Lipid extraction from Bacillus subtilis
Sampling Table
Sample name (date):
Sample No.
Cell suspension (ml)H2O from material(ml) ––– (W)（生重量 x0.9）
2-Propanol(ml)Extraction
Methanol
Chloroform (1)H2O=4.0-(W)
Phase separation
CHCl3
H20

Washing with
0.9% KCl

Empty vessel (g)Total lipid extracts(mg)

Example

No. 1

No. 2

No. 3

55555555555555555555

101010100.5
0.5

3.5

Day 1Grow B. subtilis in a 100 ml LB medium. Collect cells in a 50-ml screw-capped
glass tube by centrifugation. Wash the cells with 30 ml of chilled 1% NaCl.
Suspend washed cells in 0.5 ml chilled 1% NaCl and keep on ice. (Frozen cellpellets are thawed in running tap water until the suspension is almost thawed.
Then, samples are suspended in 1 ml chilled 1% NaCl and kept on ice.)
During the above procedures, keep 6 ml of 2-propanol in a hot water bath set at
90˚C.
To the 1 ml cell suspension, flash 5 ml of hot 2-propanol, using a 10-ml
silicon-rubber-capped glass pipette, and immediately transfer the mixture into
the hot water bath set at 90 ˚C.
↓ 5 min, 90˚C
Chill on ice.
Add methanol, chloroform, and water as calculated from the Sampling Table.
The solution must be homogenous.
↓Add 5 ml chloroform and 5 ml H2O, and mix vigorously using a vortex mixer.
↓3000 rpm x 15 min (r.t.)
Recover the lower (chloroform) layer into a fresh 30-ml screw-capped glass
tube, and then wash with an equal volume of 0.9% KCl.
↓3000 rpm x 15 min (r.t)
Recover the lower (chloroform) layer*1 into a 20 ml weighed pear-shaped flask,and evaporate the solvent on a rotary evaporator*2.
*1Special attention should be paid not to recover any material floating in theintermediate phase.
*2If you see water droplets on the bottom of flask, add a few ml of ethanol andevaporate again.
↓
Dry up the residual solvent in a vacuum desiccator, and determine the lipid dryweight.
↓Add chloroform to make a 10 mg/ml lipid solution while placing the flask on ice

to prevent solvent evaporation.
↓
Transfer the lipid solution into a 1-ml screw-capped sample tube*3.
*3Because the cap of the tube is so small, special attention should be paid toensure that the cap is screwed on tightly. Each cap should then be sealed withparafilm and stored at -30˚C. If you store the sample for more than one week,
the antioxidant BHT should be added at a final concentration of 10 µg/ml.
Correspondence: Ikuo Nishida, nishida@molbiol.saitama-u.ac.jp

Day 2
Separation of lipids
chromatographybytwo-dimensional

silicagel

thin-layer

Using a 100-µl glass micro syringe, streak 100 µl of the lipid extract (1 mg lipidequivalent) on a 1-cm line marked in the 2-cm-from-right and 2-cm-from-bottom
position of a TLC plate (20 x 20 cm, Silica gel 60, Merck 1.05721.) as shownbelow.
A view before the second
When streaking lipids

TLC development

1st

2nd

2nd

1st

Estimated weight of lipid classes in 1 mg total lipid extracts:
Pigments
0.59 mg
Glycolipids
0.2 mgNeutral phospholipids
0.12 mg
Acidic lipids
0.06 mg
1st dimension：
Acetone/Benzene/Methanol/water = 8:3:2:1（by volume)↓Air-dry the plate (you may use warm air from a hair dryer).
↓
2nd dimension：
Chloroform/Methanol/Acetic acid/Water =170:25:25:4（by volume）↓

Dry the plate using warm air from a hair dryer.
↓Spray 0.01% Purimurin/80% acetone solution over the plate.
Dry the plate using warm air from a hair dryer.
↓Mark lipid spots under long-wave UV light (360 nm), using a soft pencil (HB).
The fluorescence image must be taken on a digital camera.
↓Scrape off each spot on medicine paper, and carefully transfer all silica gel
powder into a 15-ml screw-capped glass tube*4.
*4 Special attention should be paid not to use any tubes with cracks on themouth or the screw mound.
↓Add 10 µl each of a 15:0 standard/toluene solution, using a fresh glass capillarypipette.
↓
Add 10 µl of a BHT/toluene solution, using a fresh glass capillary pipette.
(If 14:0 is expected in the lipid samples, BHT must not be added, because
14:0-Me and BHT show the same retention time on a gas chromatograph.)↓
Add 3 ml each of HCL/methanol solution. Screw on the tube and mix vigorouslywith a vortex mixer.
↓80 ˚C for 3 h on an aluminum heating block.

Day 3Chill all tubes on ice.
↓Extract FA methyl esters with 3 ml distilled hexane.
↓Evaporate hexane, using a vacuum centrifugation evaporator. Dissolve driedlipid residues in ~5 drops of hexane from a Pasteur pipette and subject to gas
chromatograph analyses.
(The hexane solution should be transferred to a 1-ml sample tube for storage ora glass tube for an automatic sample injector.)

Correspondence: Ikuo Nishida, nishida@molbiol.saitama-u.ac.jp