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56

  1. 1. MYCOLOGY<br />FUNGAL CULTURE<br />
  2. 2. CUTURE METHOD:<br />All specimens should be inoculated onto a general purpose fungal medium <br />fungi will grow very well on culture media used to isolate bacteria<br />
  3. 3. Temperature:<br />Most MOLDS grow best at: 25 – 30 OC<br />Most YEAST grow best at 35 – 37 OC<br />Most pathogenic fungi grow best: 30 to 32°C <br />EXCEPT: Sporothrixschenckii : 25 to 27°C than 30°C<br />
  4. 4. Incubation period: <br />14 days: general incubation period<br />7 days: to detect presence of yeast in the mouth, throat, or vagina<br />21 days: tissues and sterile body fluids other than blood<br />28 days: respiratory, bone marrow, blood specimens, and specimens in which dimorphic fungus are suspected<br />Plates should be checked at least TWICE during the first week, when rapidly growing isolates may appear, weekly hereafter.<br />
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  8. 8. CHROMagar<br />A selective medium for the isolation and presumptive identification of yeast and filamentous fungi and differentiation of Candida albicans, C. tropicalis and C. krusei.<br />Due to the differences in morphology and colors of the yeast colonies, this medium facilitates the detection of mixed yeast cultures in specimens.<br />It may also be used as a selective isolation medium for other yeasts and for filamentous fungi instead of Sabouraud Dextrose Agar or similar media.<br />
  9. 9. CHROMagar<br />FORMULA IN GRAMS PER LITER<br />Glucose ....................................... 20.00 <br />Peptone …….................................. 10.00<br />Chloranphenicol ............................ 0.50<br />Chromogenic Mixture.............. ........ 0.40<br />Bacteriological Agar ....................... 15.00<br />Final pH 6.1 ± 0.2 at 25°C<br />
  10. 10. CHROMagar<br />Preparation<br />Suspend 45.9 grams of the medium in one liter of distilled water. <br />Mix well and heat with frequent agitation until complete dissolution. <br />Distribute into adequate containers.<br />
  11. 11. CHROMagar<br />Approximate Formula* Per Liter Purified Water<br />Chromopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g<br />Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g<br />Chromagen Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g<br />Chloramphenicol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g<br />Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g<br />
  12. 12. CHROMagar<br />Candida albicans- green <br />Candida tropicalis - steel blue <br />Candida krusei- rose, fuzzy <br />
  13. 13. C. krusei<br />C. tropicalis<br />C. albicans<br />
  14. 14. CHLAMYDOSPORE AGAR<br />Used for differentiating Candida albicans from other species of Candida on the basis of chlamydospore formation.<br />Candida albicans always form chlamydospore on this medium. <br />The medium contains trypan blue to visualize the chlamydospore under microscopic evaluation.<br />Biotin and polysaccharide are growth factors which stimulate chlamydospore formation. <br />Potassium phosphate function as a buffer in the medium<br />
  15. 15. CHLAMYDOSPORE AGAR<br />Components (g/L)<br />Ammonium Sulphate…………………………… 1.00<br />Monopotassium Phosphate…………………… 1.00<br />Purified Polysaccharide………………………… 20.00<br />Trypan Blue………………………………………… 0.10<br />Biotin…………………………………………………. 0.000005<br />Agar…………………………………………………… 15.00<br />Final pH (at 25°C) 5.1 ± 0.2<br />
  16. 16. CHLAMYDOSPORE AGAR<br />Organisms Growth Chlamydospores<br />Candida albicans luxuriant (+)<br />Candida tropicalis luxuriant<br /> (-)<br />Candida krusei luxuriant (-)<br />Candida minosa luxuriant (-)<br />
  17. 17. LEVINE’S EMB AGAR<br />For the isolation and differentiation of Escherichia coli and Enterobacter<br />The dyes contained in this medium inhibit the growth of many accompanying Gram-positive microorganisms<br />LEVINE EMB Agar can be used to identify Candida albicans in clinical specimens, if chlorotetracycline hydrochloride is added to inhibit the entire accompanying bacterial flora<br />LEVINE EMB Agar can also be utilized for the identification of coagulase-positive staphylococci which grow characteristically as colorless "pin-point" colonies and which show good agreement with the results of the coagulase test<br />
  18. 18. LEVINE’S EMB AGAR<br />Typical Composition (g/liter)<br />Peptone …………………………………………………………….10.0<br />Lactose ……………………………………………………………..10.0<br />Di-potassium hydrogen phosphate ………………………..2.0<br />Eosin, yellowish …………………………………………………..0.4<br />Methylene blue …………………………………………………...0.065<br />Agar-agar …………………………………………………………..13.5<br />If cultivating Candida, add 100 mg tetracycline hydrochloride/litre after autoclaving and mix homogeneously. <br />The culture medium then is blue<br />
  19. 19. LEVINE’S EMB AGAR<br /> To obtain a primary culture of Candida, incubate the plates containing chlorotetracycline in a 10 % carbon dioxide atmosphere<br />Appearance:<br /> "Spidery" or "feathery“ - Candida albicans<br /> Yeast-like, round, smooth - Other Candida species; Sometimes Nocardia<br />
  20. 20. SABHI AGAR<br />Used for the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources<br />Sabouraud Dextrose Agar is a general purpose medium devised by Sabouraud for the cultivation of dermatophytes<br />Brain Heart Infusion (BHI) Agar has proven to be effective in the cultivation of a wide variety of microorganisms and is recommended for the primary recovery of fungi from clinical specimens<br />
  21. 21. SABHI AGAR<br />SABHI Agar combines the ingredients of these two formulations to provide a medium which was found to yield greater recovery of pathogenic fungi than either medium individually<br />It is recommended for the recovery of fungi from clinical specimens<br />
  22. 22. SABHI AGAR<br />Formulation:<br />SABHI Agar contains two peptones and brain heart infusion solids as sources of amino acids, nitrogen, sulfur, carbon and trace ingredients<br />Dextrose is an energy source for the metabolism of microorganisms. Sodium chloride provides essential electrolytes<br />
  23. 23. SABHI AGAR<br />Approximate Formula* Per Liter Purified Water<br />Brain Heart, Infusion from (Solids) . . . . . . . . . . . . . . . . . 4.0 g<br />Peptic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . . . 5.0 g<br />Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . 10.5 g<br />Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.0 g<br />Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g<br />Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 g<br />Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g<br />
  24. 24. Sabhi agar with Chloramphenicol and Cycloheximide<br />Selective medium for use in the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources<br />Chloramphenicol<br />Gram positive and Gram negative organisms<br />Cycloheximide<br />Most saprophytic molds<br />
  25. 25. CZAPEK’S AGAR<br />Czapek's Solution Agar is a synthetic medium widely used in mycological laboratories<br />Many moulds produce very characteristic colonies on it and may also exude pigmented substances<br />Aerial growth is often suppressed and sporulation may be enhanced<br />Some moulds, however, grow poorly on this medium and may even fail to sporulate altogether, often because of their inability to synthesize vitamins<br />As noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one <br />
  26. 26. CZAPEK’S AGAR<br />Formulation:<br />Sucrose . . . . . . . . . . . . . . . . 30 g<br />NaNO3 . . . . . . . . . . . . . . . . 3.0 g<br />K2HPO4 . . . . . . . . . . . . . . . 1.0 g<br />MgSO4.7H2O . . . . . . . . . . .0.5 g<br />KCl . . . . . . . . . . . . . . . . . . . . 0.5 g<br />FeSO4.7H2O . . . . . . . . . . . 0.01 g<br />Agar . . . . . . . .. . . . . . . . . . . . 15 g<br />Distilled water. . . . . . . . . . 1 liter<br />
  27. 27. Immunodiffusion<br />27<br />
  28. 28. Immunoelectrophoresis<br />28<br />
  29. 29. Complement Fixation<br />29<br />
  30. 30. Enzyme-linked Immunosorbent Assay<br />30<br />
  31. 31. Latex Agglutination<br />31<br />
  32. 32. Radioimmunoassay<br />32<br />
  33. 33. Immunoblotting<br />33<br />
  34. 34. END<br />Good evening!<br />

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