Inhibition of germination assay

Introduction

To assess whether, once phagocytes have ingested conidia, they are able to inhibit their growth.

Materials

  • Monocytes, macrophages
  • Conidial stock of test fungal isolates
  • Complete medium (CM; human serum 25% v/v in RPMI-1640 w/ phenol red)
  • 12-well flat-bottom tissue culture plates (Corning Inc., NY., U.S.A.)
  • Sterile round coverslips 18 mm (Homa Red Label Micro Cover glasses 18#, Thomas Scientific, U.S.A.)
  • 70% Ethanol
  • Adjustable 20-µl, 200-µl and 1000-µl pipettes and sterile disposable tips
  • Sterile disposable 1.5- and 5-ml tubes
  • Sterile 5- and 10-ml disposable plastic pipettes
  • Forceps
  • Hank's Balanced Salt Solution, without phenol red, calcium or magnesium
  • (HBSS- Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
  • Hank's Balanced Salt Solution, without phenol red, with calcium and magnesium (HBSS+ Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
  • Permount
  • Glass microscope slides

Procedure

1. Place sterile round cover slips into the wells of a 12-well tissue culture plate. The coverslips are kept in alcohol until required and flamed before being dropped into each well and centred with sterile forceps.

2. Estimate the total number of monocytes required and adjust the stock solution of effector cells in CM as appropriate.

3. Pipette 200 µl of a 5x106 monocytes/ml stock solution in CM onto each coverslip giving a final concentration of 106 monocytes per coverslip. Avoid the solution going out of the round coverslip onto the surface of the well.

4. Incubate at 37oC in 5 % CO2 for at least 45 minutes.

5. Pre-warm HBSS- and CM to 37oC.

6. Wash each coverslip 2x with 1 ml of warm (37oC) HBSS-. Adherent monocytes will remain whereas the non-adherent leukocytes will be washed off. This must be done with extreme caution, not only to avoid washing off the monocyte monolayer but also to avoid bacterial or fungal contamination.

7. If monocyte-derived macrophages are desired, add 1 ml CM and incubate at 37oC in 5 % CO2 for 2 or 3 days. Then wash once with warm HBSS-.

8. Prepare 106 conidia/ml in CM.

9. Add 1 ml to each coverslip including two blank control coverslips.

10. Incubate for 1 hour at 37oC in 5 % CO2.

11. Wash 3 times with 1 ml of warm HBSS- and cover with 1 ml CM.

12. Incubate at 37oC in 5 % CO2 for up to 8 hours.

13. Remove coverslip from 12-well plate

14. Air-dry.

15. Immerse coverslip in May-Grunwald stain for 1 minute followed by Giemsa stain for 5 to 10 minutes. Rinse in water and air-dry.

16. Mount coverslips onto glass slides (cells down) with permount.

17. Calculate the number of germinated conidia expressed as a percentage of total conidia in a total of 100 monocytes is calculated using a microscope.

Year prepared: 

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