Anaerobik Gut Funguslarının Uzun Süreli Muhafazalarında Agarlı Besi Yerlerinin ve Ön Soğutma İşleminin Etkinliğinin Araştırılması

Amaç: Anaerobik gut funguslarının (AGF) kültüre alınması ve uzun süreli muhafazası özellikle aerobik mikroorganizmalar ve prokaryotik gut mikrobiyom ile karşılaştırıldıklarında göreceli olarak düşük verimlidir. Anaerobik koşullar altında yapılan bu muhafaza işleminde hücre yapılarının düşük sıcaklıklarda uzun süreli korunması amacıyla gliserol, dimetil sülfoksit (DMSO), kan serumu, polivinilpirolidon (PVP) ve sorbitol gibi kriyoprotektanlar kullanılır. Materyal ve Metot: Bu çalışmada farklı enerji kaynakları (buğday samanı, sükroz, dekstroz, ksiloz ve glikoz) kullanılarak dağ keçisi dışkısından izolasyonu ve saflaştırılması yapılan Caecomyces GMLF77 izolatı ile kültür koleksiyonundan temin edilen Orpinomyces GMLF18 izolatı 6 aylık süreyle eğik agar ve Roll tüp agar besi yerlerinde farklı sıcaklık kombinasyonlarında muhafaza edilmiş ve izolatların yaşama oranları analiz edilmiştir. Bulgular: Caecomyces GMLF77 izolatı en yüksek yaşama oranını (%67) ksiloz içeren eğik agar içerisinde %15 gliserol varlığında doğrudan –196 °C’de (sıvı azot) muhafazaya alındığında göstermiştir. Orpinomyces GMLF18 ise glikoz içeren eğik agarlı besi yerinde, 6 aylık süreyle ön soğutmasız –196 °C’de muhafazasında %83 yaşama oranı göstermiştir. Diğer taraftan her iki izolat da kullanılan tüm besi yeri ve enerji kaynaklarında 4 °C’de ön soğutmaya tabi tutulduklarında yaşama şansı bulamamışlardır. Sonuç: Sonuçlar fungal izolatların sıvı azota aktarılmadan önce ön soğutma işlemine tabi tutulmalarının uzun süreli yaşama oranlarının artırılmasına önemli bir katkı sağlamadığını ortaya koymuştur.

Anahtar Kelimeler:

Agar, anaerobik gut fungus, kriyoprotektan, mikrobiyal muhafaza, sıvı azot

Effectiveness of Agar Nutrient and Precooling Process for Long Term Storage of Anaerobic Gut Fungi

Objective: Culturing and long term storage of anaerobic gut fungi (AGF) is a more challenging process compared to aerobic and prokaryotic gut microbiome. For prolonged storage, carried out under anaerobic conditions, various cryoprotectans such as glycerol, dimethyl sulfoxide (DMSO), blood serum, sorbitol, and polyvinylpyrrolidone (PVP) are widely used to protect the cell from possible damages of freezing. Materials and Methods: Caecomyces GMLF77 was isolated and purified from wild goat feces using various energy sources (wheat straw, sucrose, dextrose, xylose and glucose), and Orpinomyces GMLF18 was obtained from the culture collection. Both isolates were stored in agar slant and Roll tube agar media at different temperature combinations until 6 months and later survival rates of the isolates were analyzed. Results: The isolate Caecomyces GMLF77 showed the highest survival rate (67%) when it was grown in the agar slant medium containing xylose as sole energy source and stored directly at –196 °C (liquid nitrogen) under the preservation of 15% glycerol. The isolate Orpinomyces GMLF18 showed survival rate of 83% after the six-month storage period when it was grown in agar slant medium containing glucose as sole energy source and kept at –196 °C without any precooling. No survival was observed for both isolates when they were treated at 4 °C, regardless of medium type and energy source used in the experiment. Conclusion: The results suggest that precooling of fungal isolates in low temperatures just before long-term storage in liquid nitrogen has no remarkable positive effect on their survival rates.

Keywords:

Agar, anaerobic gut fungi, cryoprotectan, microbial preservation, liquid nitrogen,

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