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TECHNICAL DATA SHEET #250 REV.

5

COLUMBIA AGAR MEDIA
PRODUCTS:

Prepared Plated Media:a Columbia Agar Columbia Agar (CAB) + 5% Sheep Blood Columbia Anaerobe Agar + 5% Sheep Blood Columbia Agar (CAB) + 5% Horse Blood Columbia Agar (CAB) + 7.5% Sheep Blood and Gentamicin Columbia Anaerobe Agar + 7.5% Sheep Blood and Gentamicin Columbia Agar (CAB) + 5% Sheep Blood and Kanamycin Columbia CNA Agar + 5% Sheep Blood Columbia CNA Anaerobe + 5% Sheep Blood
a

P8233 P1350, P4155 (bi-plate) P1360 P1355 P1415 P1370 P1420 P1400 P1408

see catalog for ordering options

PURPOSE:

Columbia agar is nutrient media used for the cultivation of fastidious and nonfastidious microorganisms from clinical and nonclinical specimens. Sheep or horse blood is added to enhance the growth of bacterial species by providing the X factor (heme) necessary in the preliminary identification of hemolytic strains. The media becomes selective for streptococci and staphylococci with the addition of colistin and nalidixic acid (CNA). Various combinations of antibiotics are used as additives to further select for fastidious microorganisms. Columbia Agar (P8233) preparation meets the U.S. Pharmacopeia (USP) standards for use as an isolation media in performing microbial examination of nonsterile products.

PRINCIPLE:

Columbia agar base (CAB) is a general purpose media with the basic ingredients necessary for microorganisms to replicate and grow. The media contains peptones, which provide a mixture of nitrogeneous compounds and amino acids, and yeast and beef extracts to provide additional nitrogenous compounds, carbohydrates and vitamins. Ellner et al1 discovered peptones from both animal and vegetable protein to be complementary, and the growth of the microorganisms to be better than on the then more frequently used base media (casein hydrolysate or meat infusion media). In addition, yeast and beef extracts were added and appeared to increase the growth of Neisseria species, while cornstarch, by neutralizing the inhibitory effects of glucose, decreased the formation of a green coloration (alpha hemolysis) by beta-hemolytic streptococci. Columbia agar base (CAB) was made a selective media by adding colistin and nalidixic acid (CNA), which inhibit gram-negative microorganisms. CNA was found to be more effective in suppressing Proteus, Klebsiella, and Pseudomonas species than Phenylethyl Alcohol Agar (PEA) and did not restrict the growth of gram-positive cocci as PEA did. Green et al.6 described a media for screening stool specimens for Vancomycin Resistant Gram-Positive Cocci (VRGPC) in children that incorporates vancomycin (5 mcg/ml) and amphotericin B (8 mcg/ml) into Columbia CNA Agar. There are increasing reports of infections due to VRGPC that include Streptococcus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus species. Currently Vancomycin Resistant Enterococcus (VRE) is the most widely recognized VRGPC. Because of the emergence of other VRGPC, there is a need to screen patients for these organisms. Therefore, the addition of vancomycin and amphotericin B (fungal inhibitor) allows the laboratory to screen contaminated specimens for possible VRGPC while holding down the overgrowth of normal flora organisms. The addition of antibiotics, kanamycin or gentamicin, to Columbia Agar serves as a selective media, suppressing the growth of gram-negative and gram-positive organisms. Columbia Anaerobe Agar is recommended for the cultivation of anaerobic organisms. Supplemented with growth factors, hemin, vitamin K1, and sheep blood constituents, Columbia Anaerobe Agar supports the isolation and cultivation of obligate anaerobes.

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659 Page 

... 10................. (5) Columbia Anaerobe Agar + 7.............2 at 25°C Columbia Anaerobe Agar + 5% Sheep Blood: Same as (2) with the addition of 5.... 1.. OR 97070 • 800.2 ± 0............... colony morphology and hemolysis may not be typical.......... deterioration (shrinking.....547. 1....5% Sheep Blood and Gentamicin: Same as (2) except with an increased volume of 75.. and temperature fluctuations.............. 5....10........... heat.......0 Agar .....0 ml of Horse blood..................................0 ml Final pH 7................. 50...... PRECAUTIONS:* For in vitro diagnostic use........0 mg of Vitamin K1..... or if the expiration date has passed... Limitations: Columbia Agar with 5% Blood is a nonselective media......... 5.. Storage: Upon receipt store at 2-8°C away from direct light..0 mg of Vitamin K1................0 Yeast Enriched Peptone .....0 mg of Colistin and 10...0 Final pH 7.14. () Columbia Agar: Peptic Digest of Animal Tissue .......... Observe approved biohazard precautions.................... (2) (3) (4) Columbia Agar + 7......... (6) Columbia Agar + 5% Sheep Blood and Kanamycin: Same as (2) with the addition of 10....2 at 25°C (7) (8) (9) Columbia Agar + 5% Horse Blood: Same as (3) with 50........3 ± 0......0 mg Gentamicin......3 Columbia Agar with 5% Horse Blood is very sensitive to light.....0 Agar .... 5......TECHNICAL DATA SHEET #250 REV..............0 Cornstarch ......... biochemical and/or serologic testing are necessary for definitive identification of microorganisms.....0659 Page 2 ......3 ± 0...............3.....0 ml Sheep blood and 140... Columbia CNA Agar + 5% Sheep Blood: Same as (3) with 10..............0 Yeast Enriched Peptone ..0 Pancreatic Digest of Heart Muscle .. 5 FORMULAS: Approximate. On Columbia Agar with Blood. Columbia CNA Agar with 5% Sheep Blood inhibits many strains of coagulase-negative staphylococci...2 at 25°C Columbia Agar + 5% Sheep Blood: Peptic Digest of Animal Tissue ................ Final pH 7.......0 mg Hemin and 10.....0 g Pancreatic Digest of Casein ........................0-15........ small amounts of reducing sugars inhibit the expression of beta hemolysis........... cracking............................0 ml Sheep blood and 140.................. Columbia Anaerobe CNA Agar + 5% Sheep Blood: Same as (8) with the addition of 5......... Media should not be used if there are signs of contamination.0 Cornstarch ........0 mg Gentamicin....0 g Pancreatic Digest of Casein ............0 Sodium Chloride ..0 mg of Nalidixic acid......0 ml Kanamycin....3.... Media will perform satisfactorily 27120 SW 95th Avenue • Wilsonville................ per liter of deionized filtered water.. 5. especially Staphylococcus saprophyticus...0 Sodium Chloride ..............0 mg Hemin and 10..............5% Sheep Blood and Gentamicin: Same as (3) except with an increased volume of 75.....0 Pancreatic Digest of Heart Muscle ............0 Sheep Blood .....5..... 5.. or discoloration).... 10...

transparent to opaque. rectal. alpha-hemolytic. alpha hemolysis +Gentamicin Growth Growth Growth Growth Inhibition. white to gray in 48-72 hours Large. etc. p/c +CNA Growth N/A Growth. beta-hemolytic. Organisms isolated on selective media must be identified using appropriate biochemical tests and tested for antibiotic sensitivity if appropriate using approved CLSI (NCCLS) methods. incubate at 35°C for 18-72 hours with adequate moisture in either aerobic. domed. smooth and entire edge Small. unless another microorganism provides the V factor (satellitism). specimens should be protected from extreme heat and cold and should be delivered to the laboratory without delay.547. In general. circular. smooth.TECHNICAL DATA SHEET #250 REV. transparent. Inoculate according to standard microbiological procedures and streak the inoculum so as to obtain isolated colonies. ± hemolysis. white to golden yellow pigment Average. raised. OR 97070 • 800. Haemophilus hemolyticus and Haemophilus parahaemolyticus appear beta-hemolytic on horse blood agar and are indistinguishable from group A streptococci. ± hemolysis. p/c Inhibition. circular. gray to white Small. grayish colonies Small. beta-hemolytic.5 should be consulted. smooth and entire edge Small. Material Required but Not Provided: Standard microbiological supplies and equipment such as those products commonly used in a microbiological laboratory are not provided. beta hemolysis Growth. smooth. alpha hemolysis +Kanamycin N/A Growth N/A N/A Inhibition. a reference such as Murray et al. alpha hemolysis 27120 SW 95th Avenue • Wilsonville. additional tests are required for definitive identification. domed. Incubate under conditions that will permit growth.) onto selective media may decrease the inhibitory performance of the media. Sheep blood contains V factor-destroying enzyme (nucleotidase) which prevents the growth of Haemophilus species on sheep blood agar. raised. Interpretation: Organism: Streptococcus pyogenes Streptococcus viridans Streptococcus pneumoniae Staphylococcus aureus Staphylococcus epidermidis Corynebacteria Listeria monocytogenes Yeast Escherichia coli Colony Morphology: Small. Over inoculation of contaminated specimens (stool. round and mucoid with entire edge Average.0659 Page 3 . p/c +5% Horse Blood Growth Growth Growth. transparent to opaque. 5 if storage instructions on each package label are followed. QUALITY CONTROL:* Microorganisms Used (ATCC #): Columbia Agar Clostridium sporogenes (11437) Clostridium sporogenes (19404) Columbia Agar Base (CAB) Staphylococcus aureus (25923) Escherichia coli (25922) Streptococcus pyogenes (19615) Streptococcus pneumoniae (6305) Columbia Agar Base (CAB) Bacteroides fragilis (25285) Clostridium perfringens (13124) Fusobacterium nucleatum (25586) Peptostreptococcus anaerobius (27337) Escherichia coli (25922) Staphylococcus aureus (25923) Expected Results: Growth Growth + 5% Sheep Blood Growth Growth Growth. beta hemolysis Growth. PROCEDURE:* Specimen Collection: Information on specimen collection is found in standard reference material. opaque. Method of Use: Prior to inoculation. p/c Inhibition. In general. the media should be brought to room temperature. opaque. alpha-hemolytic. grayish colonies For other clinically significant organisms. usually white to colorless Small. capnophilic. or anaerobic atmosphere depending on the specific microorganisms to be cultured. beta hemolysis Growth.

7.0659 Page 4 . Koneman.. et al. 2007. 5 Proteus mirabilis (12453) Columbia Anaerobe Agar Bacteroides fragilis (25285) Clostridium perfringens (13124) Fusobacterium nucleatum (25586) Bacteroides levii (29147) Peptostreptococcus anaerobius (27337) Escherichia coli (25922) Staphylococcus aureus (25923) N/A + 5% Sheep Blood Growth Growth Growth Growth N/A N/A N/A N/A +CNA + 5% Sheep Blood Growth Growth Growth Growth N/A Inhibition. p/c Inhibition. 3.NF 25. 12th ed.. 28:484-488. Murray.. C. Chapter 62. et al.R. Pathol. Inc. p/c +Gentamicin Growth Growth Growth Growth Growth Inhibition. 5. Forbes. USHEW. bioMérieux. A. Mosby. Data #250 Revision Date: July 2009 27120 SW 95th Avenue • Wilsonville. opaque.. p/c User Quality Control: Check for signs of contamination and deterioration. 1980.547. OR 97070 • 800. V. et al. Lippincott. *For more detailed information. translucent. 9th ed. and bright red in color. Washington. Columbia Agar media with blood should appear firm. J. Microbial examination of nonsterile products: Tests for specified microorganisms. D.. consult appropriate references. R.. 5th ed. Color Atlas and Textbook of Diagnostic Microbiology. Journal of Clinical Microbiology. p/c Inhibition.. Louis. Bailey and Scott’s Diagnostic Microbiology. American Society for Microbiology. C. Manual of Clinical Microbiology. P.. 1997. Green. Facklam. PHS. and straw in color. Philadelphia. BIBLIOGRAPHY: 1. B. Ellner. Centers for Disease Control.. P. D. 1990. 6. United States Pharmacopeia 30 . 45:502-504. W. J. et al. Columbia media should appear firm. Atlanta.. E. R. et al. 4. 1966.TECHNICAL DATA SHEET #250 REV. 2. p/c Inhibition. 2003. Clin... St. M.. 2007. B. Isolation and Identification of Streptococci. Am.