Total Lipid Extraction

Introduction

This method has been successfully used for the extraction of total lipids from filamentous fungi (1), but is also suitable for lipid extraction from yeast and bacteria.

Materials

Vogel's minimal media (Vogel 1956)

Methanol/Choloform (2:1 v:v)

Chloroform

Ultra pure water

Equipment

Glass pipettes

Glass Bijouxs

Freeze Drier

Nitrogen gas or Vacuum Dessicator

Procedure

1) Inoculate 50 ml of Vogel's medium with freshly harvested conidia to a concentration of approximately 10⁷ /ml. Incubate with shaking 200 rpm for 18-24 h at 37^o C.

2) Collect mycelia by vacuum filtration and wash with 20 ml of 0.6 M MgSO₄

3) Freeze dry mycelia

4) Weigh out 10 mg of freeze dried mycelia in to a glass Bijoux and add 2 ml of methanol/chloroform leave for up to 4 hours with occasional mixing.

5) Remove solvent to a separate Bijoux and re-extract as in 4).

6) Collect solvent and pool with first extract.

7) Remove solvent under a stream of N₂ gas.

8) Re-dissolve lipid extract in 1ml of chloroform and wash with 1 ml of high quality water.

9) Remove top layer and use bottom (chloroform) layer to clean bijoux and dry under N₂ gas.

10) Store lipids in a vacuum dessicator until analysis

Timetable

Fungal Culture 18-24 h

Freeze drying 12-16 h

Lipid extraction 8h

Solvent removal 1h

Tips and general comments

1) Always pre-wash glassware with methanol prior to starting procedure.

2) Remove rubber cap seals from Bijouxs and avoid using plastic pipettes.Plasticisers can leach out and interfere with subsequent analysis.

3) Solvents must be of high purity; minimum HPLC grade.

4) Solvent can be removed by vacuum drying if preferred.

References

1) Birch M, Drucker DB, Riba I, Gaskill SJ, Denning D.(1998) Polar lipids of Aspergillus fumigatus, A.niger, A. nidulans A.flavus and A. terreus. MedicalMycol 36:127-134

2) Vogel HJ (1956) A convenient growth medium for Neurospora (medium N)Microbiol. Gen. Bull. 13:42-44

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