Inoculation to Medium
• The yeast are inoculated with the aid of the inoculum to the surface of the inoculum prepared from the clinical or necropsy material as it is in the same bacteria.
• In fungi isolation, a sterile bisturia is formed at 5 points on the
surface of the nutrient plantation. Later, small pieces of lesion tissue, skin scrapings or furrows are lightly soaked in the agar in these
positive areas.
• If the samples are immersed directly in the agar, splitting, breakage may occur in the agar during long-term incubations
• Yeast are passively ingested into the new medium as it is in the same bacteria.
Subculture of Fungi
If the fungi column is sporulating;
Fungus colonies usually start to produce spores from the center to the periphery, and are often sports that give a colonial characteristic color.
A substance is first dipped into the surface of a sterile agar portion to make it slightly damp and sticky, then used to collect spores from a column.
If the fungi species is a fast-breeding fungus, the inoculum is
inoculated immediately below the surface of the agar, at the exact center point on a new plate surface.
If the fungus forms small, slow-breeding colonies, the agar plate is divided into four portions and each section is separately planted.
If the fungi column is not sporulating;
• In the fungus colon, the aging and death phase begins with the hypha in the center of the colon. For this reason, the passage of the hyphen around the colon is required.
• A small agar block (5mm2) in the center is cut off on the medium to be used for passage with a sterile blister. Using the same bisturia, a similar sized and shaped agar is cut out from the side of the column to be passaged to include fungal hyphae.
• This agar block is carefully placed on the medium to be passaged in such a way that the mushroom part is above. The interrupted hypha will be regenerated and will grow towards the periphery from the block surface.
Subculture of Fungi
Microscopic Examination of Fungal Colonies
• Investigation with Dissecting microscope
• preparation of lam lamel preparation with LPCB (Lactophenol Cotton Blue ) (wet mount method)
• Adhesive tape method
• Lam Culture Technique
c-) Identification of Fungi
• Direct microscopic image of the fungus in clinical specimen
• Colony morphology and pigmentation type
• Microscopic image of macroconidia (fruiting heads) and spores in fungal colon
• Yeast morphology and budding
• Biochemical tests for yeast and less frequent and frequent fungi
• Effect specific tests such as germ tube test for Candida albicans
• Specific serological tests
• Status of contaminants!!!
Safety Conditions for Mycology
• Most of the fungi that cause disease in animals are also pathogenic to humans.
• Particular care should be taken when considering materials and cultures that are thought to contain pathogenic fungi!!!
• Particular care should be taken against pathogens that are easily aerosolized, such as Coccidioides immitis, which form highly infective arthrospores at 25 and 37 ° C.
• Dimorphic fungi such as Cryptococcus neoformans and Blastomyces dermatitidis cause very serious disease in humans.
• Ideally, all mycologic examinations should be done in the biosafety cabinet.
Treatment and Prevention
• Mammalian cells are lack of enzymes that destroyes fungal cell Wall polysaccharides. Because of this, fungi can not be eradicated by the host defence mechanisms.
• Memeli hücreleri, mantarların hücre duvarı polisakkaridlerini parçalayan enzimlere sahip değildir. Bu nedenle mantarlar, hayvanın konakçının defans mekanizmalarıyla eradike edilemezler.
• Since mammalian and fungal cells are eukaryotic, both of them have the same cell structure and also look like each other biochemically.
• Hem memeliler ve hem de mantarlar ökaryotik organizmalar olduklarından, her ikisindeki hücresel yapı, biyokimyasal olarak birbirine benzerdir.
• All eukaryotic cells’ cell membrane have sterols; in fungi these are ergosterol and in mammalian cells these are cholestrol. Thus, the invasive fungi elemination leads to severe side effects in their hosts.
• Bütün ökaryotik hücrelerin, hücre membranları steroller içerir; mantarlarda bu ergosterol iken, memelilerde ise kolesteroldür. Dolayısıyla, invaze olan mantar etkenini bozacak maddeler konakçıda da ciddi yan etkilere neden olabilmektedir.
• Although the first chemotherapeutic agent is an antifungal (oral iodids), the developing of these agents were slow in comparison to antibacterial agents.
• Her ne kadar ilk kemoterapotik ajan 1903 yılında kullanılan bir anti-mikotik (oral iodidler) iken, bu ajanların geliştirilmesi anti-bakteriyel ajanlara göre yavaş olmuştur.
• It was difficult to inhibit the invasive organism while protecting the host. This situation slows down the new drug developments.
• Konakçıya minimal zarar vererek invaze olan organizmayı inhibe etmek için, gerekli selektif toksisitenin ökaryotik hücreler için oluşturulması güç bir hedef olmuştur. Bu da yeni ilaç geliştirme çalışmalarını yavaşlatmıştır.
• Flucanozole is used for the AIDS patients to treat the cryptococcosis.
• Flukanazol bugün cryptococcosis’li AIDS hastalarının tedavisinde tercih edilen ilaçtır. Spinal sıvıyı (BOS) penetre ettiği için idealdir.
• Azolles leads to the inhibition of ergesterol synthesis.
• Azollerin genel etki mekanizması hücre duvarı sentezini etkileyen ergesterol sentezinin inhibisyonudur. Oral uygulama, düşük toksisite önemli dezavantajlarıdır.
• Ketoconazole, Fluconazole, Itraconazole, Voriconazole, Posaconazole
• Griseofulvin, is used in severe skin and nail infections. It has a very slow effect. Orally routed.
And the mechanism of its effect is due to the accumalation on stratum corneum layer and to penetrate the tissue in order to prevent the fungal invasian.
• Şiddetli deri ve tırnak infeksiyonlarında kullanılan, oldukça yavaş etkili bir ilaçtır. Oral yolla uygulanır. Etkisi, stratum corneum tabakasında birikmesi ve buradan da dokuya geçerek daha ileri fungal penetrasyonu ve üremeyiş engelleyecek şekilde bariyer oluşturması prensibine dayanır.
• 5-fluorosytosine, inhibits the RNA synthesis, mostly used in criptococcosis treatment. Orally routed.
• Alilamines, Terbinafine (lamisil). Used in dermathophyte infections.
• Echinocandins (caspofungin): New antifungal agent approved by FDA.
