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Identification of fungi.pptx
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- 2. Objective â—Ź Internalize thebasics of laboratory diagnosis in mycology â—Ź Understand the technical terms used â—Ź Familiarize with the procedures of the tests done
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- 4. Blood It is thespecimen of choice for disseminated mycoses (fungal infections that spreads throughout the body)
- 6. For blood cultureit is required that we use BHIA or BHIB BHI - Brain Heart Infusion (Agar/ Broth)
- 7. Detection is observedwithin 4 days from the start of incubation Incubation with some exception such as Histoplasma capsulatum infection which takes 2 weeks of incubation before it is detected. Direct smears are examined via germ tube, KOH and gram stain
- 8. CSF Must be processedimmediately It should be kept at room temperature or in an incubator at 25-30C
- 10. CSF is filteredthrough a 0.45um pore size membrane filter attached to a syringe Filter is removed and placed on to culture medium Culture is then examined daily and observed for growth
- 11. If CSF volumereceived is less than 2ml, it should be centrifuged, and then sediments are divided equally into several parts of agar surface without any antibacterial or antifungal additives Direct smears are examined via germ tube, KOH and gram stain
- 12. Skin, hair andnails Skin scrapings are used wherein it the method makes use of a sterile scalpel’s blunt end and suspected lesions are scraped. For hair, strands are plucked with forceps and are placed into a sterile petri plate prior to culture.
- 16. Specimens then areplated or placed into a tube slant with Mycobiotic agar (agar optimal for fungal growth and inhibits bacterial growth) Mycobiotic agar contains any of the following: gentamicin, cyclohexamide, and chloramphenicol Direct smears are examined via germ tube, KOH and gram stain
- 17. Respiratory secretions Most commonspecimen in the study of mycology since fungal infections primarily focuses on the respiratory tract
- 19. 0.5ml of specimenis inoculated into the media Antibacterial agents are added to the media for optimal recovery of fungus and prevent bacterial growth Direct smears are examined via germ tube, KOH and gram stain
- 20. Tissue, bone marrowand sterile body fluids Soft or hard tissues are minced or grind prior to culture, with 1ml of tissue recovered for culture Bone marrow is directly plated to the media Body fluids are centrifuged, decanted and sediments are inoculated to the medium Direct smears are examined via germ tube, KOH and gram stain
- 22. Urine Urine samples arecentrifuged and decanted. Sediments are inoculated using a loop or dropper into the media. Antibacterial agents are added to prevent bacterial contamination Direct smears are examined via germ tube, KOH and gram stain
- 24. Gram stain Fungi canbe detected using some staining agents such as Gram stain. But fungi (yeast & mold) cannot be classified as Gram positive or negative using such staining agent.
- 25. Reagents/Supplies • Crystal Violet,the primary stain • Iodine, the mordant • A decolorizer made of alcohol (95%) • Safranin, the counterstain
- 31. Procedure • Take aclean, grease free slide. • Prepare the smear of suspension on the clean slide with a loopful of sample. • Air dry and heat fix • Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. • Flood the gram’s iodine for 1 minute and wash with water. • Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water. • Add safranin for about 1 minute and wash with water. • Air dry, Blot dry and Observe under Microscope.
- 34. Interpretation Presence of manyyeast cells or fungal spores under the microscope is indicative of fungal infection
- 36. KOH The KOH (Potassiumhydroxide) procedure is a method used to examine specimens for yeast. KOH serves as an enzymatic agent that breaks down debris in a specimen, such as epithelial cells and WBCs, to view yeast or pseudohyphae
- 37. Principle The KOH mountis used to aid in detecting fungal elements in specimens containing keratinous material, such as skin, nails, or hair and other debris. The KOH dissolves the background keratin, unmasking the fungal elements to make them more apparent
- 38. Reagents/Supplies • Personal protectiveequipment • Sharps container • Biological waste container and bag • Sterile microscope slides • Sterile pipettes • Glass coverslips • Potassium hydroxide (KOH)
- 40. Procedure ● Mix thespecimen well. ● Using a sterile pipette, remove one drop or 10 microliters of the specimen from the tube. ● Place one drop (10 µL) of the specimen on a clean microscope slide with the patient’s identification number. ● Without touching the specimen, add one drop (10 µl) of 10% Potassium hydroxide (KOH) directly to the drop of specimen on the slide. ● Place a coverslip on the drops on the slide. ● Place the slide on a brightfield microscope, focus using low power (10X), and scan at least 10 fields using high power (40X). ● Examine for budding yeast or yeast with pseudohyphae. ● Record results based on your laboratory’s criteria.