Fungal culture methods and media are described. Most molds grow best at 25-30°C while most yeasts grow best at 35-37°C. General incubation is 14 days but it can be as short as 7 days for detecting oral yeast or as long as 28 days for tissue specimens. ChromAgar differentially identifies Candida species by color. Chlamydospore agar induces chlamydospore formation in C. albicans. Sabhi agar is recommended for recovering fungi from clinical specimens. Czapek's agar is a synthetic medium used to observe fungal morphology and pigment production.

1. MYCOLOGY FUNGAL CULTURE

2. CUTURE METHOD: All specimens should be inoculated onto a general purpose fungal medium fungi will grow very well on culture media used to isolate bacteria

3. Temperature: Most MOLDS grow best at: 25 – 30 OC Most YEAST grow best at 35 – 37 OC Most pathogenic fungi grow best: 30 to 32°C EXCEPT: Sporothrixschenckii : 25 to 27°C than 30°C

4. Incubation period: 14 days: general incubation period 7 days: to detect presence of yeast in the mouth, throat, or vagina 21 days: tissues and sterile body fluids other than blood 28 days: respiratory, bone marrow, blood specimens, and specimens in which dimorphic fungus are suspected Plates should be checked at least TWICE during the first week, when rapidly growing isolates may appear, weekly hereafter.

8. CHROMagar A selective medium for the isolation and presumptive identification of yeast and filamentous fungi and differentiation of Candida albicans, C. tropicalis and C. krusei. Due to the differences in morphology and colors of the yeast colonies, this medium facilitates the detection of mixed yeast cultures in specimens. 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.

9. CHROMagar FORMULA IN GRAMS PER LITER Glucose ....................................... 20.00 Peptone …….................................. 10.00 Chloranphenicol ............................ 0.50 Chromogenic Mixture.............. ........ 0.40 Bacteriological Agar ....................... 15.00 Final pH 6.1 ± 0.2 at 25°C

10. CHROMagar Preparation Suspend 45.9 grams of the medium in one liter of distilled water. Mix well and heat with frequent agitation until complete dissolution. Distribute into adequate containers.

11. CHROMagar Approximate Formula* Per Liter Purified Water Chromopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.0 g Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.0 g Chromagen Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.0 g Chloramphenicol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

12. CHROMagar Candida albicans- green Candida tropicalis - steel blue Candida krusei- rose, fuzzy

13. C. krusei C. tropicalis C. albicans

14. CHLAMYDOSPORE AGAR Used for differentiating Candida albicans from other species of Candida on the basis of chlamydospore formation. Candida albicans always form chlamydospore on this medium. The medium contains trypan blue to visualize the chlamydospore under microscopic evaluation. Biotin and polysaccharide are growth factors which stimulate chlamydospore formation. Potassium phosphate function as a buffer in the medium

15. CHLAMYDOSPORE AGAR Components (g/L) Ammonium Sulphate…………………………… 1.00 Monopotassium Phosphate…………………… 1.00 Purified Polysaccharide………………………… 20.00 Trypan Blue………………………………………… 0.10 Biotin…………………………………………………. 0.000005 Agar…………………………………………………… 15.00 Final pH (at 25°C) 5.1 ± 0.2

16. CHLAMYDOSPORE AGAR Organisms Growth Chlamydospores Candida albicans luxuriant (+) Candida tropicalis luxuriant (-) Candida krusei luxuriant (-) Candida minosa luxuriant (-)

17. LEVINE’S EMB AGAR For the isolation and differentiation of Escherichia coli and Enterobacter The dyes contained in this medium inhibit the growth of many accompanying Gram-positive microorganisms 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 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

18. LEVINE’S EMB AGAR Typical Composition (g/liter) Peptone …………………………………………………………….10.0 Lactose ……………………………………………………………..10.0 Di-potassium hydrogen phosphate ………………………..2.0 Eosin, yellowish …………………………………………………..0.4 Methylene blue …………………………………………………...0.065 Agar-agar …………………………………………………………..13.5 If cultivating Candida, add 100 mg tetracycline hydrochloride/litre after autoclaving and mix homogeneously. The culture medium then is blue

19. LEVINE’S EMB AGAR To obtain a primary culture of Candida, incubate the plates containing chlorotetracycline in a 10 % carbon dioxide atmosphere Appearance: "Spidery" or "feathery“ - Candida albicans Yeast-like, round, smooth - Other Candida species; Sometimes Nocardia

20. SABHI AGAR Used for the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources Sabouraud Dextrose Agar is a general purpose medium devised by Sabouraud for the cultivation of dermatophytes 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

21. SABHI AGAR 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 It is recommended for the recovery of fungi from clinical specimens

22. SABHI AGAR Formulation: SABHI Agar contains two peptones and brain heart infusion solids as sources of amino acids, nitrogen, sulfur, carbon and trace ingredients Dextrose is an energy source for the metabolism of microorganisms. Sodium chloride provides essential electrolytes

23. SABHI AGAR Approximate Formula* Per Liter Purified Water Brain Heart, Infusion from (Solids) . . . . . . . . . . . . . . . . . 4.0 g Peptic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . . . 5.0 g Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . 10.5 g Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.0 g Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25 g Agar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.0 g

24. Sabhi agar with Chloramphenicol and Cycloheximide Selective medium for use in the cultivation of pathogenic and nonpathogenic fungi from a variety of clinical and nonclinical sources Chloramphenicol Gram positive and Gram negative organisms Cycloheximide Most saprophytic molds

25. CZAPEK’S AGAR Czapek's Solution Agar is a synthetic medium widely used in mycological laboratories Many moulds produce very characteristic colonies on it and may also exude pigmented substances Aerial growth is often suppressed and sporulation may be enhanced Some moulds, however, grow poorly on this medium and may even fail to sporulate altogether, often because of their inability to synthesize vitamins As noted above, the addition of agar to this medium makes it, in reality, a semi-synthetic one

26. CZAPEK’S AGAR Formulation: Sucrose . . . . . . . . . . . . . . . . 30 g NaNO3 . . . . . . . . . . . . . . . . 3.0 g K2HPO4 . . . . . . . . . . . . . . . 1.0 g MgSO4.7H2O . . . . . . . . . . .0.5 g KCl . . . . . . . . . . . . . . . . . . . . 0.5 g FeSO4.7H2O . . . . . . . . . . . 0.01 g Agar . . . . . . . .. . . . . . . . . . . . 15 g Distilled water. . . . . . . . . . 1 liter

27. Immunodiffusion 27

28. Immunoelectrophoresis 28

29. Complement Fixation 29

30. Enzyme-linked Immunosorbent Assay 30

31. Latex Agglutination 31

32. Radioimmunoassay 32

33. Immunoblotting 33

34. END Good evening!