BD™ Bromcresol Purple Lactose Agar

INSTRUCTIONS FOR USE – READY-TO-USE PLATED MEDIA PA-256501.06 Rev.: Nov 2012

BD Bromcresol Purple Lactose Agar

INTENDED USE BD Bromcresol Purple Lactose Agar is a differential, non-selective medium for use in enumerating bacteria in urine. It supports the growth of urinary pathogens and contaminants but prevents undue swarming of Proteus species due to its lack of electrolytes. The medium allows the differentiation of the bacteria recovered into lactose fermenters and non-fermenters.

PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Bromcresol Purple Lactose Agar is a modification of the medium devised by Wurtz.1,2 Beef extract has been added to improve its productivity. The medium is an alternative to CLED Agar, but includes a different pH indicator. As in CLED Agar, the absence of electrolytes reduce the swarming of Proteus species and thus render the medium suitable for the counting of bacteria in urine. In BD Bromcresol Purple Lactose Agar peptone and beef extract provide nutrients. Due to lactose as the only sugar included into this medium and bromcresol purple as a pH indicator, it allows the differentiation of lactose fermenters, which produce acids from this sugar and change the color of the medium surrounding colonies to yellow, from lactose nonfermenters (blue- violet).1 Due to the absence of sodium, the swarming of Proteus spp. is reduced or absent with most of the strains of Proteus.

REAGENTS BD Bromcresol Purple Lactose Agar Formula* Per Liter Purified Water Peptone 5,0 g Beef extract 3,0 Lactose 10,0 Bromcresol Purple 0,025 Agar 15,0 pH 7.0 +/- 0,3 *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. 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 aerobically for 18 to 24 hours at 35 to 37° C.

PA-256501.06 - 1 - Strains Growth Results Escherichia coli Growth good to excellent; medium-sized yellow colonies, ATCC 25922 yellow medium surrounding colonies Enterobacter cloacae Growth good to excellent; medium-sized light yellow ATCC 13047 colonies, yellow to bluish medium surrounding colonies Proteus vulgaris ATCC 8427 Colorless to blue-gray colonies, swarming partially to completely inhibited; blue-violet medium Enterococcus faecalis Growth good to excellent; small pale violet to yellow ATCC 29212 colonies; yellow medium surrounding colonies Staphylococcus saprophyticus Growth fair to excellent; small white colonies; yellow ATCC 15305 medium surrounding colonies Uninoculated Blue-violet

PROCEDURE Materials Provided BD Bromcresol Purple Lactose Agar (90 mm Stacker plates). Microbiologically controlled.

Materials Not Provided Ancillary culture media, reagents and laboratory equipment as required.

Specimen Types This medium is mainly used for enumerating and differentiating bacteria in urine. Midstream or catheter urine, or urine collected by suprapubic bladder puncture can be used (see also PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE). Observe aseptic techniques for collecting urine specimens. Urine must be directly streaked onto the medium not later than 2 hours after collection or must be kept refrigerated (not longer than 24 hours) to avoid overgrowth of the infectious agents or contaminants before inoculation of this medium.3,4 The medium may also be used for differentiation of isolates from other specimens into lactose fermenters and nonfermenters.

Test Procedure Collect a sample of the undiluted, well-mixed urine using a calibrated loop (0.01 or 0.001 ml). Ensure proper loading of the loop with the specimen. Inoculate the sample down the middle of the plate in a single streak from which additional spreading of the inoculum is performed. 4 ,5 Incubate plates in ambient air at 35 ± 2°C for 24 to 48 h. For differentiation of isolates from other specimens into lactose fermenters and nonfermenters, streak the isolate (which must be a pure culture) onto the medium to obtain isolated colonies and incubate as described for urine specimens.

Results After incubation, the plates are read for growth and the appropriate color reactions. Colonies of lactose fermenting organisms will be yellow, surrounded by a yellow halo in the medium. Colonies of lactose non-fermenters will be colorless to gray-blue and will not produce a color change of the medium.

Calculation and Interpretation of Results Count the number of colonies (cfu) on the plate. If a 0.01 ml loop was used, each resultant colony is representative of 100 CFU/ml; if a 0.001 ml loop was used, each colony corresponds to 1000 CFU/ml of urine.3 Midstream and catheter urine: Current guidelines indicate that for a single isolate a density of 105 cfu/ml indicates infection, <105 cfu/ml indicates urethral or vaginal contamination, and between 104 to 105 CFU/ml needs to be re-evaluated based on clinical information.3,4 Contaminant bacteria usually appear in low numbers which vary in colonial morphology. Urine collected by suprapubic bladder puncture: Since the bladder is sterile in non-infected individuals, any cfu detected indicates an infection.

PA-256501.06 - 2 - Urinary pathogens will usually yield high counts having uniform colonial morphology and must be subcultured directly to routine media for identification and susceptibility testing.4,5

PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE BD Bromcresol Purple Lactose Agar is suitable for the isolation and counting of many aerobically growing microorganisms, such as Enterobacteriaceae, Pseudomonas and other non-fermenting Gram negative rods, enterococci, staphylococci, Candida species, and many others from urine specimens.

Streptococci and other organisms requiring blood or serum for growth may only be insufficiently recovered on this medium or may need extended incubation. Therefore, the specimen should also be cultivated onto a blood agar plate if such organisms are expected. Genitourinary pathogens such as Neisseria gonorrhoeae, Gardnerella vaginalis, Chlamydia, Ureaplasma, or other fastidious organisms do not grow on this medium. Consult the references for the appropriate detection techniques of these organisms.3-5 Although a differentiation according to lactose fermentation and certain diagnostic tests may be performed directly on this medium, biochemical and, if indicated, immunological testing using pure cultures is necessary for complete identification.

Performance Results In an internal evaluation, the medium was tested with twenty strains, 17 of which were Enterobacteriaceae and three were Gram positive cocci. All strains produced excellent growth and allowed differentiation into lactose fermenters and nonfermenters. Swarming of Proteus was significantly to completely reduced to allow counting of colonies and purification of mixed cultures. Details are shown in the Table:

Species Growth Results on BD Bromcresol Purple Lactose Agar Enterococcus faecalis Growth good to excellent; small, yellowish to white colonies; Enterococcus faecium yellow medium surrounding colonies Escherichia coli, lactose positive Growth good to excellent; medium-sized to large yellow colonies; yellow medium surrounding colonies Escherichia coli, lactose negative Growth good to excellent; medium-sized to large gray colonies; blue-violet medium Morganella morganii Growth good to excellent; medium-sized gray colonies; blue- violet medium Proteus mirabilis Growth good to excellent; medium-sized to large gray colonies; no swarming; blue-violet medium Proteus penneri Growth good to excellent; medium-sized to large gray colonies; small swarming zones surrounding individual colonies; blue-violet Proteus vulgaris medium Providencia alcalifaciens Growth good to excellent; medium-sized gray colonies; blue- violet medium Providencia stuartii Growth good to excellent; medium-sized gray-white colonies; blue-violet medium Salmonella Abony Salmonella Enteritidis Growth good to excellent; medium-sized to large gray-white Salmonella Saintpaul colonies; blue-violet medium Salmonella Typhimurium Shigella boydii Shigella flexneri (2 strains) Growth good to excellent; medium-sized gray colonies; blue- Shigella sonnei (2 strains) violet medium Staphylococcus aureus

PA-256501.06 - 3 - REFERENCES 1. Wurtz. 1897. Techniques bactériologiques. Masson, Paris. 2. Atlas, R.M.: Handbook of Microbiological Media (L.C. Parks, editor).1993. CRC Press, Boca Raton, USA. 3. Thomson, R.B., and J.M. Miller. 2003. Specimen collection, transport, and processing: bacteriology. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C. 4. Clarridge, J.E., M.T. Pezzlo, and K.L. Vosti. 1987. Cumitech 2A, Laboratory diagnosis of urinary tract infections. Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C. 5. Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). 2003. Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.

PACKAGING/AVAILABILITY BD Bromcresol Purple Lactose Agar Cat. No. 256501 Ready-to-use Plated Media, cpu 20

FURTHER INFORMATION For further information please contact your local BD representative.

Becton Dickinson GmbH Tullastrasse 8 – 12 D-69126 Heidelberg/Germany Phone: +49-62 21-30 50 Fax: +49-62 21-30 52 16 [email protected] http://www.bd.com http://www.bd.com/europe/regulatory/

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