BD BBL Mycoslide. INSTRUCTIONS FOR USE READY-TO-USE DIPSLIDE MEDIA DA Rev.: June 2003

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1 INSTRUCTIONS FOR USE READY-TO-USE DIPSLIDE MEDIA DA Rev.: June 2003 BD BBL Mycoslide INTENDED USE BBL Mycoslide is a three-sided dipslide containing media for the detection of fungi, including yeasts, from urine, sputum, throat washes, stool specimens, and a variety of specimens collected on swabs. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Filamentous fungi and yeasts are frequent pathogens, causing many types of local and systemic infections in immunocompetent and immunocompromised individuals. 1 One of the most frequent fungal pathogens is Candida albicans. In immunocompromised patients, C. albicans and the recently described C. dubliniensis are frequently causing localized and systemic infections. The three main body sites involved in local infections are the respiratory tract, the genitourinary tract, and the intestinal tract. Quantitative determination of the yeasts isolated from these body sites is important since they may be present in low numbers in normal flora but increase significantly during infections. 1-5 BBL Mycoslide consists of a three-sided agar carrier, covered with two fungal media and a medium for the detection of the bacteria present in the specimen. The whole agar carrier is dipped into liquid specimens or is inoculated by streaking specimens from swabs. After incubation, the isolates may be counted and used for further differentiation and identification tests and for susceptibility testing. Medium 1 is Malt agar for the detection of all types of fungi, including moulds and yeasts. Malt extract contains all necessary nutrients to support growth of fungi. The low ph of the medium results in a partial to inhibition of contaminating bacteria but is well tolerated by fungi. This medium is used for the quantitative determination of fungal pathogens. Medium 2 is Malt Agar supplemented with cycloheximide which is an inhibitor of saprophytic moulds and certain yeasts. Most pathogenic fungi, including Candida albicans, are not inhibited on this medium. However, Cryptococcus neoformans and certain Candida species other than C. albicans which may also play a role as infectious agents, are inhibited by cycloheximide. Medium 3 is CLED (Cystine-Lactose-Electrolyte-Deficient) Agar, a differential culture medium for use in isolating and enumerating bacteria in urine. It may also be used for isolating bacteria from other specimens if they are not too fastidious. On BBL Mycoslide, the medium is mainly used to determine the accompanying bacterial flora. BBL Mycoslide is used for the detection, isolation and enumeration of fungi from a variety of body sites and specimen types. It can also be used as a transport medium from the physician s office to analytical laboratories. REAGENTS Formulas* Per Liter Purified Water BBL Mycoslide Medium 1: Malt Agar Medium 2: Malt Agar with Cycloheximide Medium 3: CLED Agar Malt Extract 30.0 g Malt Extract 30.0 g Gelatin Peptone 4.0 g Agar 15.0 Agar 15.0 Casein Peptone 4.0 ph 4.1 +/- 0.2 Cycloheximide 1.0 Beef Extract 3.0 ph 4.1 +/- 0.2 Lactose 10.0 L-Cystine Bromthymol Blue 0.02 Agar 15.0 ph 7.3 +/- 0.3 DA

2 *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. Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures, biohazards, and disposal of used product. When using BBL Mycoslide 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 Mycoslide in the dark at 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 C. Once opened, slides must be used immediately. USER QUALITY CONTROL Prepare suspensions matching the McFarland standard 0.5 of the strains mentioned below. For each organism, prepare a tube (wide enough to allow insertion of a BBL Mycoslide agar carrier) with 20 to 25 ml of sterile saline and add 1 ml of the above suspension of the strain. Open a BBL Mycoslide, remove the agar carrier, dip it briefly into the test strain suspension, and proceed as described under Test Procedure for liquid specimens. Incubate for 2 to 5 days at 35 ± 2 C. Inspect agar surfaces for growth as described under Results and Interpretation. Strains Medium 1 Malt Agar Medium 2 Malt Agar with Cycloheximide Medium 3 CLED Agar Candida albicans Growth Growth Growth ATCC Aspergillus niger Growth Inhibition partial to Growth ATCC E. coli Inhibition partial to Inhibition partial to Growth ATCC Pseudomonas aeruginosa Inhibition partial to Inhibition partial to Growth ATCC Enterococcus faecalis Inhibition partial to Inhibition partial to Growth ATCC Staphylococcus aureus Inhibition partial to Inhibition partial to Growth ATCC Uninoculated Light amber Light amber Greenish-yellow PROCEDURE Materials Provided BBL Mycoslide. Microbiologically controlled. Materials Not Provided Ancillary culture media, reagents and laboratory equipment as described. Specimen Types BBL Mycoslide is suitable for the isolation and enumeration of fungi from urine (mid-stream or catheter urine, or urine collected by suprapubic bladder puncture), sputum, throat washes, stool, and a variety of other specimens (e.g., vaginal swabs). Do not use to detect bacterial infections.. DA

3 Collection and Preparation of Specimens Observe aseptic techniques for collecting specimens. Use standard procedures for collection. 1,6 Urine must be fresh or not older than 2 hours. Alternatively, urine specimens may be kept refrigerated (not longer than 24 hours) to avoid overgrowth of the infectious agents or contaminants before inoculation. Sputum: One part of fresh coughed or induced sputum is added to one part of a 0.25% trypsin solution and shaken with sterile glass beads for 45 minutes at 37 C. Alternatively, the sputum may be homogenized in a Stomacher for 1 minute. If only a small amount of sputum is available, it may be used without pretreatment. Throat washes: Centrifuge the fluid for 10 minutes at 2000 x g. Discard the liquid phase, take up the sediment in 5 ml trypsin solution (0.25%) or sterile saline and shake with sterile glass beads for 15 minutes at 37 C. Stool specimens, freshly passed or kept refrigerated for not longer than 48 hours. Homogenize 1g stool in 100 ml 0.25% trypsin solution or sterile saline with sterile glass beads for 15 minutes at 37 C. Swabs: Collect the specimen (e.g., from the throat or vagina) on a swab and apply directly without pretreatment as described below. If there is a delay between collection and inoculation, transport the swab in appropriate transport media. 1,6 Test Procedure 1. Visually inspect agar surfaces with BBL Mycoslide tube closed. Discard slides with shrinkage of the media or contamination. 2. Label the tube with patient name, specimen number, and date of inoculation. 3. Unscrew cover and remove the slide from the plastic tube, without touching the agar surfaces. Inoculation with liquid specimens (e.g., urine or throat washes): 1. Briefly dip the slide three times into liquid specimens (e.g., urine or pretreated sputum, throat washes, etc.) so that the agar layers are ly inoculated by the liquid. Do not leave the slide in the liquid for more than 10 seconds since this might wash out the ingredients from the media and/or loosen the gel from the plastic carrier. 2. If only a small volume of specimen is available, collect it with a sterile pipette or syringe and rinse the three media ly. 3. Let excess material drip off by holding the tip of the slide against the inside rim of the tube. 4. The last drops can be eliminated from the tip of the slide with a tissue paper. Carefully return the slide to the plastic tube and close ly. 5. Incubate the slide for 48 hours at 30 ± 2 C or forward it to a special laboratory after inoculation. Longer incubation may be necessary for growth of filamentous fungi, e.g., from lower respiratory tract infections. Inoculation with specimens on swabs: Carefully streak the swab onto the three culture media of the slide. Results and Interpretation After incubation, carefully remove the slide from the tube for interpretation. Observe aseptic techniques during the whole inspection procedure. It is recommended to wear surgical gloves during inspection. Do not touch the agar surfaces! Medium 1 (Malt Agar): This medium allows detection of the total count of all fungi. Filamentous fungi usually produce colonies with a rough surface (aerial mycelium). The colonies are often pigmented (black, gray, blue-green, yellowish, and others). Yeast species produce white to cream, shiny to dull colonies that usually have a smooth surface and resemble bacterial colonies. Further tests are needed for a identification of the pathogens. 1 For quantitative determination, compare the actual growth on the slide with the reference pictures provided in the Interpretation Guide at the end of this document. In case of heavy growth and large numbers of colonies 10 5 ), the agar surfaces may be ly covered by a confluent lawn of growth. Refer to the table and to the references for the clinical significance of pathogen counts for the specimens from various body sites: 1-5 DA

4 Specimen Type Number of yeast colonies Normal 1) Questionable 2) Clinically significant Midstream or catheter urine 10 3 /ml /ml >10 5 /ml Urine collected by suprapubic bladder Any appearance of fungi is clinically significant puncture Sputum 10 3 /ml /ml >10 5 /ml Throat washes >10 4 Stool 10 3 /g /g >10 5 /g 3) Vaginal specimens Any appearance of fungi is clinically significant 1) Low counts may be significant in pretreated patients or in patients with chronic infections. 2) Re-evaluate if possible. 3) A therapy of patients with high yeast counts in stool must be carefully evaluated. High yeast counts in stool often occur in patients pretreated with antibacterial antimicrobials. After the antibacterial therapy has been d, the yeast count usually decreases again to normal. In immunocompromised patients, even low counts may indicate an infection. 1,3 Medium 2 (Malt Agar with cycloheximide): This medium ensures the suppression of those filamentous fungi that are sensitive to cycloheximide (e.g., moulds like Aspergillus). Certain yeasts are also inhibited by this additive. Cycloheximide resistance is an important characteristic for the differentiation of fungi: Organisms 1) Clinical significance and frequency Cycloheximide-resistant Candida albicans Important pathogen; frequent C. dubliniensis Often found in HIV patients C. guilliermondii Rare, low C. kefyr (=C. pseudotropicalis) Rare, low Other species Rare Cycloheximide-sensitive Candida tropicalis Rare, low C. krusei Relatively frequent, known pathogen C. parapsilosis Rare, known pathogen Candida (Torulopsis) glabrata Relatively frequent, known pathogen Cryptococcus neoformans Rare; important pathogen Aspergillus, Penicillium, and other fungi Aspergillus may produce lower respiratory tract or other infections. These fungi may also occur as contaminants. 1) Note that growth or inhibition on this medium is not limited to the species listed in this Table. The organisms mentioned occur rather frequently in clinical specimens. For additional information, consult the references. 1,7 Medium 3 (CLED Agar) is used for the quantitative determination of the bacterial count present in the specimen. The bacterial counts may be important in the assessment of the relationship between bacterial and fungi growth, e.g., in stools. Note that further tests, such as biochemical and/or microscopic procedures are necessary to ly identify the pathogens isolated on this dipslide. 1 Most of these methods are only available in diagnostic laboratories and usually cannot be performed in a physician s office. Therefore, all dipslides with doubtful or clearly positive results should be sent to an analytical laboratory performing mycological tests. After use and prior to discarding, all used tubes and other contaminated material must be autoclaved or incinerated. For details, see GENERAL INSTRUCTIONS FOR USE document. DA

5 PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE BBL Mycoslide is used for the detection, isolation, and enumeration of fungi, including yeasts, from urine, sputum, throat washes, stool specimens, and a variety of other specimens. The media contained on this dipslide are commonly used standard media for the isolation of the 1, 8-10 respective fungal and bacterial groups. Occasionally, the growth of bacteria on the fungal culture media (medium 1 and 2) of this dipslide is not ly suppressed. In cases of doubt, a methylene blue or Gram stain from suspicious colonies should be performed. Most less fastidious bacterial species will grow on medium 3 (CLED Agar). However this medium does not support the growth organisms like pathogenic Neisseria species, Haemophilus spp., and others. Beta-hemolytic streptococci do not grow well on this medium. Therefore, blood agar or chocolate agar must be included if a bacterial infection with such organisms is suspected. On BBL Mycoslide, CLED Agar is mainly used to determine the accompanying bacterial flora. For exact diagnosis of bacterial infections, it is recommended to use other systems and media. BBL Urotube systems should be used for diagnosing bacterial infections from urine specimens. Specimens for the detection of bacterial infections from other body sites must be processed on plated culture media as appropriate for the specimen. The number of species that may grow on the media of this dipslide is large and is not restricted to those mentioned in the Results and Interpretation section. Appropriate identification of many organisms can only be provided by analytical laboratories. Therefore, slides should be sent to such laboratories after inoculation or after inoculation and incubation. BBL Mycoslide is intended to be used for primary isolation and counting. For further tests, and especially if mixed cultures occur on the media of this system, subcultures onto appropriate plated media must be made to obtain pure cultures which are mandatory for identification and susceptibility testing. 1 Dipslides must not be used for performing disc susceptibility tests. REFERENCES 1. Fromtling, R.A. (section editor) Mycology. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8 th ed. American Society for Microbiology, Washington, D.C. 2. Halle, E., et al Genitalinfektionen Teil I und II. In: Mauch, H., Lüttiken, R., and S. Gatermann (eds.): MiQ - Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik, vol. 10 and 11. Urban & Fischer, Munich, Germany. 3. Kist, M., et al Infektionen des Darmes. In: Mauch, H., Lüttiken, R., and S. Gatermann (eds.): MiQ - Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik, vol. 9. Urban & Fischer, Munich, Germany. 4. Podbielski, A., et al Infektionen des Mundes und der oberen Atemwege. In: Mauch, H. and R. Lüttiken (eds.): MiQ - Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik, vol.13. Urban & Fischer, Munich, Germany. 5. Mauch, H., et al Infektionen der tiefen Atemwege Teil II. In: Mauch, H., Lüttiken, R., and S. Gatermann (eds.): MiQ - Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik, vol. 8. Urban & Fischer, Munich, Germany. 6. Sutton, D.A Specimen collection, transport, and processing: mycology. 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. 7. Hazen, K.C New and emerging yeast pathogens. Clin. Microbiol. Rev. 8: MacFaddin, J. D Media for isolation-cultivation-identification- maintenance of medical bacteria, vol. 1, p Williams & Wilkins, Baltimore, MD. 9. Larone, D.H. 1995: Medically important fungi - a guide to identification. 3 rd edition. ASM Press, Washington. 10. Atlas, R.M Handbook of Microbiological media. CRC Press, Boca Raton, FL. DA

6 PACKAGING/AVAILABILITY BBL Mycoslide Cat. No slides FURTHER INFORMATION For further information please contact your local BD representative. BD Diagnostic Systems Tullastrasse 8 12 D Heidelberg/Germany Phone: , Fax: Reception_Germany@europe.bd.com BD Diagnostic Systems Europe Becton Dickinson France SA 11 rue Aristide Bergès Le Pont de Claix/France Tel: Fax: BD, BD logo and BBL are trademarks of Becton, Dickinson and Company. Urotube and Mycoslide are trademarks of Becton Dickinson GmbH. ATCC is a trademark of the American Type Culture Collection Becton, Dickinson and Company INTERPRETATION GUIDE (Malt Agar, Medium 1) For the quantitative determination of cell counts (see Results and Interpretation section for clinically important counts) DA