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Phenotypic Profiling of Candida glabrata in Liquid Media

FI Fabian Istel
MT Miha Tome
SJ Sabrina Jenull
Karl Kuchler Karl Kuchler

Published: Apr 5, 2017 DOI: 10.21769/BioProtoc.2228 Views: 10194

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Abstract

Here, we describe a method for a large-scale liquid screening approach in C. glabrata. This liquid media method offers several distinct advantages over solid media approaches. This includes growth measurement on a plate reader instead of comparing growth by eye-sight. Furthermore, the liquid method requires lower amounts of antifungals and offers a higher sensitivity. While this method has been optimized for C. glabrata it might be used for other Candida species and yeast-like fungi as well.

Keywords: Candida glabrata search Liquid media screening search Antifungals search Large-scale screening search Antifungal tolerance search Antifungal resistance search

Materials and Reagents

  1. Filtered pipet tips 1, 250 µl XL (Starlab, TipOne®, catalog number: S1122-1830 )
  2. Pin pad, 96 RePad (Singer Instruments, catalog number: RP-MP-2L )
  3. Pipet tips 300 µl (Starlab, TipOne®, catalog number: S1110-8810 )
  4. Filtered pipet tips 200 µl (Starlab, TipOne®, catalog number: S1120-8810 )
  5. 96-well plates (Starlab, CytoOne®, catalog number: CC7682-7596 ) (200 µl/well)
  6. Aluminum foil, self-adhesive (Starlab, catalog number: E2796-9792 )
  7. Breathable foil (Sigma-Aldrich, catalog number: Z380059 )
  8. Petri dishes (92 mm) (SARSTEDT)
  9. Gas-permeable material
  10. Culture tube (for at least 5 ml of culture)
  11. Strains to be screened
    Examples: Deletion mutants of C. glabrata (Schwarzmüller et al., 2014) or clinical isolates of C. glabrata
  12. Glycerol
  13. Compound(s) used for the screening
    Example: Fluconazole (Discovery Fine Chemicals, catalog number: 86386-73-4 )
  14. Control strains (see the ‘Notes’ section for further details)
  15. DMSO
  16. (Optional) Ethanol
  17. Sterile water (double distilled)
  18. BactoTM peptone (BD, BactoTM, catalog number: 211820 )
  19. BactoTM yeast extract (BD, BactoTM, catalog number: 212720 )
  20. BactoTM agar (BD, BactoTM, catalog number: 214030 )
  21. Glucose (Merck Millipore, catalog number: 108337 )
  22. YPD media (yeast extract peptone dextrose) (see Recipes)
  23. Solid YPD media (see Recipes)

Equipment

  1. Yeast replica robot (Singer Instruments, model: RoToR HDA )
  2. Plate reader (Victor3V) (PerkinElmer, model: Victor3V Multilabel Plate Reader)
  3. Electronic pipette 1,200 µl (Gilson, model: Pipetman Concept, catalog number: F31015 )
  4. Electronic pipette 5,000 µl (Gilson, model: Pipetman Concept, catalog number: F31016 )
  5. Electronic pipette 12 x 300 µl (Gilson, model: Pipetman Concept, catalog number: F31044 )
  6. Plate mixer (Eppendorf, model: MixMate® , catalog number: 5353000014)
  7. Rotary shaker for culture flasks (Eppendorf, New BrunswickTM, model: Innova® 44 )
  8. Incubator (Heraeus Instruments, catalog number: B6120 )
  9. (Optional) 48-well replica stamp (V&P Scientific, catalog number: VP 408H )
  10. (Optional) 96-well replica stamp (V&P Scientific, catalog number: VP 407 )
  11. (Optional) Library copier (guide for stamps) (V&P Scientific, catalog number: VP 381 )

Procedure

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Antimicrobial assay > Antifungal assay

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