TECHNICAL DATA SHEET #250 REV.

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 see catalog for ordering options

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

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 

TECHNICAL DATA SHEET #250 REV. 5

FORMULAS:
Approximate, per liter of deionized filtered water.

(1) Columbia Agar: Peptic Digest of Animal Tissue ........................ 5.0 g Pancreatic Digest of Casein........................... 10.0 Yeast Enriched Peptone....................................5.0 Pancreatic Digest of Heart Muscle ...................3.0 Cornstarch ....................................................... 1.0 Sodium Chloride............................................... 5.0 Agar........................................................ 10.0-15.0 Final pH 7.3 0.2 at 25C (2) Columbia Agar + 5% Sheep Blood: Peptic Digest of Animal Tissue ........................ 5.0 g Pancreatic Digest of Casein............................. 5.0 Yeast Enriched Peptone..................................10.0 Pancreatic Digest of Heart Muscle ...................3.0 Cornstarch ....................................................... 1.0 Sodium Chloride............................................... 5.0 Agar.................................................................14.0 Sheep Blood................................................... 50.0 ml Final pH 7.3 0.2 at 25C (3) Columbia Anaerobe Agar + 5% Sheep Blood: Same as (2) with the addition of 5.0 mg Hemin and 10.0 mg of Vitamin K1. (4) Columbia Agar + 7.5% Sheep Blood and Gentamicin: Same as (2) except with an increased volume of 75.0 ml Sheep blood and 140.0 mg Gentamicin. (5) Columbia Anaerobe Agar + 7.5% Sheep Blood and Gentamicin: Same as (3) except with an increased volume of 75.0 ml Sheep blood and 140.0 mg Gentamicin.

(6) Columbia Agar + 5% Sheep Blood and Kanamycin: Same as (2) with the addition of 10.0 ml Kanamycin. Final pH 7.2 0.2 at 25C (7) Columbia Agar + 5% Horse Blood: Same as (3) with 50.0 ml of Horse blood. (8) Columbia CNA Agar + 5% Sheep Blood: Same as (3) with 10.0 mg of Colistin and 10.0 mg of Nalidixic acid. (9) Columbia Anaerobe CNA Agar + 5% Sheep Blood: Same as (8) with the addition of 5.0 mg Hemin and 10.0 mg of Vitamin K1.

PRECAUTIONS:*

For in vitro diagnostic use. Observe approved biohazard precautions. Storage: Upon receipt store at 2-8C away from direct light. Media should not be used if there are signs of contamination, deterioration (shrinking, cracking, or discoloration), or if the expiration date has passed. Limitations: Columbia Agar with 5% Blood is a nonselective media; biochemical and/or serologic testing are necessary for definitive identification of microorganisms. On Columbia Agar with Blood, colony morphology and hemolysis may not be typical; small amounts of reducing sugars inhibit the expression of beta hemolysis. Columbia CNA Agar with 5% Sheep Blood inhibits many strains of coagulase-negative staphylococci, especially Staphylococcus saprophyticus.3 Columbia Agar with 5% Horse Blood is very sensitive to light, heat, and temperature fluctuations. Media will perform satisfactorily

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659 Page 

TECHNICAL DATA SHEET #250 REV. 5

if storage instructions on each package label are followed. Haemophilus hemolyticus and Haemophilus parahaemolyticus appear beta-hemolytic on horse blood agar and are indistinguishable from group A streptococci; additional tests are required for definitive identification. Sheep blood contains V factor-destroying enzyme (nucleotidase) which prevents the growth of Haemophilus species on sheep blood agar, unless another microorganism provides the V factor (satellitism). Over inoculation of contaminated specimens (stool, rectal, etc.) onto selective media may decrease the inhibitory performance of the media. Organisms isolated on selective media must be identified using appropriate biochemical tests and tested for antibiotic sensitivity if appropriate using approved CLSI (NCCLS) methods.

PROCEDURE:*

Specimen Collection: Information on specimen collection is found in standard reference material. In general, specimens should be protected from extreme heat and cold and should be delivered to the laboratory without delay. Method of Use: Prior to inoculation, the media should be brought to room temperature. Inoculate according to standard microbiological procedures and streak the inoculum so as to obtain isolated colonies. Incubate under conditions that will permit growth. In general, incubate at 35C for 18-72 hours with adequate moisture in either aerobic, capnophilic, or anaerobic atmosphere depending on the specific microorganisms to be cultured. Interpretation: Organism: Colony Morphology: Streptococcus pyogenes Small, beta-hemolytic, transparent to opaque, domed, smooth and entire edge Streptococcus viridans Small, alpha-hemolytic, transparent to opaque, domed, smooth and entire edge Streptococcus pneumoniae Small, alpha-hemolytic, round and mucoid with entire edge Staphylococcus aureus Average, hemolysis, opaque, circular, smooth, raised, white to golden yellow pigment Staphylococcus epidermidis Average, hemolysis, opaque, circular, smooth, raised, usually white to colorless Corynebacteria Small, grayish colonies Listeria monocytogenes Small, beta-hemolytic, transparent, gray to white Yeast Small, white to gray in 48-72 hours Escherichia coli Large, grayish colonies For other clinically significant organisms, a reference such as Murray et al.5 should be consulted. Material Required but Not Provided: Standard microbiological supplies and equipment such as those products commonly used in a microbiological laboratory are not provided.

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, alpha hemolysis +Gentamicin Growth Growth Growth Growth Inhibition, p/c Inhibition, p/c +5% Horse Blood +CNA Growth Growth Growth N/A Growth, beta hemolysis Growth, beta hemolysis Growth, alpha hemolysis Growth, alpha hemolysis +Kanamycin N/A Growth N/A N/A Inhibition, p/c Inhibition, p/c

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659 Page 

TECHNICAL DATA SHEET #250 REV. 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, p/c Inhibition, p/c +Gentamicin Growth Growth Growth Growth Growth Inhibition, p/c Inhibition, p/c

User Quality Control: Check for signs of contamination and deterioration. Columbia media should appear firm, translucent, and straw in color. Columbia Agar media with blood should appear firm, opaque, and bright red in color.

BIBLIOGRAPHY:

1. Ellner, P. D., et al. 1966. Am. J. Clin. Pathol. 45:502-504. 2. Facklam, R. R., Isolation and Identification of Streptococci, USHEW, PHS, Centers for Disease Control, Atlanta, 1980. 3. Forbes, B. A., et al., Bailey and Scotts Diagnostic Microbiology, 12th ed., C. V. Mosby, St. Louis, 2007. 4. Koneman, E. W., et al., Color Atlas and Textbook of Diagnostic Microbiology, 5th ed., J. B. Lippincott, Philadelphia, 1997. 5. Murray, P.R., et al., Manual of Clinical Microbiology, 9th ed., American Society for Microbiology, Washington, D. C., 2003. 6. Green, M., et al. 1990. Journal of Clinical Microbiology, 28:484-488. 7. United States Pharmacopeia 30 - NF 25, Chapter 62, Microbial examination of nonsterile products: Tests for specified microorganisms, 2007. *For more detailed information, consult appropriate references. bioMrieux, Inc. Data #250 Revision Date: July 2009

27120 SW 95th Avenue Wilsonville, OR 97070 800.547.0659 Page