Fungal media

Mycology,Fungi, media used, Sabourads Dextrose Agar, Potato Dextrose Agar, Hichrome Agar, Dichloran Glycerol Agar, growth of fungi

1. MEDIA FOR FUNGI M.MEENAKSHI, ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, SRI RAMAKRISHNA COLLEGE OF ARTS AND SCIENCE FOR WOMEN, COIMBATORE

2. MEDIA FOR FUNGI Fungal culture The most direct and usually conclusive means of establishing the diagnosis of a fungal infection is to grow the fungus from a patient sample. Numerous different samples can yield a fungus, including blood, cerebrospinal fluid, pus, urine, tissue, respiratory samples (sputum, bronchoscopy lavage), pleural, pericardial or peritoneal fluid, skin scraping, hair, nail clippings, oral or vaginal samples. Processing of these samples may involve centrifugation or softening/ liquidisation to allow spreading of the sample onto an agar medium. A number of laboratory manuals and specific describe the methods for doing this although remarkably few comparative studies comparing one method with another have been done.

3. FUNGAL CULTURE

4. FUNGAL SPECIMENS IN DIFFERENT MEDIA

5. MEDIA FOR FUNGI

6. THE IMPORTANCE OF MEDIA SELECTION The yield of most fungi is improved by direct culture of samples on so-called ‘fungal media’. For some fungi, cultures are always or almost always negative on bacterial media, examples being Histoplasma, Mucorales and Coccidioides spp. The culture of Aspergillus spp. on bacterial media is ~30% less effective than on fungal media General purpose media that are commonly used for fungal culture are Sabouraud dextrose, malt extract and less commonly brain heart infusion medium. To prevent contamination of the medium by bacteria, chloramphenicol is used, but prevents the growth of Actinomyces , which others grows well on Sabouraud dextrose agar. For reducing the frequency of environmental fungal growth, cycloheximide is added, but this reduces the yield of many opportunistic fungi including Aspergillus spp., Cryptococcus neoformans and Mucorales isolates. Therefore if cycloheximide is used, one agar plate not containing it should also be used in parallel.

7. COMMON FUNGAL CULTURE MEDIA

8. MEDIA…… SABOURAUD AGAR OR SABOURAUD DEXTROSE AGAR (SDA) It is a type of agar growth medium containing peptone. It is used to cultivate dermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia . It has utility for research and clinical care. It was created by, and is named after, Raymond Sabouraud in 1892. The formulation was later adjusted by Chester W. Emmons when the pH level was brought closer to the neutral range and the dextrose concentration lowered to support the growth of other microorganisms. The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth Typical composition Sabouraud agar is commercially available and typically contains: [5] 40 g/L  dextrose 10 g/L  peptone 20 g/L  agar pH 5.6

9. USES OF SDA Medical usefulness Clinical laboratories can use this growth medium to diagnose and further speciate fungal infections, allowing medical professionals to provide appropriate treatment with antifungal medications. Histoplasma and other fungal causes of atypical pneumonia can be grown on this medium. Fig 1- Candida albicans Fig 2- Trichophyton Fig 3- Sporothrix

10. POTATO DEXTROSE AGAR   Potato dextrose agar  (BAM Media M127) and  potato dextrose  broth are common microbiological growth media made from  potato  infusion, and  dextrose .  Potato dextrose agar  ( abbreviated " PDA ") is the most widely used medium for growing fungi and bacteria. value ingredients & conditions 1000ml water 4g (from 200g infused potato) potatoes (sliced washed unpeeled) 20g Dextrose 20g agar powder 5.6±0.2 final pH 25°C temperature

11. POTATO DEXTROSE AGAR   ….. Potato infusion can be made by boiling 200 grams (7.1 oz ) of sliced (washed but unpeeled) potatoes in ~ 1 litre (0.22 imp gal; 0.26 US gal) distilled water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is 1 litre (0.22 imp gal; 0.26 US gal). 20 grams (0.71 oz ) dextrose and 20 grams (0.71 oz ) agar powder is then added and the medium is sterilized by autoclaving at 15 pounds per square inch (100 kPa ) for 15 minute A similar growth medium, Potato dextrose broth (abbreviated "PDB") is formulated identically to PDA, omitting the agar. Common organisms that can be cultured on PDB are yeasts such as Candida albicans and Saccharomyces cerevisiae and molds such as Aspergillus niger

12. Fig 1-Aspergillus MORPHOLOGY OF ISOLATED FUNGAL STRAINS GROWN ON POTATO DEXTROSE AGAR PLATES

13. MALT EXTRACT AGAR Malt Extract Agar Malt Extract Agar  is used as a general purpose growth media to isolate and cultivate yeasts and molds from clinical samples, as well as a wide range of environmental sources. ... P80 and W80) to inhibit bacterial overgrowth while permitting successful selective isolation of fungi and yeasts . Dextrose:  20.0gm Peptone:  6.0gm Malt Extract:  20.0gm

14. USES Identification of fungi is performed by observing various aspects of colony morphology, characteristic microscopic structures, rate of growth, media which supports the organisms growth, and source of the specimen. Yeasts are identified by various biochemical tests. Consult the listed references for information regarding the identification and further testing of fungi and yeast cultures

15. CORN MEAL AGAR Corn Meal Agar is a well- established mycological medium used for the cultivation of fungi and to study chlamydospores production of Candida species (6). Corn Meal Agar is a general purpose medium used for the cultivation of fungi and for the study of Candida species for chlamydospore production . Formula adjusted, standardized to suit performance parameters Directions .Suspend 17 grams in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. If desired add 1% polysorbate 80. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates. Ingredients Gms / Litre Corn meal, infusion 50.000 Agar 15.000 Final pH ( at 25°C) 6.0±0.2

16. USES OF CORN MEAL AGAR Candida albicans produces mycelium-bearing ball-like clusters of budding cells and the characteristic thick-walled round chlamydospores9. The addition of 0.001g % w/v Trypan blue to Corn Meal Agar provides a contrasting background for the observation of characteristic morphological features of yeast cultures10. FIG 1- YEAST IDENTIFICATION ON CORN MEAL AGAR

17. DICHLORAN GLYCEROL AGAR Recommended for selective isolation of xerophilic moulds from food and clinical samples Dichloran Glycerol Medium was formulated by Hocking and Pitt (2) and is recommended for isolation and enumeration of xerophilic moulds from dried and semidried foods Ingredients Gms / Litre 5.000 Dextrose (Glucose) 10.000 1.000 0.500 0.002 Chloramphenicol 0.100 Agar 15.000 Final pH ( at 25°C) Gms /litre Peptone 5.000 Potassium dihydrogen phosphate 1.000 Magnesium sulphate 0.5 Dichloran 0.002 Chloramphenicol 0.1 Agar 15.00 Dextrose 10.000 Final Ph 5.6

18. MEDIA FOR SELECTIVE CULTURE AND PRESUMPTIVE IDENTIFICATION There are some additional specialised media that can be used directly on specimens to allow separation of similar looking colonies including bird seed agar to identify Cryptococcus spp. in respiratory cultures from HIV patients or chromogenic agar for the direct, partial identification of Candida spp . ChromAgar used to identify : 1=Candida albicans ; 2= Candida krusei ; 3=Candida tropicalis 1=C glabrata ; 2 = C albicans 3=C krusei; 4=C tropicalis

19. INCUBATION 21 days The standard temperature for  incubation of fungi  is 30ºC and cultures should be  incubated  in a humidified environment for 21 days. They should be inspected daily for at least a week, and at least 3 times weekly thereafter. Some  fungi  are very slow to grow, notably Histoplasma spp., and may need longer  incubation  times.

20. INCUBATION

21. OTHER MEDIA Dermatophyte agar. RPMI agar. Sabouraud 2 agar. Sabouraud Chloramphenicol Actidione agar. Sabouraud Dextose agar. Sabouraud Chloramphenicol 2 agar. Sabouraud Gentamicine Chloramphenicol 2. Sabouraud liquid medium.