BD Modified CNA Agar BD Modified CNA Agar with Crystal Violet

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1 INSTRUCTIONS FOR USE READY-TO-USE PLATED MEDIA PA Rev.: June 2003 BD Modified CNA Agar BD Modified CNA Agar with Crystal Violet INTENDED USE BD Modified CNA Agar is a selective medium for the isolation of Gram positive bacteria from clinical specimens. BD Modified CNA Agar with Crystal Violet is used for the isolation of streptococci and enterococci and inhibits staphylococci and Gram negative bacteria. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. The nutritional composition of Trypticase Soy Agar (TSA) has made it a popular medium, both unsupplemented and as a base for media containing blood. 1 Trypticase Soy Agar II (TSA II) is an improved version, containing growth factors to provide clearer hemolytic zones than Trypticase Soy Agar (TSA) when supplemented with 5% sheep blood. It provides excellent growth and of beta hemolytic streptococci and other organisms. Ellner and colleagues found that a medium containing colistin and nalidixic acid in a Columbia agar base enriched with 5% sheep blood would support the growth of staphylococci, hemolytic streptococci and enterococci while inhibiting the growth of Proteus, Klebsiella and Pseudomonas species. 2 Columbia Agar base has a relatively high carbohydrate content and, therefore, beta-hemolytic streptococci may produce a greenish hemolytic reaction that may be mistaken for alpha. Trypticase Soy Agar II gives clearer hemolytic reactions but usually smaller colonies. In BD Modified CNA Agar and in BD Modified CNA Agar with Crystal Violet, Trypticase Soy Agar II is the base medium which provides nutrients and is supplemented with colistin and nalidixic acid to provide selectivity for Gram positive bacteria. Sheep blood and additional calf serum enhance the growth and allow the detection of hemolytic reactions. In addition to these ingredients, BD Modified CNA Agar with Crystal Violet contains crystal violet which is an efficient inhibitor of staphylococci. Thus, this medium is used for the isolation of streptococci and enterococci which are not inhibited by this dye. REAGENTS Formulas* Per Liter Purified Water BD Modified CNA Agar Pancreatic Digest of Casein 14.5 g Papaic Digest of Soybean Meal 5.0 Sodium Chloride 5.0 Agar 14.0 Growth Factors 1.5 Colistin 0.01 Nalidixic Acid 0.01 Calf Serum 50.0 ml Sheep Blood, defibrinated 5% ph 7.3 +/- 0.2 BD Modified CNA Agar with Crystal Violet, in addition to the ingredients listed above, contains 2 mg of crystal violet per liter of medium. *Adjusted and/or supplemented as required to meet performance criteria. PRECAUTIONS. For professional use only. Do not use plates if they show evidence of microbial contamination, discoloration, drying, cracking or other signs of deterioration. PA

2 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. USER QUALITY CONTROL Inoculate representative samples with the following strains (for details, see GENERAL INSTRUCTIONS FOR USE document). Incubate at 35 ± 2 C for 18 to 24 hours, preferably in an aerobic atmosphere enriched with carbon dioxide. Strains BD Modified CNA Agar BD Modified CNA Agar with Crystal Violet Streptococcus pneumoniae ATCC 6305 greenish-grey colonies; Growth fair to excellent; greyish-greenish colonies; Streptococcus pyogenes ATCC Staphylococcus aureus ATCC Enterococcus faecalis ATCC alpha small whitish colonies with beta medium-sized white to yellow colonies with or without beta whitish colonies with greyish zone; no alpha small blue-grey colonies; beta Inhibition complete Growth fair to excellent; small bluish colonies; no Proteus mirabilis ATCC Inhibition partial to complete; no swarming Inhibition partial to complete; no swarming Uninoculated Red (blood color) Red (blood color), bluish hue PROCEDURE Materials Provided BD Modified CNA Agar or BD Modified CNA Agar with Crystal Violet, both provided in 90 mm Stacker plates. Microbiologically controlled. Materials Not Provided Ancillary culture media, reagents and laboratory equipment as required. Specimen Types BD Modified CNA Agar is a selective medium for many Gram positive bacteria in aerobic bacteriology and can be used for all types of bacteriological specimens. BD Modified CNA Agar with Crystal Violet is a selective medium for streptococci and enterococci, and is inhibitory to staphylococci. It is mainly used for the detection of streptococci in specimens from body sites where these organisms play a major role as infectious agents, e.g., for the detection of Group A streptococci (=Streptococcus pyogenes) in specimens from the upper respiratory tract, but it may also be used for the detection of these and other streptococci and enterococci from other clinical specimens. For additional information on these media, see PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE. PA

3 Test Procedure Streak the specimen as soon as possible after it is received in the laboratory onto BD Modified CNA Agar or BD Modified CNA Agar with Crystal Violet. 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. In order to provide detection of all pathogens contained in the specimen, BD Columbia Agar with 5% Sheep Blood and, depending on the type of specimen, additional selective media must be included. 3,4 Since many pathogens require carbon dioxide on primary isolation, plates should be incubated in an aerobic atmosphere containing approximately 3 to 10% CO 2. Incubate plates at C for 18 to 24 hours or longer, if necessary. Results Typical colonial morphology on BD Modified CNA Agar is as follows: Streptococci (non-group D) Small, white to grayish. May be hemolytic, depending on the species Enterococci (Group D) Small to medium-sized, but larger than group A streptococci; gray Staphylococci Medium-sized, white to gray or cream to yellow. May be hemolytic, depending on the species Micrococci Large, white to gray or yellow to orange Corynebacteria Small to large, white to gray or yellow Candida spp. Small, white Listeria monocytogenes Small, gray, with weak beta Gram-negative bacteria Partial to complete inhibition On BD Modified CNA Agar with Crystal Violet, staphylococci and Gram negative bacteria will be partially to completely inhibited, while streptococci and enterococci will produce the same growth reactions as on BD Modified CNA Agar. However, there is a tendency that the colonies take on a bluish coloration due to accumulation of the crystal violet. Other Gram positive bacteria may or may not grow, depending on their sensitivity to the inhibitors. For details on the interpretation of growth on primary isolation plates, consult the references. 4-7 PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE BD Modified CNA Agar is used for the isolation of Gram positive bacteria, e.g., streptococci, staphylococci, coryneforms, Listeria spp and others. 2 BD Modified CNA Agar with Crystal Violet is used for the isolation of streptococci and enterococci. Staphylococci are inhibited on this medium. Other Gram positive bacteria may or may not grow on the medium, depending on the species and their sensitivity to the inhibitors. Most Gram negative bacteria such as Enterobacteriaceae are inhibited on these media. Performance Results In an internal evaluation, strains of the following species were tested for growth and hemolytic reactions on these media. BD Columbia CNA Agar with 5% Sheep Blood was used as a growth reference. Plates were incubated at 35 to 37 C for 20 hours in an aerobic atmosphere enriched with CO 2. Results are shown in Table 1. Limitations of the Procedure Although they are Gram positive bacteria, aerobic sporeformers (Bacillus spp. and related genera) may be inhibited on these media. These media are not inhibitory to fungi. The number and types of bacterial species occurring as infectious agents is very large. Therefore, before the media are routinely used for rarely isolated or newly described microorganisms, their suitability must first be tested by the user by cultivating pure cultures of the organism in question. PA

4 Although certain diagnostic tests may be performed directly on these media, biochemical and, if indicated, immunological testing using pure cultures is necessary for complete identification. Table 1: Performance Results Species BD Modified CNA Agar BD Modified CNA Agar with Crystal Violet Growth, alpha Growth, weak alpha BD Columbia CNA Agar with 5% Sheep Blood Growth, alpha Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Growth, alpha Streptococcus sanguis Growth, weak alpha Enterococcus faecalis Growth Growth Growth Staphylococcus aureus No growth Staphylococcus epidermidis Growth No growth Growth Listeria monocytogenes Growth, weak beta No growth Escherichia coli No growth No growth No growth Proteus mirabilis Very weak growth, Very weak growth, swarming inhibited swarming inhibited Pseudomonas aeruginosa No growth No growth No growth Growth, weak alpha Growth, very weak beta Very weak growth, swarming inhibited REFERENCES 1. Vera, H.D., and D.A. Power Culture media, p In: E.H. Lennette, A. Balows, W.J. Hausler, Jr., and J.P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 2. Ellner, P.D., C.J. Stoessel, E. Drakeford, and F. Vasi A new culture medium for medical bacteriology. Am. J. Clin. Pathol. 45: Forbes, B.A. and P.A. Granato Processing specimens for bacteria. In: Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C. 4. Baron, E. J, L. R. Peterson, and S. M. Finegold Bailey & Scott s diagnostic microbiology, 9th ed., p Mosby-Year Book, Inc. St. Louis, MO. 5. Isenberg, H. D. (ed.) Interpretation of aerobic bacterial growth on primary culture media, Clinical microbiology procedures handbook, vol.1, p American Society for Microbiology, Washington, D.C. 6. Ruoff, K. L., R.A. Whiley, and D. Beighton Streptococcus. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8 th ed. American Society for Microbiology, Washington, D.C. 7. Teixeira, L.M., and R.R. Facklam Enterococcus. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8 th ed. American Society for Microbiology, Washington, D.C. PACKAGING/AVAILABILITY BD Modified CNA Agar with 5% Sheep Blood Cat. No Ready-to-use Plated Media, cpu 20 BD Modified CNA Agar with Crystal Violet and 5% Sheep Blood Cat. No Ready-to-use Plated Media, cpu 20 PA

5 FURTHER INFORMATION For further information please contact your local BD representative. BD Diagnostic Systems Tullastrasse 8 12 D Heidelberg/Germany Phone: Fax: [email protected] BD Diagnostic Systems Europe Becton Dickinson France SA 11 rue Aristide Bergès Le Pont de Claix/France Tel: Fax: BD, BD logo, Trypticase and Stacker are trademarks of Becton, Dickinson and Company ATCC is a trademark of the American Type Culture Collection 2003 Becton, Dickinson and Company PA