Culture Media for Mycological Examination Dr Kedar karki Culture Media • Dixon's Agar for Malassezia furfur • Czapek Dox Agar for Aspergillus & Penicillium • Bird Seed Agar • Cornmeal Glucose Sucrose Yeast Extract Agar for Zygomycetes • Sabouraud's Dextrose Agar for Dermatophytes Culture Media • Potato Dextrose Agar • Brain Heart Infusion Agar (BHIA) with 5% Sheep Blood • Cornmeal Agar • Malt Extract Agar • Sabouraud's Dextrose Agar for yeasts and moulds Culture Media • • • • Lactrimel Agar for dermatophytes Hair Perforation Test for dermatophytes Urease Agar with 0.5% Glucose Trichophyton Agars Nos 2-7 Culture Media • Rice Grain Medium for Microsporum • Littman Oxgall Agar • Sabouraud's Dextrose Agar with 5% NACL Culture Media • Vitamin Free Agar (Trichophyton Agar No 1) • 1% Peptone Agar • CGB Agar Czapek Dox Agar for Aspergillus & Penicillium • For the routine cultivation of fungi, especially Aspergillus, Penicillium, and non-sporulating moulds. Czapek Dox Agar • • • • Czapek Dox Agar (Oxoid CM97) 45.4 g Distilled water 1000 ml 1. Soak the ingredients in small amount of water. 2. Bring remaining water to boil, add to soaking ingredients and bring to the boil again, stirring continuously. • 3. Dispense for slopes as required. • 4. Autoclave at 121C for 10 minutes, remove and slope or pour for plates as required. Sabouraud's Dextrose Agar for Dermatophytes • Sabouraud's Dextrose Agar with Cycloheximide, Chloramphenicol, Gentamycin and Yeast Extract. For the primary isolation and cultivation of dermatophytes. Sabouraud's Dextrose Agar (Oxoid CM41) Cycloheximide (Actidione) Chloramphenicol Yeast extract Gentamicin (40 mg/ml) 65 g 0.5 g 1x250 mg capsule 5g 0.65 ml 1000 ml Distilled water Method: • 1. Soak all ingredients, except Gentamicin, in 100 ml water. • 2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring. • 3. Add the Gentamicin. Mix well. • 4. Dispense for slopes if required. • 5. Autoclave at 121C for 10 minutes. Remove and slope, or pour for plates as required. Potato Dextrose Agar. • For the routine cultivation and identification of fungi. • Potato dextrose agar (Oxoid CM139) 39 g • Distilled water 1000 ml Method: • 1. Soak potato dextrose agar in small amount of the water in a stainless steel jug. • 2. Boil remaining water, add to soaking ingredients, bring to the boil, stirring constantly. • 3. Dispense for slopes as required. • 4. Autoclave at 121C for 15 minutes. Remove and slope or pour for plates as required. Brain Heart Infusion Agar (BHIA) with 5% Sheep Blood • For the primary isolation and cultivation of yeasts and moulds. For the primary isolation and cultivation of yeasts and moulds. Brain-heart infusion agar (Oxoid) Sheep blood Gentamicin (40 mg/ml) Chloramphe nicol 52 g 50 ml 0.65 ml 1x250 mg capsule Distilled water 1000 ml Method: • 1. Soak BHI and antibiotics in 100 ml water. • 2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring. • 3. Autoclave at 121C for 15 minutes, and aseptically add the blood to the cooled (50C) medium before dispensing for slopes, or for plates as required. Sabouraud's Dextrose Agar for yeasts and moulds • Sabouraud's Dextrose Agar with Chloramphenicol and Gentamycin. For the primary isolation and cultivation of yeasts and moulds. Sabouraud's Dextrose Agar (Oxoid CM41) 65 g 1x250 mg capsule 0.65 ml 1000 ml Chloramphenicol Gentamicin (40 mg/ml) Distilled water Method: • 1. Soak all ingredients, except Gentamicin, in 100 ml water. • 2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring. • 3. Add the Gentamicin. Mix well. • 4. Dispense for slopes if required. • 5. Autoclave at 121C for 10 minutes. Remove and slope, or pour for plates as required. 1% Peptone Agar • For the cultivation and differentiation of fungi Ingredients: • Bacto Peptone (BD) 5 g • Bacto-Agar (BD) 10 g • Distilled water 500 ml Method: • 1. Soak agar and peptone in small amount of water • 2. Boil remaining water, add this to soaking ingredients and bring to boil again. • 3. Dispense for slopes. • 4. Autoclave for 10 minutes at 121C, then slope on racks. Malt Extract Agar. • For the routine cultivation and identification of fungi. Ingradiants • Malt Extract (Oxoid L39) 20 g • Bacto agar (BD) 20 g • Distilled water 1000 ml Method: • 1. Dissolve malt extract in a plastic beaker and pH the solution to pH 6.5 with NaOH. • 2. Soak agar in small quantity of solution. Bring remaining solution to the boil, stirring constantly. • 3. Add to soaking agar. Bring to boil, stirring constantly. Method: • 4. Dispense for slopes as required. • 5. Autoclave at 121C for 10 minutes, remove and slope or pour for plates as required. Urease Agar with 0.5% Glucose • • • • Ingredients: Urea, broth base (Oxoid CM71) 0.9 g Distilled water 91 ml Bacto Agar (BD) 1.5 g Method: • 1. Place ingredients into 100ml bottle. • 2. Autoclave at 115C for 20 minutes. • 3. Cool to 50C. Method: • 4. Add aseptically 5ml of sterile urea solution (Oxoid SR20). • 5. All 4ml of 10% Sterile Glucose solution. • 6. Dispense for slopes, and slope on rack. • 7. Incubate overnight. Rice Grain Slopes • To induce sporulation and for differentiation of M. audouinii, M. canis and M. distortum. • Method: • 1. Place approximately 1/2 teaspoon rice grains into wide neck 20ml vials. • 2. Add 8ml distilled water to each vial. • 3. Lid, then slope on racks ensuring rice grains are evenly distributed. • 4. Autoclave racks at 121C for 15 minutes. Antifungal Susceptibility Testing • NCCLS and Sensititre YeastOne Methods • Neo-Sensitab and Etest Methods • Antifungal Susceptibility Profiles Antifungal Susceptibility Testing • Notes on the disk diffusion and ETEST methods. Introduction • The disk agar gradient method can accurately and reproducibly determine the susceptibility of fungi to antifungal agents eg fluconazole. Improvements include the use of RPMI glucose agar, inoculum density adjustment with a 0.5 McFarland Standard, and incubation at 35C for 24 hours. These changes standardise the test to current NCCLS guide-lines and make it relatively comparable to standard bacterial test methods. This method and interpretive criteria apply to rapidly growing yeast species, defined as strains producing growth within a 24-48 hour incubation period. Antifungal agents available for testing: Etest (AB Biodisk) ALS Australia Tel: 02 9576 5258 Neo-sensitabs (Rosco). Dutec Diagnostics Australia Tel: 02 9798 9066 Amphotericin B 10 ug. Ketoconazole 15 ug Itraconazole 8 ug Amphotericin B Ketoconazol e Itraconazole Fluconazole 25 ug Voriconazole 1.0 ug 5-Fluorocytosine 1 and 10 ug Fluconazole Voriconazole 5Fluorocytosi ne Store both disks and Etest refrigerated with desiccant. Disk Method • 1. Melt and pour RPMI-Glucose Agar plates [media may be stored in 60 ml aliquot's and then melted in a microwave to pour plates when required]. For a standard screen using disks of all the above antifungal agents we typically use 3 plates set up as follows (Additional plates may need to be poured for Etest and other investigational purposes. Note susceptibility tests for Ampohtericin may be unreliable on RPMI media; the use of Antibiotic Medium 3 may enhance the detection of resistance, but this medium is not standardised and substantial lot-to-lot variability is possible.): Disk Method • 1. Amphotericin B 20 ug. • 2. Voriconazole 1.0 ug, Itraconazole 8 ug and Fluconazole 25 ug • 3. 5-Fluorocytosine 1.0 ug/ml for yeasts 10 ug for Aspergillus. Disk Method • 2. Prepare an inoculum by suspending a single isolated colony in about 5 ml of normal saline or sterile water. Agitate to achieve a smooth suspension on the Vortex mixer. Adjust the suspension with saline or water to approximate a density of a #0.5 McFarland Turbidity Standard [we use a bioMerieux Densimat]. For filamentous fungi, which do not sporulate profusely, add one drop of the wetting agent Tween 20 to the sterile distilled water and then break up the mould by shaking it with small glass beads or by grinding it into a suitable suspension. Disk Method • 3. Moisten a sterile cotton (not dacron) swab in the adjusted inoculum suspension. Express excess moisture by rolling the swab on the inside of tube above fluid level. Streak the surface of an RPMI-glucose agar plate in 4 different directions (at 90 degree angles) to cover the entire surface. Let the surface of RPMI-glucose agar plates dry at 35 C with lids ajar until no droplets of moisture are on the agar surface. Disk Method • 4. Using a pair of flamed sterilised forceps apply the disks containing the antifungal agent to be tested onto the surface of the inoculated agar plate and press lightly to insure complete contact with agar. • 5. Incubate plates at 35C for 24-48 hours or until sufficient growth has occurred. Plates should be read as early as possible after 24 hours incubation and results recorded in the susceptibility book. Etest showing a trailing end point. Sensititre® YeastOne™ Test Panel Sensititre® YeastOne™ Test Panel • This is a microtitre broth dilution method based on the NCCLS M27-A2 standard described above. Each test consists of a disposable microtitre plate, which contains dried serial dilutions of six antifungal agents, Amphotericin B (range 0.008-16 mg/ml), Fluconazole (range 0.125-256 mg/ml), Itraconazole (range 0.008-16 mg/ml), Ketoconazole (range 0.008-16 mg/ml) and 5Fluorocytosine (range 0.03-64 mg/ml), Voriconazole (range 0.008-16 mg/ml) in individual wells (Fig.2). The wells also contain Alamar Blue as a colorimetric indicator, which greatly improves the end point readability by a colour change from blue to pink. Results are expressed as an MIC and comparative studies against the NCCLS method have shown favorable results Fungitest® Fungitest® • (This is a modified microtitre broth breakpoint test also based on the NCCLS M27-A2 standard described above. Each plate has six antifungal agents at two different concentrations, Amphotericin B (2 and 8 mg/ml), Fluconazole (8 and 64 mg/ml), Itraconazole (0.5 and 4 mg/ml), Ketoconazole (0.5 and 4 mg/ml), Miconazole (0.5 and 8 mg/ml) and 5-Fluorocytosine (2 and 32 mg/ml) in individual wells, plus control growth and no growth wells. The wells also contain a redox indicator to improve the end point readability by a colour change from blue to pink (Fig. 3). Results are expressed as Susceptible, Intermediate or Susceptible Dose Dependent or Resistant with MIC’s given relative to the break points e.g. Susceptible MIC <0.5 mg/ml Etest® • This is an agar diffusion method using a strip with a predefined concentration gradient of the antimicrobial agent being tested. This gradient allows for an MIC determination to be made. Etests have been extensively used for susceptibility testing of bacteria and they are also available for antifungal agents, including Amphotericin B (range 0.002-32 mg/ml), Ketoconazole (range 0.002-32 mg/ml), Itraconazole (range 0.002-32 mg/ml), Fluconazole (range 0.016-256 mg/ml), Voriconazole (range 0.002-32 ug/ml) and 5Fluorocytosine (range 0.002-32 mg/ml) (fig. 3). Etest® • The two difficulties with Etests against fungi have always been which medium to use and with the interpretation of the end point. The later problem remains but it is now recommended that modified RPMI1640 agar supplemented with 0.2% glucose be used in line with the NCCLS standard Etests • Etests are simple to perform and the methodology is similar to that used in bacteriology. Individual antifungals may be tested and there is reasonable correlation with the NCCLS standard. Etest end points are often difficult to read due to the trailing effect seen with some strains [about 20% of yeasts (Fig. 4)]. As a result MIC's may tend to be higher than expected so it is important to repeat any “resistant” results in parallel with a control stain or to have them confirmed by another method. Etest and Neo-Sensitab test. Neo-Sensitabs®( • This is a simple agar diffusion method using tablets to determine the susceptibility of fungi to antifungal agents (fig.3). Once again there have been problems with which media to use and with the interpretation of the end points. Most users have now changed over to modified RPMI-1640 agar supplemented with 0.2% glucose and have adopted the basic NCCLS guidelines to create a more standardised test. Neo-Sensitabs®( • However, recent studies have used MuellerHinton agar supplemented with 2% glucose and 0.5 mcg/ml methylene blue as the medium and a Biomic plate reader to electronically read and interpret zones sizes16. A large number of antifungals is available in tablets; Amphotericin B (10 ug), Ketoconazole (15 ug), Itraconazole (8 ug), Fluconazole (25 ug), Voriconazole (1.0 ug), 5-Fluorocytosine (1 and 10 ug), Clotrimazole (10 ug), Miconazole (10 ug), Econazole (10 ug), Natamycin (10 ug) and Nystatin (50 ug). Neo-Sensitabs®( • Neo-Sensitabs are cheap, agar diffusion tests are easy to set up and show potential for the screening of large numbers of isolates for resistance. However, individual disk zone sizes are often not able to differentiate between Susceptible and Susceptible Dose Dependent isolates and the correlation between zone size and MIC is more variable10. Once again, resistant isolates need to be confirmed by using one of the appropriate NCCLS methodology. Antifungal Susceptibility Profiles Dermatophytes Fl u S S S Itr a S S S Ket o S S S Organism Trichophyton sp. Epidermophyton floccosum Microsporum sp. Terb S S S Griseo S S S Vori S S S S = Susceptible; SDD = Susceptible but Dose Dependent; R = Resistant; V = Variable; I = Intermediate; N = No data. Yeasts Am B S 0.5 ug/m l S 0.5 ug/m l Organism Flu Vori Itra Keto Terb 5FC Cas po Candida albicans S 0.5-1 ug/m l SDD 4-8 ug/m l S 0.06 ug/m l S 0.06 ug/m l S 0.25 ug/m l S 0.25 ug/m l S 0.25 ug/m l S 0.25 ug/m l V 0.03>128 ug/ml S 1.0 ug/m l S 1.0 ug/m l S Candida famata N S Candida glabrata S 0.5 ug/m l SDD 2-32 ug/m l S 1-2 ug/m l SDD 0.5-1 ug/m l V 1-4 ug/m l R >128 ug/ml S 0.5 ug/m l S Candida guilliermond ii S 0.5 ug/m l SDD 4-8 ug/m l S 0.250.5 ug/m l S 0.5 ug/m l S 0.5-1 ug/m l V 6.25-100 ug/ml S 0.12 5-0.2 ug/m l S Candida krusei S 0.5-1 ug/ml R 32-64 ug/ml S 0.5-1 ug/ml SDD 0.5 ug/ml I 1.0 ug/ml R 50->100 ug/ml V 8-32 ug/ml S Candida lusitaniae V 0.5-2 ug/ml S 0.5-1 ug/ml S 0.06 ug/ml S 0.25 ug/ml S 0.25 ug/ml N S 0.125 ug/ml S Candida parapsilosis S 0.5-1 ug/ml S 1-2 ug/ml S 0.06 ug/ml S 0.125-0.5 ug/ml S 0.5 ug/ml S 0.25-2 ug/ml S 1.0 ug/ml S Candida tropicalis S 0.5 ug/ml SDD 1-8 ug/ml S 0.25 ug/ml S 0.25 ug/ml V 0.5-1 ug/ml V 10-128 ug/ml S 1.0 ug/ml S S Cryptococcus neoformans 0.5 -1 ug/ ml SDD 2-8 ug/ ml S 0.2 5 ug/ ml SDD 0.5 ug/ ml S 0.2 5 ug/ ml S 0.25 ug/ml S 2-8 ug/ ml R Malassezia furfur S S S S S S* S N S Saccharomyces cerevisiae 0.5 ug/ ml S 2-8 ug/ ml S 0.0 3 ug/ ml S 0.5 -1 ug/ ml S 0.5 ug/ ml N S 0.1 25 ug/ ml N S S S S Trichosporon beigellii 1.0 ug/ ml 2-8 ug/ ml 0.1 25 ug/ ml 0.1 25 ug/ ml S 0.5 ug/ ml V 0.05128 ug/ml I 816 ug/ ml N Moulds Organis m AmB Flu Vori Itra Keto Terb 5FC Caspo Aspergi llus fumigat us Aspergi llus flavus Pseudal lescheri a boydii [=Sced osporiu m apiospe rmun]. Scedos porium prolifica ns Fusariu m sp Bipolari s sp S 1.0 ug/ml S 1-2 ug/ml R >100 ug/ml R >100 ug/ml S 0.5 ug/ml S 0.5-1 ug/ml SDD 0.5 ug/ml SDD 0.5-1 ug/ml R 8 .0 ug/ml R 8.0 ug/ml S 0.5 ug/ml S 0.5 ug/ml R >64 ug/ml R > 64 ug/ml S S V 1-16 ug/ml V 8-64 ug/ml S 0.5 ug/ml V 0.5-4 ug/ml V 1-4 ug/ml R 10-100 ug/ml R > 64 ug/ml N R >16 ug/ml I 1-4 ug/ml S 0.25 ug/ml R > 64 ug/ml R > 64 ug/ml R > 64 ug/ml S 0.5-1 ug/ml S 1-4 ug/ml S 0.25-0.5 ug/ml R >8 ug/ml R >8 ug/ml S 0.5 ug/ml R >16 ug/ml R > 16 ug/ml S 0.5 ug/ml R >100 ug/ml R 0.5-128 ug/ml S 0.5-1 ug/ml R >64 ug/ml R >64 ug/ml R > 64 ug/ml N N N Clado phial opho ra sp Exop hiala sp Phial opho ra sp Paeci lomy ces sp Penic illium sp S 1.0 ug/ml S 0.25 ug/ml S 0.5-1 ug/ml V 0.516 ug/ml S 0.5-1 ug/ml V 4-64 ug/ml R 64 ug/ml R 16-64 ug/ml R 64 ug/ml V 4-64 ug/ml S 1.0 ug/ml S 0.25 ug/ml S 0.25 ug/ml S 0.5-1 ug/ml S 0.5 ug/ml S 0.125 ug/ml S 0.5 ug/ml S 0.5 ug/ml V 0.5- 8 ug/ml S 0.5 ug/ml V S 0.251 ug/ml S 0.062 ug/ml S 1.0 ug/ml V 1-64 ug/ml S 0.5-4 ug/ml S 0.25 ug/ml V 4-64 ug/ml N V 0.5-2 ug/ml V 0.5 ug/ml N N V 0.564 ug/ml V 0.5 ug/ml N N N S 1.0 ug/ml N AmB = Amphotericin B; Flu = Fluconazole; Vori = Voriconazole; Itra = Itraconazole; Keto = Ketoconazole; Terb = Terbinafine [* topical only]; 5FC = 5-Fluorocytosine; Griseo =Griseoflvin.