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Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

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Monitoring the proliferation of T. reesei and A. nidulans spores by light microscopy and flow cytometry. (A) Spherical size-monodispersed alginate microcapsules fabricated in the microencapsulator. (B−C) Pictures of encapsulated spores during germination. In this test the spores were encapsulated in 400-μm, 3% alginate capsules. After encapsulation, the beads were incubated in shaking flasks and samples were recovered after different time of incubation. (B) Trichoderma reesei. (C) A. nidulans. (D−E) Flow cytometry analysis of encapsulated spores T. reesei. Aliquots were analyzed by COPAS SELECT flow cytometry allowing the measurement of different optical parameters: size (time of flight), optical density (EXT), green self-fluorescence and red self-fluorescence signals. The germination of spores is associated with an increase in density. These measurements are represented in the graphs showing the increase of EXT over time (left) and the EXT distribution within the bead (right). A.U., arbitrary unit; EXT, extinction.
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fig2: Monitoring the proliferation of T. reesei and A. nidulans spores by light microscopy and flow cytometry. (A) Spherical size-monodispersed alginate microcapsules fabricated in the microencapsulator. (B−C) Pictures of encapsulated spores during germination. In this test the spores were encapsulated in 400-μm, 3% alginate capsules. After encapsulation, the beads were incubated in shaking flasks and samples were recovered after different time of incubation. (B) Trichoderma reesei. (C) A. nidulans. (D−E) Flow cytometry analysis of encapsulated spores T. reesei. Aliquots were analyzed by COPAS SELECT flow cytometry allowing the measurement of different optical parameters: size (time of flight), optical density (EXT), green self-fluorescence and red self-fluorescence signals. The germination of spores is associated with an increase in density. These measurements are represented in the graphs showing the increase of EXT over time (left) and the EXT distribution within the bead (right). A.U., arbitrary unit; EXT, extinction.

Mentions: Fungal spores were microencapsulated in calcium alginate beads (400 µm) in a Cellena microencapsulator device (Ingeniatrics) following the manufacturer’s instructions as previously described (Gomez-Herreros et al. 2012) (Figure 1A). Microencapsulation conditions were adjusted to obtain single-spore capsules, which ensures that all the mycelial cells growing inside the microcapsules derived from one single spore and consequently, they are clonal. This procedure gave microcapsules with a regular spherical shape and homogenous size, which contained one spore inside the beads (Figure 2A). Spore suspensions were adjusted at 0,160 units at O.D. 600 nm, and 10 µL of each suspension was mixed with 290 µL of water and 2.7 mL of alginate 3%. The sample was then injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply (Figure 1A). The stationary jet broke up by capillary instability into homogeneous droplets, which gel in a continuously stirred 3% calcium chloride solution at room temperature. The capsules were stored in the same solution at 4° with agitation for at least 1 hr or until they were used, then they were filtered and washed with distilled water to remove excess of calcium chloride. Then, microcapsules were inoculated with a sterile spoon into liquid media and incubated under the appropriate conditions.


Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Monitoring the proliferation of T. reesei and A. nidulans spores by light microscopy and flow cytometry. (A) Spherical size-monodispersed alginate microcapsules fabricated in the microencapsulator. (B−C) Pictures of encapsulated spores during germination. In this test the spores were encapsulated in 400-μm, 3% alginate capsules. After encapsulation, the beads were incubated in shaking flasks and samples were recovered after different time of incubation. (B) Trichoderma reesei. (C) A. nidulans. (D−E) Flow cytometry analysis of encapsulated spores T. reesei. Aliquots were analyzed by COPAS SELECT flow cytometry allowing the measurement of different optical parameters: size (time of flight), optical density (EXT), green self-fluorescence and red self-fluorescence signals. The germination of spores is associated with an increase in density. These measurements are represented in the graphs showing the increase of EXT over time (left) and the EXT distribution within the bead (right). A.U., arbitrary unit; EXT, extinction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232552&req=5

fig2: Monitoring the proliferation of T. reesei and A. nidulans spores by light microscopy and flow cytometry. (A) Spherical size-monodispersed alginate microcapsules fabricated in the microencapsulator. (B−C) Pictures of encapsulated spores during germination. In this test the spores were encapsulated in 400-μm, 3% alginate capsules. After encapsulation, the beads were incubated in shaking flasks and samples were recovered after different time of incubation. (B) Trichoderma reesei. (C) A. nidulans. (D−E) Flow cytometry analysis of encapsulated spores T. reesei. Aliquots were analyzed by COPAS SELECT flow cytometry allowing the measurement of different optical parameters: size (time of flight), optical density (EXT), green self-fluorescence and red self-fluorescence signals. The germination of spores is associated with an increase in density. These measurements are represented in the graphs showing the increase of EXT over time (left) and the EXT distribution within the bead (right). A.U., arbitrary unit; EXT, extinction.
Mentions: Fungal spores were microencapsulated in calcium alginate beads (400 µm) in a Cellena microencapsulator device (Ingeniatrics) following the manufacturer’s instructions as previously described (Gomez-Herreros et al. 2012) (Figure 1A). Microencapsulation conditions were adjusted to obtain single-spore capsules, which ensures that all the mycelial cells growing inside the microcapsules derived from one single spore and consequently, they are clonal. This procedure gave microcapsules with a regular spherical shape and homogenous size, which contained one spore inside the beads (Figure 2A). Spore suspensions were adjusted at 0,160 units at O.D. 600 nm, and 10 µL of each suspension was mixed with 290 µL of water and 2.7 mL of alginate 3%. The sample was then injected with a syringe pump through a capillary feed tube inside a chamber and pressurized by a continuous air supply (Figure 1A). The stationary jet broke up by capillary instability into homogeneous droplets, which gel in a continuously stirred 3% calcium chloride solution at room temperature. The capsules were stored in the same solution at 4° with agitation for at least 1 hr or until they were used, then they were filtered and washed with distilled water to remove excess of calcium chloride. Then, microcapsules were inoculated with a sterile spoon into liquid media and incubated under the appropriate conditions.

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

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Related in: MedlinePlus