The collected samples were immediately delivered to the microbiological laboratory. The samples were then inoculated using the dilution method (dilutions −1 to −6) or qualitative culture method (swabs only) on the following media: McConkey (Graso, Biotech Starogard Gdański, Poland [21 ]), Columbia (Lab-Agar, Biomaxima, Lublin, Poland [22 ]), Scheadler (Scheadler-Agra, Biomaxima, Lublin, Poland [22 ]), Bile Esculine Azide (Lab-Agar, Biomaxima, Lublin, Poland), MRS Agar (Oxoid, Brno, Czech Republic [23 ]) or Sabouraud Agar (Biomaxima, Lublin, Poland [22 ]). Media were aerobically incubated at 37 °C (McConkey, Columbia, Bile Esculine Azide and Sabouraud) for 24 h, or anaerobically at 37 °C (M.R.S and Scheadler) for 48 h. After incubation, the phenotypically grown colonies were counted and reported, with the results being presented as colony-forming units (CFU) per mL (CFU/mL). After isolation, the microorganisms were identified through MALDI TOF MS mass spectrometry (MALDI Biotyper, Bruker [24 (link)]).
Sabouraud agar
Manufactured by Biomaxima
Sourced in Poland
Sabouraud Agar is a microbiological culture medium used for the isolation and identification of fungi, particularly yeast and mold species. It provides the necessary nutrients and growth conditions for the cultivation of these microorganisms.
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4 protocols using sabouraud agar
1
Microbial Enumeration and Identification
2
Oral Microbiome Analysis via MALDI-TOF
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The samples were immediately delivered to the microbiological laboratory, where they were inoculated by the dilution method (dilutions −1 to −6) or qualitative culture method (swabs only) on the following media: McConkey (Graso, Biotech), Columbia (Lab-Agar, Biomaxima), Scheadler (Scheadler-Agra, Biomaxima), Bile Esculine Azide (Lab-Agar, Biomaxima), MRS Agar (Oxoid), Sabouraud Agar (Biomaxima). Media were aerobically incubated at 37°C (McConkey, Columbia, Bile Esculine Azide) or anaerobically at 37°C (M.R.S and Scheadler, GENbag Atmosphere Generators [BioMérieux, France] for 48 h). After incubation, the phenotypical colonies were counted and reported, and results were presented as CFU/ml (colony forming unit). After isolation, the microorganisms were identified by MALDI TOF mass spectrometry (Vitek MS Home bioMérieux).
Multiple analyses were performed to identify factors associated with oral health status, biodiversity and composition of oral commensal and potentially pathogenic bacterial microbiota, and in-hospital mortality.
Multiple analyses were performed to identify factors associated with oral health status, biodiversity and composition of oral commensal and potentially pathogenic bacterial microbiota, and in-hospital mortality.
3
Clinical Candida albicans Strain Isolation
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Two references strains of yeasts from American Type Culture Collection (ATCC): Candida albicans ATCC 2091 and Candida albicans ATCC 10231 were included. Moreover, 30 clinical isolates of C. albicans were used. These microorganisms were isolated from oral cavity of hospitalized patients with hematological malignancies (from the collection of clinical strains deponded in Department of Pharmaceutical Microbiology of Medical University in Lublin, Poland). The Ethical Committee of the Medical University of Lublin approved the study protocol (No. KE-0254/75/2011). The isolates were identified by standard diagnostic methods: microscopic, macroscopic, and biochemical microtest, e.g., API 20 C AUX, ID 32 C, API Candida (bioMèrieux, France) on the basis of assimilation of various substrates (Biernasiuk et al. 2018 ; Łączkowski et al. 2018 (link)). Strains were stored as glycerol stock at −70 °C. For research purposes, fungal cultures were conducted at 35 °C for 24 h on Sabouraud agar (BioMaxima S.A., Lublin, Poland).
4
Candida Species Identification Protocol
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The reference strains of fungi from American Type Culture Collection (ATCC) were included. These fungi belonged to yeasts: Candida albicans ATCC 2091, Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030 and Candida krusei ATCC 14243. Moreover, 60 clinical isolates of Candida spp.: C. albicans (30 isolates) and non-albicans Candida spp. (NAC) (30 isolates: C. tropicalis, C. glabrata, C. famata, C. parapsilosis, C. krusei, C. guilliermondii, and C. lusitaniae) were used. These microorganisms were isolated from the oral cavity of hospitalized patients with hematological malignancies (from the collection of clinical strains deposited in the Department of Pharmaceutical Microbiology of Medical University in Lublin, Poland). The Ethical Committee of the Medical University of Lublin approved the study protocol (No. KE-0254/75/2011). The isolates were identified by standard diagnostic methods—microscopic, macroscopic, and biochemical microtest, e.g., ID 32 C (bioMèrieux S.A., Warsaw, Poland)—on the basis of assimilation of various substrates [37 (link)]. Strains were stored as glycerol stock at −70 °C. For research purposes, fungal cultures were conducted at 35 °C for 24 h on Sabouraud agar (BioMaxima S.A., Lublin, Poland).
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