria have been added. Chloramphenicol is a broad-spectrum antibiotic inhibitory to a wide range of gram-negative and gram-positive bacteria but may have an inhibitory effect on several pathogenic fungi.4 Antimicrobials like penicillin, gentamicin, and streptomycin, or a combination of these, have been shown to be effective in inhibiting bacteria without affecting fungal growth.2-5 These media are used for the isolation of fungi from clinical specimens or materials suspected to contain bacterial contaminants. Sabouraud Glucose Agar is one of the media used in the Microbial Limit Tests in the USP & EP. 6,12 REAGENTS Formulas: see Table 1 PRECAUTIONS . For professional use only. Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking or other signs of deterioration. Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures, biohazards, and disposal of used product. STORAGE AND SHELF LIFE On receipt, store plates in the dark at 2 to 8° C, in their original sleeve wrapping until just prior to use. Avoid freezing and overheating. The plates may be inoculated up to the expiration date (see package label) and incubated for the recommended incubation times. Plates from opened stacks of 10 plates can be used for one week when stored in a clean area at 2 to 8° C. PA-254039.08 - 1 - Table 1: REAGENTS Formulas* Per Liter Purified Water Product Name BD Sabouraud BD Sabouraud BD Sabouraud BD Sabouraud Agar Glucose Agar Agar with Agar with with Penicillin and Gentamicin and Chloramphenicol Ingredients Streptomycin Chloramphenicol Pancreatic Digest of 5.0 g 5.0 g 5.0 g 5.0 g Casein Peptic Digest of 5.0 5.0 5.0 5.0 Animal Tissue Glucose 40.0 40.0 40.0 40.0 Agar 15.0 15.0 15.0 15.0 Penicillin G - 60000 IU - - Streptomycin - 0.06 g - - Gentamicin - - 0.04 - Chloramphenicol - - 0.4 0.4 pH pH 5.6 +/- 0.2 5.4 +/- 0.2 5.6 +/- 0.2 5.6 +/- 0.2 *Adjusted and/or supplemented as required to meet performance criteria. USER QUALITY CONTROL Inoculate representative samples with the following strains (for details, see GENERAL INSTRUCTIONS FOR USE document). See footnote for incubation. Product Name BD Sabouraud BD Sabouraud BD Sabouraud BD Sabouraud Glucose Agar Agar with Agar with Agar with Penicillin and Gentamicin and Chloramphenicol Test Strains Streptomycin Chloramphenicol **Candida Growth good to albicans excellent Growth good to excellent ATCC 10231 **Saccharomyces Growth good to cerevisiae excellent Growth good to excellent DSM 1333 ** Aspergillus Growth good to brasiliensis excellent Growth good to excellent ATCC16404 ** Penicillium Growth good to roquefortii excellent Growth good to excellent ATCC 9295 **Trichophyton Growth good to mentagrophytes excellent Growth good to excellent ATCC 9533 *Staphylococcus No inhibition aureus Inhibition complete ATCC 25923 *Escherichia coli No inhibition ATCC 25922 Inhibition complete Uninoculated Light amber Incubation: *48 h 35-37° C / **</= 5 days, 20-25° C, aerobically PROCEDURE Materials Provided BD Sabouraud Glucose Agar, BD Sabouraud Agar with Chloramphenicol, BD Sabouraud Agar with Gentamicin and Chloramphenicol, or BD Sabouraud Agar with Penicillin and Streptomycin, all provided in 90 mm Stacker plates. Microbiologically controlled. PA-254039.08 - 2 - Materials Not Provided Ancillary culture media, reagents and laboratory equipment as required. Specimen Types The products described in this document are isolation media for fungi and can be used (alternatively or as a combination of two of these media) for all clinical specimens (see also PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE). The media are also used in many areas of industrial microbiology and in hygiene monitoring. Test Procedure Streak the specimen as soon as possible after it is received in the laboratory. The streak plate is used primarily to isolate pure cultures from specimens containing mixed flora. Alternatively, if material is being cultured directly from a swab, roll the swab over a small area of the surface at the edge; then streak from this inoculated area. • If the specimen consists of skin scrapings, hairs, or nails, place the material in the center of the media surface. If possible, larger particles should be slightly pressed onto the surface by means of sterile forceps to provide contact with the medium. • For the detection of dermatophytes, also include BD Dermatophyte Agar or BD Mycosel Agar. • If BD Sabouraud Agar with Chloramphenicol, BD Sabouraud Agar with Gentamicin and Chloramphenicol, or BD Sabouraud Agar with Penicillin and Streptomycin are used, also include a plate of BD Sabouraud Glucose Agar. • Eventually, a nonselective medium such as BD Columbia Agar with 5% Sheep Blood should also be inoculated to provide an indication of bacterial pathogens present in the specimen. If used for the detection of yeasts (e.g., Candida species) in clinical specimens, incubate for 48 hours at 30 to 35° C. If filamentous fungi, including dermatophytes are suspected, incubate for up to one week at 25 to 30° C. Dermatophytes occasionally need 3 weeks or longer to produce growth. If used for hygiene monitoring, incubate for up to 7 days at 20 to 25° C. If incubation is longer than 3 days, provide adequate moisture. Plates may be sealed with adhesive plastic tape to avoid desiccation. For details on growth temperature and incubation, consult the references.5,7-10 Results After sufficient incubation, the plates may show isolated colonies in streaked areas and confluent growth in areas of heavy inoculation. Examine plates for fungal colonies exhibiting typical color and morphology. Biochemical tests and microscopical and serological procedures should be performed to confirm findings. 6-9 Since the number of pathogenic fungi is large, no details on their appearance is given here. Consult the references.2,3, 5-9 PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE The media presented in this document are standard media for the isolation and cultivation of fungi from all types of clinical and non-clinical specimens.4-11 Due to the wide growth temperature range of fungi, it may be necessary to inoculate several plates of the same medium and incubate them at different temperatures. Consult the Test Procedure section and appropriate references.5, 7-10 Since BD Sabouraud Glucose Agar is only weakly selective, bacteria will grow on the medium, especially after an extended incubation period. If bacterial contamination of the specimens, materials, or areas under investigation is suspected, media with a higher selectivity, e.g., BD Sabouraud Agar with Chloramphenicol, BD Sabouraud Agar with Gentamicin and Chloramphenicol, or BD Sabouraud Agar with Penicillin and Streptomycin must be used for the culture of the specimen or sample. Nocardia and Actinomyces are filamentous bacteria (no fungi!) and, therefore, do not grow on all Sabouraud media containing bacterial inhibitors. PA-254039.08 - 3 - REFERENCES 1. Sabouraud, R. 1892. Contribution a l'etude de la trichophytie humaine. Etude clinique, microscopique et bacterioloqique sur la pluralité des trichophytons de l'homme. Ann. Dermatol. Syphil. 3: 1061-1087. 2. Haley, L.D., J. Trandel, and M.B. Coyle. 1980. Cumitech 11, Practical methods for culture and identification of fungi in the clinical microbiology laboratory. Coordinating ed., J.C. Sherris. American Society for Microbiology, Washington, D.C. 3. Ajello, L., L.K. Georg, W. Kaplan, and L. Kaufman. 1963. CDC laboratory manual for medical mycology. PHS Publication No. 994, U.S. Government Printing Office, Washington, D.C. 4. MacFaddin, J.F. 1985. Media for isolation-cultivation- identification-maintenance of medical bacteria. vol. I. Williams & Wilkins, Baltimore. 5. Sutton, D.A. 2003. Specimen Collection, transport, and processing: mycology. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C. 6. U.S. Pharmacopeial Convention, Inc. The U.S. Pharmacopeia /The national formulary. Current edition. U.S. Pharmacopeial Convention, Inc., Rockville, Md. USA 7. Larone, D.H. 2002. Medically important fungi: a guide to identification. 4th ed. American Society for Microbiology, Washington, D.C. 8. Summerbell, R.C. 2003. Trichophyton, Microsporum, Epidermophyton, and agents of superficial mycoses. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C. 9. Kwon-Chung, K.J., and J.E. Bennett. 1992. Medical mycology. Lea & Febiger, Philadelphia.