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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
COMPARATIVE EVALUATION OF DIFFERENT METHODS FOR SPORE'S ISOLATION FROM TRICHOPHYTON RUBRUM
CULTURES
1Khushboo Arya, 2Sana Akhtar Usmani, 3Shikha Chandra, 4Saumya Chaturvedi, 5Deeksha
Jattan, 6Nitin Bhardwaj, 7Ashutosh Singh
1Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 2Research Scholar- Department of Biochemistry, University of Lucknow,
Lucknow, Uttar Pradesh, India 226024, 3Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 4Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 5Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 6Department of Zoology and Environmental Science, Gurukula Kangri (Deemed University), Haridwar, 249404, Uttarakhand, India, 7Assistant Professor - Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024. https://doi.org/10.5281/zenodo.13829100
Abstract. Trichophyton rubrum, despite being the most prevalent dermatophyte, presents significant challenges to comprehending its behaviour, virulence, host-pathogen interaction, and survival mechanisms. These challenges arise from its distinctive morphological features and the production of a limited number of spores. However, concerted research efforts have been directed toward addressing these obstacles. Countless researchers have devoted their endeavours to establishing robust spore isolation methods. This study provides a comparative analysis of these methods inclusive of modifications as a result of these collective researches.
Keywords: Dermatophytes, Trichophyton, spores, isolation
Introduction
Based on the previous studies, the isolation of Trichophyton rubrum spores can present several difficulties owing to the unique features [1,2]. The main factors contributing to the difficulties are restricted sporulation, selective growing circumstances, spore type, delayed sporulation, and contamination hazards. Under typical laboratory conditions, T. rubrum shows restricted sporulation, mainly in mycelial form, with minimal production of conidia on culture media [3]. The limited availability of spores can impede both experimental and identification procedures. The spore generation of T. rubrum is linked to particular growth parameters, including as temperature, pH, and nutritional composition [4,5]. Researches explain that under specific stress circumstances, such as nutrition deprivation or exposure to light, optimal sporulation may take place. However, reproducing this process consistently in a laboratory environment can be problematic. T. rubrum predominantly produces fragile and tiny microconidia, which present difficulties in manipulation, isolation, and visualization when compared to more durable dermatophytes spores. Even under ideal circumstances, T. rubrum frequently displays delayed sporulation, which extends the isolation process. Such delay can further complicate the handling of T. rubrum spores. The handling of T. rubrum spores also presents contamination hazards from other fungal species or bacterial pathogens, requiring meticulous aseptic procedure and frequent subculturing to minimize these obstacles. To summarize, extracting T. rubrum spores necessitate
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
detailed methodologies and a comprehensive knowledge about growth and sporulation behaviour of T. rubrum.
Methods of spore's isolation
Hole puncher method
In the isolation of spores for dermatophytes such as T. rubrum, the 'hole puncher method' of spore isolation is a usually employed technique. It is a straightforward and efficient method for isolating spores (conidia) from fungal colonies cultivated on solid media (Fig 1A). The procedure uses a sterile punch to gather minute sections of the fungal colony, where spores are densely packed, for subsequent experimental procedures [6,7].
Briefly, the T. rubrum culture inoculum was effectively spread using an L-shaped spreader on the Potato dextrose Agar (PDA) plate. Further, evenly spaced holes in the PDA plate were made using a sterile punch, maintaining a distance of 2cm between each hole. After 3 days holes were filled with PBS buffer and mixed well and taken out the spores in the PBS buffer from the hole. Plate was again incubated further to another 3 days to harvest the spores again.
This method is used for collecting a limited number of spores for the selective collection of spores from high-density areas, ensuring a higher yield. When conducted properly, it minimizes the risk of contamination.
A BCD
Figure 1: T. rubrum spores' isolation from the solid and liquid cultures: A: Growth of T. rubrum on PDA plate (solid culture sample); B: Growth of T. rubrum in PDB (Liquid culture sample); C: Mixed population of spores and hyphae from the result of poor spore isolation; D: Isolated spores under hemocytometer.
Centrifugation method
This is another method frequently used to isolate spores in more significant amounts. This method can be used for both liquid and solid medium. For solid medium (Fig 1A), the T. rubrum culture is inoculated on PDA plated and incubated for two weeks for growth. A well-grown plate is then thoroughly scraped by a sterile scraper in the PBS buffer and centrifuged at 3000 rpm for
5 minutes [8]. The resulting pallet is collected, resuspended in the buffer, and filtered by Mira cloth, which ensures the collection of spores as a filtrate.
The liquid medium (Fig 1B) centrifugation method has the same steps as the additional step, in which the culture scraped from the PDA plate was again incubated in PDB for 3 to 4 days for more sporulation before centrifugation, resulting in a higher yield of spores but generally provide spores with impurity of debris and hyphae as shown in Fig 1C.
Filtration method
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
This method, known for its effectiveness, is the most reliable for spore isolation. The use of Tween 20 effectively detaches the spores from the mycelium. The process begins with a well-grown culture plate, opened under laminar airflow (LAF), and treated with a 1 mL PDB solution of 0.01% Tween 20 for 5 minutes.
The treated culture was scraped gently without scraping the mycelia by L shaped spreader and filtered with Whatman filter paper 40 number [9,10]. Filtrate (mostly spores) was collected in the eppendorf, and number of spores were counted by hemocytometer (Fig1D). This method gives comparatively pure spores but in less amount.
Conclusion
Methods can be modified to achieve more spores in their pure form and according to required parameter and purposes. Physiological features play important role in sporulation [11]. Therefore, by paying close attention to growth conditions, utilizing sporulation-promoting media, and implementing contamination prevention measures, successful isolation of T. rubrum spores can be achieved, serving both research and diagnostic purposes effectively.
Acknowledgements: AS thanks support from ICMR (No.52/08/2019-BIO/BMS), DST-PURSE program (SR/PURSE Phase 2/29(C)), UP Higher Education (No. 10/2021/281/-4-Sattar-2021-04(2)/2021 and No. 39/2024/242/Sattar-4-2024-001-4(33)/2023), DBT (BT/PR38505/MED/29/1513/2020) and DST (CRG/2022/001047) and the University of Lucknow.
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