READY-TO-USE DIPSLIDE MEDIA DA-212115.03 Rev.: Aug 2005
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INSTRUCTIONS FOR USE – READY-TO-USE DIPSLIDE MEDIA DA-212115.03 Rev.: Aug 2005 BD™ BBL UROTUBE • BBL UROTUBE M • BBL UROTUBE E • BBL UROTUBE E. coli • BBL UROTUBE SXT INTENDED USE BBL UROTUBE products are dipslides used for the isolation and determination of the colony count of microorganisms isolated from urine specimens. The surfaces of the dipslides are coated with two or three different culture media. All BBL UROTUBE products contain CLED Agar for the determination of the total count of bacteria and/or yeasts, and MacConkey Agar for the detection of Gram negative bacteria, e.g., Enterobacteriaceae. The third medium, if available, is dependent on the respective product. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Dipslides are widely used today as a screening technique for the enumeration and isolation of microorganisms from routine urine specimens.1-5 After the dipslide is immersed into a properly collected fresh urine specimen and the slide is subsequently incubated, the colony number on the surface of CLED Agar is compared to a reference picture. CLED Agar was developed by Sandys in 1960 to prevent the swarming of Proteus by restricting the electrolytes in the culture medium which was modified later several times for use in urine culture.6 It was designated as Cystine-Lactose-Electrolyte-Deficient (CLED) medium and reported to be ideal for dip-inoculum techniques and for urinary bacteriology in general.6,7 The nutrients in CLED Agar are supplied by the gelatin and casein peptones, and beef extract. Lactose is included to provide an energy source for organisms capable of utilizing it by a fermentative mechanism. Bromthymol blue is used as a pH indicator to differentiate lactose fermenters from lactose-nonfermenters. Organisms which ferment lactose will lower the pH and change the color of the medium from green to yellow. The cystine permits the growth of "dwarf colony" coliforms.3 Electrolyte sources are reduced in order to minimize the swarming of Proteus species. The number of colonies on the CLED Agar is directly proportional to the number of bacteria per ml of the urine specimen. CLED Agar is included on all BBL UROTUBE dipslide products as medium 1. The second medium is MacConkey Agar. It is only slightly selective since the concentration of bile salts, which inhibits gram-positive micro-organisms, is low in comparison with other enteric plating media. This medium is recommended for use with clinical specimens likely to contain mixed microbial flora, such as urine, because it allows a preliminary grouping of enteric and many other gram-negative bacteria in lactose fermenters and lactose nonfermenters.8,9 In MacConkey Agar, peptones provide nutrients. Crystal violet inhibits Gram positive bacteria, especially enterococci and staphylococci. Differentiation of enteric micro-organisms is achieved by the combination of lactose and the neutral red pH indicator. Colorless or pink to red colonies are produced depending upon the ability of the isolate to ferment the carbohydrate. MacConkey Agar is included on all UROTUBE dipslide products as medium 2. A third medium is included to detect the presence of characteristic bacterial groups and yeasts. The type of medium depends on the product in use: BBL UROTUBE, in addition to media 1 and 2 mentioned above, contains Cetrimide Agar, a widely used medium for the selective isolation of Pseudomonas aeruginosa.10,11 Gelatin peptone and casein provide nutrients. Potassium and magnesium salts enhance the pigment formation of P. aeruginosa. Glycerol is an energy and carbon source. Cetyl trimethyl ammonium bromide (=cetrimide) is a detergent that selectively inhibits most Gram negative rods other than P. aeruginosa. DA-212115.03 Page 1 of 10 BBL UROTUBE M, in addition to media 1 and 2 mentioned above, contains Malt Agar, a partially selective medium for the isolation of yeasts, e.g. Candida albicans. Malt extract and lactic acid provide nutrients and maintain the pH at a low level which provides selectivity to inhibit many bacterial species.11 BBL UROTUBE E, in addition to media 1 and 2 mentioned above, contains Enterococcus Agar, a selective differential medium for Enterococcus species.11 In Enterococcus Agar, casein and yeast extract provide nutrients. Sodium chloride maintains osmotic stability. The combination of citrate, azide, kanamycin, polymyxin B, nalidixic acid, and neomycin inhibit most bacteria other than Enterococcus species. Esculin is a substrate for beta-glucosidase, characteristically found in Enterococcus species. One of the products of enzymatic hydrolysis, esculetin, reacts with ferric ions to produce a brown to black precipitate in the medium surrounding Enterococcus colonies. BBL UROTUBE E. coli, in addition to media 1 and 2 mentioned above, contains Beta- Glucuronidase (=BGLU) Agar, a differential medium for the specific detection of E. coli and Proteus species. The medium contains nitrophenyl-ß-glucuronide, a chromogenic substrate to detect beta-glucuronidase activity which is typically found in Escherichia coli, while many other bacteria, including Proteus, Providencia, and Morganella, are negative.11,12 Upon degradation of the substrate, yellow nitrophenol is accumulated in the medium and the colonies, while glucuronidase-negative colonies are colorless to grayish on a colorless medium. Yellow growth from this medium can be subjected to an indole test which, if positive, confirms the identification of E. coli. The indole formation on this medium is enhanced by the addition of the indole precursor (tryptophan). Additionally, this amino acid is the substrate for the enzyme tryptophan deaminase (TDA) which is characteristically found in Proteus, Providencia, and Morganella species. This enzyme can be easily detected by performing a TDA test with ferric chloride. For further confirmation, an indole test may be performed from colorless growth which is positive in Proteus vulgaris, Providencia species, and Morganella morganii, too. BBL UROTUBE SXT, in addition to media 1 and 2 mentioned above, contains antagonist-free susceptibility test medium (PDM Medium) with trimethoprim/sulfamethoxazole (SXT), for the determination of resistance or sensitivity against SXT. Growth of any bacteria on this medium means resistance to SXT. After inoculation, or after inoculation and incubation, the dipslides may also be used as transport media from the physician’s office to analytical laboratories. Growth from the media on the dipslides may be used for identification and susceptibility testing of the isolates. REAGENTS Formulas* Per Liter Purified Water Medium 1 and 2 are common to all BBL UROTUBE dipslides. Medium 1: CLED Agar Medium 2: MacConkey Agar Peptic Digest of Casein 4.0 g Peptic Digest of Casein 17.0 g Selected Peptones 4.0 Meat Peptone 3.0 Meat Extract 3.0 Lactose 10.0 Lactose 10.0 Sodium Chloride 5.0 Cystine 0.128 Bile Salts Mixture 1.5 Bromthymol Blue 0.02 Neutral Red 0.03 Agar 15.0 Crystal Violet 0.001 pH 7,3 ± 0,2 Agar 13.5 pH 7.1 ± 0.2 Medium 3: The type of medium 3 (if available) depends on the product in use (see PRINCIPLES AND EXPLANATION OF THE PROCEDURE). BBL UROTUBE, in addition to media 1 and 2 mentioned above, contains Cetrimide Agar: Peptic Digest of Gelatin 16.0 g Glycerol 8.0 g Hydrolysed Casein 10.0 Cetyl Trimethyl Ammonium Bromide 0.3 DA-212115.03 Page 2 of 10 Potassium Sulfate 10.0 Agar 13.0 Magnesium Chloride 1.4 pH 7.1 ± 0.2 BBL UROTUBE M, in addition to media 1 and 2 mentioned above, contains Malt Agar: Malt Extract 30.0 g Agar 18.0 g Lactic Acid 6.3 pH 4.0 ± 0.4 BBL UROTUBE E, in addition to media 1 and 2 mentioned above, contains Enterococcus Agar: Peptic Digest of Casein 20.0 g Kanamycin 0.02 g Yeast Extract 5.0 Polymyxin B 0.002 Sodium Chloride 5.0 Nalidixic acid 0.0075 Citrate 1.5 Neomycin 0.002 Sodium Azide 0.15 Agar 15.0 Esculin 1.0 pH 7.1 ± 0.2 Ferric Ammonium Citrate 0.5 BBL UROTUBE E. coli, in addition to media 1 and 2 mentioned above, contains Beta- Glucuronidase (=BGLU) Agar: Peptone 10.0 g Sodium Chloride 5.0 g Nitrophenyl-ß-Glucuronide 0.1 Agar 15.0 Tryptophan 0.3 pH 7.2 ± 0.2 BBL UROTUBE SXT in addition to media 1 and 2 mentioned above, contains antagonist-free medium (PDM Medium) with trimethoprim/sulfamethoxazole (SXT). PDM Medium 32.3 g Trimethoprim 0.003 g Sulfamethoxazole 0.04 pH7.3 ± 0.3 *Formulas may be adjusted and/or supplemented as required to meet performance criteria. PRECAUTIONS . For professional use only. Do not use dipslides if they show evidence of microbial contamination, discoloration, drying, cracking or other signs of deterioration. Observe aseptic techniques and biohazard protection measures throughout the whole procedure. Wear protective gloves when collecting and handling specimens and positive slides containing infectious agents. Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures, biohazards, and disposal of used product. When using these products as a transport medium from a physician’s office to a diagnostic laboratory, observe local regulations for shipping infectious specimens. STORAGE AND SHELF LIFE On receipt, store BBL UROTUBE products in the dark at 15 – 20° C in the original package until just prior to use. Avoid freezing, overheating, desiccation, and temperature fluctuations. The slides may be inoculated up to the expiration date (see package label) and incubated for the recommended incubation times. Unopened slides from opened packages can be used up to the expiry date when stored in a clean area at 15 – 20° C. Once opened, slides must be used immediately. USER QUALITY CONTROL Prepare suspensions of the test strains mentioned below in physiological saline, matching the McFarland 0.5 standard (appr. 5 x 107 CFU/ml). Dilute the suspensions to a CFU of 104 to 105 per ml.