Bile Chrysoidin Glycerol Agar with MUG

Transcription

1 INSTRUCTIONS FOR USE READY-TO-USE PLATED MEDIA PA Rev.: Mar 2013 Bile Chrysoidin Glycerol Agar with MUG INTENDED USE Partially selective medium for the isolation and differentiation of Enterobacteriaceae and several other Gram negative rods and for the identification of E. coli from urine and other clinical specimens. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Bile Chrysoidin Glycerol Agar with MUG is a partially selective differential medium which includes approximately the same spectrum of organisms as MacConkey Agar. However, due to several biochemical reactions, it allows the morphological and color-based differentiation of a larger variety of colonies. Peptones and yeast extract are nutrients. Ox gall is a selective agent to inhibit Gram positive bacteria except enterococci. Thiosulfate together with ferric ammonium citrate is the indicator system for the hydrogen sulfide production (blackening of colonies). Bromthymol blue is a ph indicator. When glycerol is used, acid formation (yellow coloration) results; when urea is degraded by urease, alkaline products will occur (green to blue green coloration). The addition of MUG when viewed under UV light ( nm) allows the detection of beta-glucuronidase, an enzyme typical for E. coli. REAGENTS BD Bile Chrysoidin Glycerol Agar with MUG Formula* Per Liter Purified Water Peptones 12.0 g Yeast Extract 5.0 g Sodium Chloride 5.0 g Ox gall 8.0 g Sodium Thiosulphate 1.0 g Bromthymol Blue 0.12 g Ferric Ammonium Citrate 2.0 g Urea 1.0 g Glycerol 20.0 ml Chrysoidin 12.5 mg 4-Methylumbelliferyl-ß-D-glucuronide 0.1 g (=MUG) Agar 14.0 g ph 7.5 ± 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. PA Page 1 of 5

2 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 plates at C in an aerobic atmosphere. Stämme Escherichia coli ATCC Citrobacter freundii ATCC 8090 Proteus mirabilis ATCC Salmonella Typhimurium ATCC Shigella flexneri ATCC Pseudomonas aeruginosa ATCC Enterococcus faecalis ATCC Uninoculated Wachstum Yellow to greenish, occasionally orange to brownish colonies, fluorescence under UV (ß-Glucuronidase positive); medium is green Yellow colonies, partly with black centers. Medium is yellowish to green Colonies yellowish to green, often with a black center; swarming inhibited or strongly reduced. Medium is green Colonies yellowish to green, often with a black center. Medium is green Green to blue-green colonies. Medium is green Green to blue-green colonies; medium is dark green Small yellow colonies; medium is yellow Clear, green PROCEDURE Materials Provided BD Bile Chrysoidin Glycerol Agar with MUG (90 mm Stacker plates). Microbiologically controlled. Materials Not Provided Ancillary culture media, reagents and laboratory equipment as required. Specimen Types and Collection of Specimens This medium can be used for all types of clinical and nonclinical specimens (see also PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE). If used for urine, the medium may also be used for quantitative determinations of the viable count of Gram negative bacteria. For the collection and transport of specimens the usually used procedures apply. To detect all pathogenic bacteria present in the clinical specimen and eventually also normal flora, the specimen must be streaked on a nonselective optimal medium, e.g. BD Columbia Agar with 5% Sheep Blood, and eventually on other selective media. Test Procedure Inoculate the medium by streaking for isolation. If urines are applied, a defined volume or a dilution of the specimen may be spreaded over the whole surface. Incubate the inoculated plates for 18 to 24 hours at C. Results On BD Bile Chrysoidin Glycerol Agar with MUG frequently isolated bacterial species show the following growth characteristics: Organismus Wachstum E. coli Yellow to yellow-green; fluorescence under UV light Citrobacter freundii Yellow, often with a black center Proteus mirabilis Yellowish to greenish-yellow, often with a black center, swarming reduced or inhibited Salmonella spp. Yellow to yellow-green, often with a black center, or whole colony black PA Page 2 of 5

3 Klebsiella spp. Yellow Enterobacter spp. Yellow to yellow green Shigella spp. Blue to blue-green, rarely yellowish. Strains of S. sonnei may fluoresce under UV Pseudomonas aeruginosa Dark green to blue-green Enterococcus spp. Small, yellow Staphylococcus spp. Weak growth or completely inhibited Most Enterobacteriaceae will produce medium-sized to large colonies. With the exception of E. coli, further biochemical tests are needed for a complete identification of the isolates. PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE BD Bile Chrysoidin Glycerol Agar with MUG can be used for the isolation and presumptive differentiation of Gram negative rods (e.g. Enterobacteriaceae) and several nonfermenters, e.g. Pseudomonas aeruginosa. The addition of MUG allows the detection of E. coli when viewed under UV light ( = 360 nm) since MUG-positive colonies will fluoresce. Rare strains of E. coli, however, are MUG-negative and do not fluoresce under UV light. Like E. coli, Shigella sonnei may produce fluorescing colonies. Additionally, enterococci may grow on this medium. Staphylococci are usually inhibited, but may break through. For the final identification of isolates other than E. coli, additional biochemical and eventually serological tests must be performed. Some strains of enterococci and Hafnia alvei show reduced growth on this medium. It was shown in internal studies that this is also true for the competitor medium and is, therefore, no weakness of BD Bile Chrysoidin Glycerol Agar with MUG. Performance Evaluations In the internal validation at least three lots of the medium were tested with strains of the following species: Acinetobacter anitratus Proteus mirabilis Acinetobacter lwoffi Proteus vulgaris Cedecea davisiae Providencia alcalifaciens Citrobacter freundii Pseudomonas aeriginosa Citrobacter koseri (diversus) Pseudomonas putida Enterobacter aerogenes Salmonella Arizonae Enterobacter cloacae Salmonella Enteritidis Enterobacter sakazakii Salmonella Typhimurium Escherichia coli Serratia marcescens Escherichia coli, lactose negative Shigella flexneri Hafnia alvei Shigella sonnei Klebsiella pneumoniae Yersinia enterocolitica Morganella morganii Enterococcus faecalis Pantoea agglomerans Staphylococcus aureus All Gram negative bacteria and Enterococcus faecalis produced growth in the expected colors. Staphylococcus aureus was partially to completely inhibited. Additionally, two external performance evaluations were conducted. In these studies, the medium produced by BD (= test medium) was compared to the usually used Bile Chrysoidin- Glycerol Agar with MUG (= reference medium; either home-made or from a different supplier). In the first study, 157 clinical specimens (104 urine specimens, 19 wound swabs, 12 blood cultures, 18 bronchial secretions, and one each of pus, ear swab, abscess and vaginal swab) PA Page 3 of 5

4 were tested. 169 strains were isolated on the test medium and on the reference medium (Table 1). The appearance of the isolates on both media was comparable or identical. In no case isolates were only recovered on one of the media only. There were 3 MUG-negative E. coli strains isolated on both media which, accordingly, did not yield fluorescent colonies. In the second study, 103 clinical specimens (11 tracheal secretions, 10 sputa, 15 bronchial secretions or washings, 32 wound swabs, 4 ear swabs, 2 punitions, 2 skin swabs and 27 urine specimens were streaked on the test and the reference medium. In this study, all isolates were detected on both media and their appearance was identical or comparable on both, too. Additionally, ten known positive specimens were cultivated on the test and the reference medium. Of the 103 clinical specimens, 55 did not produce growth on both media. From the positive specimens, 75 strains were isolated (Table 1). As a result from both studies, there were no differences between the test and the reference medium, neither in sensitivity, nor in the appearance of the individual species (detailed data are on file). Table 1: Species isolated on the test medium in the two external performance evaluations Spezies Study 1* Study 2* Acinetobacter baumannii 0 1 Aspergillus niger 0 1 Citrobacter freundii 2 1 Citrobacter koseri 1 0 Enterobacter cloacae 9 0 Enterobacter cloacae 0 3 Enterococcus faecalis 0 3 Enterococcus faecium 0 4 Enterococcus sp. 1 7 Escherichia coli Escherichia hermannii 1 0 Klebsiella oxytoca 3 3 Klebsiella pneumoniae 10 6 Morganella morganii 1 0 Proteus mirabilis 15 6 Proteus vulgaris 4 0 Providencia stuartii 0 1 Pseudomonas aeruginosa Salmonella Enteritidis 0 1 Serratia fonticola 0 1 Serratia marcescens 4 0 Staphylococcus sp. 0 3 Staphylococcus, koagulase-negativ 1 0 Stenotrophomonas maltophilia 2 0 Vibrio cholerae 1 0 * Numbers indicate numbers of isolates. REFERENCES 1. Ziesché, K. et al. (1985) Der Galle-Chrysoidin-Glycerol (GCG)-Nährboden in seiner Anwendung zur Diagnostik gramnegativer aerober Bakterien, besonders der Enterobacteriaceae. Zeitschr. Gesamte Hygiene 31: 516- PRODUCT AVAILABILITY BD Bile Chrysoidin Glycerol Agar with MUG Cat. No Prepared Plated Media, 120 plates FURTHER INFORMATION For further information please contact your local BD representative. PA Page 4 of 5

5 Becton Dickinson GmbH Tullastrasse 8 12 D Heidelberg/Germany Phone: Fax: Reception_Germany@europe.bd.com ATCC is a trademark of the American Type Culture Collection BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD PA Page 5 of 5