Conidiocidal assay

Introduction

To evaluate the phagocytes' efficiency in killing phagocytosed conidia.

Materials

  • Effector cells (monocytes or neutrophils)
  • Conidial stock of test fungal isolates
  • Sabouraud Dextrose Agar plates (SDA)
  • Complete Medium (CM; human serum 25% v/v + RPMI-1640 w/ phenol red)
  • 25% (v/v) Triton X solution in distilled H2O
  • 96-well flat-bottom cell culture cluster (Corning Inc., NY., U.S.A.)
  • Adjustable 20-7micro;l, 200-µl and 1000-µl pipettes and sterile disposable tips
  • Adjustable 200-µl 12-channel pipette and sterile disposable tips
  • Sterile disposable 1.5-ml tubes
  • Sterile disposable 5- and 10-ml plastic pipettes
  • Glass spreaders
  • 70% Ethanol
  • Sterile distilled water
  • 0.9% (w/v) normal saline

Procedure

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

2. In a 96-well plate seed 200,000 effector cells and 200,000 conidia.  The final volume in each well is made up to 196 µl with CM (see table below).

Well number Effector cells Conidia CM
1 + + +
2 + + +
3 - + +
4 - + +
5 - + +
6 - + +

4. At time zero, add 4 µl of 25% Triton X to wells number 5 and 6 and resuspend thoroughly by pipetting several times. Take 10 µl from well number 5 and 6, which should contain 10,000 conidia and resuspend in 990 µl of sterile distilled water.

5. Mix well and resuspend 10 µl in 990 µl of sterile 0.9% normal saline which should contain 1 conidia/µl.

6. Plate out 100 µl in duplicate on SDA plates using an alcohol flamed sterile glass spreader.

7. Incubate SDA plates at 37°C until colonies can be seen and counted.

8. Incubate the 96-well plate for 6 hours (depending on the fungal species) at 37°C in 5% CO2.

9. Add 4 µl of 25% Triton X to wells number 1-4 and resuspend thoroughly by pipetting several times. Take 10 µl from each, which should contain 10,000 conidia and resuspend in 990 µl of sterile distilled water.

10. Mix well and resuspend 10 µl in 990 µl of sterile 0.9% normal saline which should contain 1 conidia/µl.

11. Plate out 100 µl in duplicate on SDA plates using an alcohol flamed sterile glass spreader.

12. Incubate SDA plates at 37°C until colonies can be seen and counted.

13. Count the number of colonies on each plate at least two times, one when colonies can be seen, and another 24 hours later.

Year prepared: 

2003