TECHNICAL DATA SHEET #640 REV.

BURKHOLDERIA (PSEUDOMONAS) CEPACIA ISOLATION MEDIA

PRODUCTS:
Plated Media: Pseudomonas cepacia Agar (PC) Burkholderia (Pseudomonas) cepacia Selective Agar (BCSA) OFPBL P2282 P1226 303433

PURPOSE:
Pseudomonas cepacia (PC) Agar, Burkholderia (Pseudomonas) cepacia Selective Agar (BCSA), and OFPBL are used for the selective isolation of Burkholderia (Pseudomonas) cepacia from clinical and non-clinical specimens. They are primary isolation media for sputum samples from patients with cystic brosis (CF) and specimens from patients with chronic granulomatous disease (CGD) as well as environmental uids.

SUMMARY AND EXPLANATION:

Burkholderia (Pseudomonas) cepacia is an opportunistic pathogen generally associated with nosocomial infections. Due to its ability to survive for extended period of time in hostile environments, it is found in such widely varied and inhibitory items such as equipment, medications, mouthwash and disinfectants. Nosocomial infections caused by this organism include bacteremia, urinary infections, and respiratory infections. However, the most serious implication is when identied in patients with CF, it has signicant impact on prognosis and quality of life. It has also been shown to be a primary cause of bacteremia, pneumonia, and death in the CGD patient population.3 Therefore, it is critical that isolation and proper identication be expeditious and accurate. Recovery of this organism on general media poses problems due to overgrowth of common isolates such as Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. This necessitated the development of more selective recovery media, which inhibit the growth of Pseudomonas aeruginosa, while supporting the slow-growing Burkholderia (Pseudomonas) cepacia. PC Agar was developed by Gilligan et al. for improved recovery of B. cepacia.4 Inhibitory agents are added for selectivity, while the phenol red color indicator facilitates detection. OFPBL (Oxidation-fermentation basal media with polymyxin B, bacitracin, lactose) is a differential media that is less selective and detects the presence of B. cepacia through the color change of the indicator, bromthymol blue. Most recently, Henry et al.5 formulated BCSA, a highly selective and differential media, to improve the speed and accuracy of isolating the B. cepacia complex, as well as provide quantitative recovery.5,6 It has been noted in reference texts that this media is more sensitive and selective than either PC Agar or OFPBL.2,8

PRINCIPLE:
This media contains various enzymatic digests of proteinaceous substrates, inorganic salts, carbohydrates and other necessary nutrients to provide the growth factors for the target organisms. Selective ingredients are added to the media to inhibit common contaminants while supporting growth of the B. cepacia. PC agar contains crystal violet for inhibition of gram-positive cocci (esp. enterococci and staphylococci), bile salts for inhibition of most other gram-positive cocci, and the combination of ticarcillin and polymyxin B for gram-negative inhibition. OFPBL incorporates polymyxin B and bacitracin (for the inhibition of gram-positive organisms and Neisseria).9 BCSA incorporates crystal violet, polymyxin B, gentamicin, and vancomycin with the addition of sucrose for additional carbohydrate support. Phenol Red is the pH indicator in PC and BCSA media to facilitate the detection of B. cepacia. Metabolism of pyruvate in PC produces alkaline end products which raise the pH of the media and cause the color of the indicator to change, resulting in a pink to pink-red appearance. Where heavy growth of B. cepacia occurs, the pink color intensies. In BCSA, acid production from the oxidation of carbohydrates creates a yellow zone in the media in the area surrounding the growth, whereas peptone utilization results in a pink zone. Absorption of the crystal violet dye will cause colonies to appear purple to purple-gray. Bromthymol Blue is the pH indicator incorporated in OFPBL to facilitate the detection of B. cepacia. Lactose metabolism creates acid end products which lower the pH resulting in a yellow color change. Colonies of B. cepacia will appear to have a yellow color.

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TECHNICAL DATA SHEET #640 REV. 5

FORMULAS:
Approximate, per liter of deionized ltered water.

(1) Pseudomonas cepacia Agar (PC)

Pyruvate ............................................ 5.0 g Proteose Micro Peptone .................. 1.0 Bile Extract (Oxgall) ......................... 1.5 Ammonium Sulfate........................... 1.0 Dibasic Potassium Phosphate ......... 1.4 Monobasic Potassium Phosphate ... 4.4 Agar .............................................. 15.0 Magnesium Sulfate ........................... 0.2 Ferrous Sulfate ............................... 10.0 mg Crystal Violet .................................... 1.0 Phenol Red ..................................... 20.0 Ticarcillin ....................................... 100.0 Polymyxin B ........................... 300,000.0 units Final pH 6.8 0.2 at 25C

(2) Burkholderia (Pseudomonas) cepacia Selective Agar (BCSA) Peptic Digest of Casein .................10.0 g Yeast Extract ....................................1.5 Sodium Chloride ..............................5.0 Sucrose .........................................10.0 Lactose ..........................................10.0 Crystal Violet .............................. 0.002 Phenol Red ....................................0.08 Agar ...............................................14.0 Gentamycin....................................10.0 mg Polymyxin B ............................ 600,000 units Vancomycin ................................... 2.5 mg Final pH 7.0 0.1 at 25C (3) OFPBL Pancreatic Digest of Casein ..................2.0 g Sodium Chloride ....................................5.0 Dipotassium Phosphate.........................0.3 Agar .....................................................15.0 Bromthymol Blue ................................. 0.03 Lactose ................................................10.0 Bacitracin ............................................. 200 units Polymyxin B .................................. 300,000 Final pH 6.8 0.2 at 25C

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: PC Agar is not a differential media; other microbes can utilize pyruvate, produce alkaline by-products, and cause the media to turn pink to hot-pink in color. Organisms such as B. gladioli, and others may also grow on OFPBL Agar and resemble B. cepacia (yellow colonies). Therefore, pure culture colonies must have biochemical testing performed to denitively identify B. cepacia. PC Agar may not be as selective for Burkholderia cepacia in environmental uids that contain high levels of naturally occurring gramnegative bacteria, e.g., Alcaligenes and Achromobacter species, Pseudomonas mesophilica, and Pseudomonas stutzeri.1 B. cepacia growth on BCSA may be oxidase negative on initial isolation. While highly selective against gram-negative organsisms, breakthrough growth of some gram-negative rods may be observed. (B. gladioli and Chryseobacterium indologenes, in particular, may demonstrate poor growth recovery.) The B. cepacia complex represents nine distinctive genomovars (or genomic species); growth of the organism on BCSA may exhibit variable morphology. Any selective media may show some inhibition of target organisms. It is therefore recommended that a non-inhibitory media be utilized along with the selective media for any culture. It is highly recommended that any rst time isolate of the B. cepacia complex from a CF patient be sent to a recognized reference lab for identication conrmation.

PROCEDURE:
Specimen Collection: Information on specimen collection is found in standard reference material.2,7,8 In general, specimens should be protected from extreme heat, cold, or desiccation and should be delivered to the laboratory without delay. If a delay is unavoidable, the use of a buffered holding media has proven effective in the recovery of most microorganisms.

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TECHNICAL DATA SHEET #640 REV. 5

Method of Use: Allow medium to warm to room temperature, surface should be smooth and shiny without excess moisture. Directly inoculate the specimen or the transport swab onto the selective agar and, using standard microbiological procedures, streak so as to obtain isolated colonies. Incubate aerobically at 35C 72-96 hours and examine daily. A denitive identication is accomplished through biochemical testing. Materials Required But Not Provided: Standard microbiological supplies and equipment such as those commonly found in a microbiological laboratory are not provided.

QUALITY CONTROL:
Microorganisms Used : (ATCC#) PC Burkholderia cepacia (25416) Escherichia coli (25922) Pseudomonas aeruginosa (27853) Stenotrophomonas maltophilia (49129) Staphylococcus aureus (25923) Grey-pink colony w/pink zone Expected Results: BCSA Purple colony w/yellow zone OFPBL Yellow colony w/yellow zone

Inhibition:partial to complete

Inhibition:partial to complete

N/A

Inhibition:partial to complete

Inhibition:partial to complete

N/A

Grey-pink colony w/pink zone

Purple colony w/purple zone

Inhibition:partial to complete

Inhibition:partial to complete

Inhibition:partial to complete

Inhibition:partial to complete

User Quality Control: Check for signs of contamination and deterioration. PC Agar and BCSA should appear rm, translucent, and light pink/salmon in color. OFPBL media should appear rm, opalescent, and green in color.

BIBLIOGRAPHY:
1. 2. 3. 4. 5. 6. 7. 8. 9. Carson, L. A., et al. 1988, Comparative evaluation of selective media for isolation of Pseudomonas cepacia from cystic brosis patients and environmental sources. J. Clin. Microbiol., 26:2096-2011. Forbes, B. A., Sahm, D.F., and Weissfeld, A.S., Bailey and Scotts Diagnostic Microbiology, 10th ed., C. V. Mosby, St. Louis, 1998. Gilligan, P. H. and P. Vandamme, 2003. Misc. Gram Negative Bacteria, pp 729-748. In Murray, P. R., et al., Manual of Clinical Microbiology, 8th ed., American Society for Microbiology, Washington D.C., 2003. Gilligan, P. H., et al., 1985, Isolation medium for the recovery of Pseudomonas cepacia from respiratory secretions of patients with cystic brosis. J. Clin. Microbiol., 22:5-8. Henry, D.A., et al. 1997. J. Clin. Microbiol. 35:614-619. Henry, D.A., et al. 1999. J. Clin. Microbiol. 37:1004-1007. Isenberg, H. D. (ed.), Clinical Microbiology Procedures Handbook, vol. 1, p. 3.1.1-3.10.1, American Society for Microbi olgy, Washington, D.C., 1992. Murray, P. R., et al., Manual of Clinical Microbiology, 8th ed., American Society for Microbiology, Washington D.C., 2003. Welch, D.F., et al., 1987. Selective and differential medium for recovery of Pseudomonas cepacia from the respiratory tracts of patients with cystic brosis. J. Clin. Microbiol. 25:1730-1734.

bioMrieux, Inc. Data #640 Revision Date: April 2009

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