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 

..0 Sodium Chloride ...... biochemical and/or serologic testing are necessary for definitive identification of microorganisms.............. colony morphology and hemolysis may not be typical..... Storage: Upon receipt store at 2-8°C away from direct light...0 Sodium Chloride ...0 Agar ..0 mg of Vitamin K1.......... 50.............. 5.......... heat........ or discoloration).............0 mg Gentamicin... 10..0 mg of Colistin and 10.2 at 25°C (7) (8) (9) Columbia Agar + 5% Horse Blood: Same as (3) with 50...........TECHNICAL DATA SHEET #250 REV.0 g Pancreatic Digest of Casein . deterioration (shrinking...... 5.0 ml Sheep blood and 140. especially Staphylococcus saprophyticus........0-15........ PRECAUTIONS:* For in vitro diagnostic use............0 ml Sheep blood and 140.... 5 FORMULAS: Approximate.............0 mg of Vitamin K1...........0 mg Hemin and 10................. (2) (3) (4) Columbia Agar + 7.... or if the expiration date has passed...0 ml of Horse blood.....0 Pancreatic Digest of Heart Muscle ..........0 mg Hemin and 10.14.......... cracking...0 ml Final pH 7.......3 Columbia Agar with 5% Horse Blood is very sensitive to light...... Limitations: Columbia Agar with 5% Blood is a nonselective media.....0659 Page 2 .5% Sheep Blood and Gentamicin: Same as (2) except with an increased volume of 75...............0 ml Kanamycin............................2 ± 0...........0 Cornstarch ..2 at 25°C Columbia Anaerobe Agar + 5% Sheep Blood: Same as (2) with the addition of 5..0 Yeast Enriched Peptone ........3 ± 0.........3......... Columbia CNA Agar with 5% Sheep Blood inhibits many strains of coagulase-negative staphylococci....................................... (6) Columbia Agar + 5% Sheep Blood and Kanamycin: Same as (2) with the addition of 10.... (5) Columbia Anaerobe Agar + 7..................0 mg Gentamicin......... Columbia CNA Agar + 5% Sheep Blood: Same as (3) with 10....0 Yeast Enriched Peptone .....0 mg of Nalidixic acid...3 ± 0.. Observe approved biohazard precautions.............0 Sheep Blood .............10......... and temperature fluctuations...... Final pH 7................ 5.547. Media will perform satisfactorily 27120 SW 95th Avenue • Wilsonville.............0 Final pH 7..5% Sheep Blood and Gentamicin: Same as (3) except with an increased volume of 75..... Media should not be used if there are signs of contamination...............2 at 25°C Columbia Agar + 5% Sheep Blood: Peptic Digest of Animal Tissue .........3...0 Pancreatic Digest of Heart Muscle . 5. On Columbia Agar with Blood.............. OR 97070 • 800........ 1.........................0 Agar ............. 10.....0 g Pancreatic Digest of Casein .............5........... Columbia Anaerobe CNA Agar + 5% Sheep Blood: Same as (8) with the addition of 5..... per liter of deionized filtered water.......... 1.... small amounts of reducing sugars inhibit the expression of beta hemolysis. () Columbia Agar: Peptic Digest of Animal Tissue ...0 Cornstarch .. 5......

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

Koneman. Chapter 62. 7. translucent. 2. bioMérieux. B. USHEW. 28:484-488. Pathol. 6. 9th ed. Journal of Clinical Microbiology.. 45:502-504. 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. PHS. American Society for Microbiology.. 12th ed.. Facklam. J. 3. Louis. B. 4. C.. Mosby.547. opaque. Murray. W. p/c User Quality Control: Check for signs of contamination and deterioration. et al. and straw in color. consult appropriate references.TECHNICAL DATA SHEET #250 REV. et al.NF 25. R. Isolation and Identification of Streptococci. A. Ellner. M. E.. p/c Inhibition. Forbes. 5th ed. et al. p/c Inhibition. United States Pharmacopeia 30 . Lippincott.. p/c +Gentamicin Growth Growth Growth Growth Growth Inhibition. Columbia media should appear firm. 2007. 1980. C. 1966.. Bailey and Scott’s Diagnostic Microbiology. et al. BIBLIOGRAPHY: 1. and bright red in color. D. St. p/c Inhibition. Manual of Clinical Microbiology.R. Green. 2003. Washington. 2007. R.0659 Page 4 . 5. V. Centers for Disease Control. *For more detailed information... P. Philadelphia. 1990. Am. OR 97070 • 800. P. Atlanta. et al.. J.. Color Atlas and Textbook of Diagnostic Microbiology. Microbial examination of nonsterile products: Tests for specified microorganisms.. Data #250 Revision Date: July 2009 27120 SW 95th Avenue • Wilsonville. 1997. D. Columbia Agar media with blood should appear firm. Clin.. Inc.

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