Growth and Storage of Aspergillus fumigatus

Introduction

Aspergillus fumigatus is easily cultured and grows quickly. It is harder to get it to grow filamentously in liquid culture as it readily pellets.

Materials

  • 260 ml vented tissue culture flasks
  • Loops
  • Glass beads
  • PBS (Phosphate buffered Saline): make up using a PBS tablet, autoclave and add Tween 80 to 0.05 %.
  • Potato dextrose agar / broth
  • Sabourauds dextrose agar / broth
  • Modified Vogel's minimal medium : Vogel's salts in 1% glucose
  • Glycerol

Procedures

a) Growth on tissue culture plates

1) Pour 50 ml of either potato dextrose agar or Sabourauds dextrose agar into a 260 ml tissue culture plate and leave to set.

2) Place a loop of spores from a stock spore solution into the middle of a culture plate. Incubate at 30 to 37°C for 5-10 days with periodic checking. Examine microscopically for any sign of contamination.

3) Place a few glass beads and 8 ml of PBS into the flask and close the lid tightly. Shake the flask vigorously back and forth until most of the spores have been dislodged. Remove the spores using a pasteur pipette and store at 4°C. If necessary spores can be filtered through 2 layers of Whatman 105 lens tissue to remove hyphal fragments. A yield of 2-4 x 108/ml should be obtained.

b) Long term storage

1) Aliquot out 150 - 300 mµl glycerol into cryotubes and add 700 - 850 µl stock spore solution in PBS. Mix and store at - 80°C or in liquid nitrogen.

2)Stocks can also be maintained for months by plating stocks onto agar slopes in 5 ml bottles and storing the slopes at 4°C after the cultures have grown.

c) Overnight culture in liquid medium

1) Inoculate 50 ml of Vogel's minimal medium, Saubouraud dextrose broth or potato dextrose broth to a final concentration of 107 spores / ml and incubate with shaking at 200 rpm until late exponential phase (18-24 h) at 37°C.

2) Dry down the mycelium onto Whatmann 54 paper using a Buckner funnel and a side-arm flask attached to a vacuum pump and wash with 0.6 M MgSO4.

3. To freeze dry, transfer the mycelium to a universal tube, freeze at - 80°C for a few hours, loosen the lid and freeze dry overnight. Seal the tube with parafilm and store at room temperature.

Tips and general comments

1) We grow A.fumigatus on solid medium in tissue culture flasks rather than on standard microbiological plates for 3 reasons: a) to control the spread of spores; spore cross-contamination is less likely with narrow-necked flasks b) the large surface area also means that fewer flasks are required to give a good yield of spores c) it is easier to harvest spores without creating an aerosol.

2) If vented flasks are not used, make sure the lids are loose and sealed with parafilm to enable aerobic growth. This will facilitate conidiation.

3) To limit pellet formation during liquid culture, we suggest using a high spore inoculum and disrupting the flow of the liquid by placing the flasks at an angle to the horizontal.

4) Spore viability is reduced when storage is at -80°C because of temperature fluctuations in freezers. It is preferable to store stocks in a liquid nitrogen ewer.

5) If spores in PBS are to be kept at 4°C for a few weeks, then a sensible precaution would be to add penicillin and streptomycin to final concentrations of 100 IU/ml and 100µg/ml respectively and to keep a close watch for contamination.

FGSC link: Media for culture of Aspergillus nidulans

References

Vogel, H.J. (1956) A convenient growth medium for Neurospora (medium N). Microbiol. Gen. Bull. 13, 42 - 44

Year prepared: 

2003