lipidsandbioassay

BCH3033L  LIPIDS AND BIOASSAY (REVISED 7/00 brc)

I.  Analysis of total lipid extracts from plants for lipid classes.
II. Preparation of plant extracts for assay of photoactivated antimicrobial compounds.

This week each group will prepare a lipid extract for 
chromatography next week.  Each group will also prepare a 
methanol extract from a plant and assay it for antimicrobial 
activity.  The assay will be scored next week.

A.  General Information on Lipid Biochemistry (CHAPTER 8 GARRETT & GRISHAM)

    Lipids may be defined as a heterogeneous group, of compounds 
that are synthesized by living cells and that are sparingly soluble 
in water but are soluble in nonpolar solvents; they can be 
extracted from tissues by nonpolar solvents.

There are many classification schemes of lipids in use but it is usually
more useful to consider lipids by their functions. Triglycerides (fats
and oil) serve as storage and transport forms of metabolic energy.
Waxes, hydrocarbons and fatty alcohols form protective surfaces of
plant and animal tissues. Lipids that are membrane components are
usually amphipathic and complex (phospholipids, sphingolipids,
glycolipids, etc.). Finally, there are a large group of low
molecular weight, derived lipids (steroids, vitamins, carotenoids,
quinones, etc.) that are diverse in their functions as hormones,
vitamins, photopigments and other specialized roles.

In the first part of the experiment, you will obtain a lipid fraction from
either a plant. Analysis of lipids will be by two dimensional TLC of the
extract for the different lipid classes.

B. EXPERIMENTAL PROCEDURE

Experimental work in lipid biochemistry requires some special 
precautions:

Use a hood whenever possible!

    To pipette highly volatile solutions (e.g., ether) use a 
propipette.  Draw the solution up into the pipette and let it drain 
down several times to saturate the space above the liquid; 
otherwise, the solution will tend to drip from the tip of the 
pipette.  Be gentle when using either the large propipette or a 
Pasteur pipette bulb; when solvent reaches the bulb, contaminants 
are introduced and the rubber of the bulb deteriorates.  Never 
invert a pipette with fluid in it!  Since many of the solutions 
used in these experiments are highly volatile, solutions should 
always be kept stoppered (with aluminum foil or tightly fitting 
glass stoppers) when not in immediate use.  This will prevent an 
accumulation of toxic and/or highly flammable vapors in the air as 
well as keeping the concentration of the solutions reasonable 
constant.  

    Lipids are highly prone to oxidation and polymerization, processes 
induced by both atmospheric oxygen and light.  Solutions to be 
stored should be gassed briefly with nitrogen and stored in the 
dark at -20 OC. 

MOST IMPORTANT, solvents are highly flammable; do not use near open 
flames.

C.  PROTOCOL

PART A. Extraction of Total Lipids.
Lipids are released and extracted from intact cells by grinding in
relatively, nonpolar, organic solvents (hexane-isopropanol).
Tissue is re-extracted several times and tissue debris is removed
by centrifugation. The supernatants are combined and non-lipid
contaminants which are water soluble are removed by partitioning
against an aqueous salt solution.

1. Each group will be given a sample to extract. Weigh 5.0 g of
liver, egg yolks, peanuts, avocado, etc.,record the weight to the
nearest 0.1 g, and transfer to a mortar.

2. Add about an equal amount of acid-washed sand and grind with a pestle.

3. Add 5 ml of hexane-isopropanol (3:2) and grind to a smooth paste;
then add 5 more mls of solvent and continue grinding.

4. Transfer to a Sorvall centrifuge tube and prepare a balance tube by weight.

5. Centrifuge at 10,000 x g for 5 minutes. Decant the supernatant fluid into a Sorvall centrifuge tube.

6. Re-extract the pellet from the original extract with 10 ml of
hexane-isopropanol (3:2). Centrifuge and combine the supernatant fluids.

7. Add 5 mL of 15% Na2SO4 to the combined supernatants and vortex
for 1 min. Centrifuge at 10k x g for 5 min to separate the phases.

8. Pipette off organic layer (top) and transfer to a 50 mL
centrifuge tube. Evaporate to dryness under a gentle stream of air
IN THE HOOD.

9. Resuspend lipid residue in 1 ml of CHCl3. Transfer
the total lipid extract to a screw cap glass tube, label it with
what was extracted (fish, egg, etc.), group number and section.
Give it to the TA to be stored in the freezer for analysis by two
dimensional TLC next week.

WEEK TWO: LIPIDS
Thin layer chromatography analysis of total lipid
extract from plants

To separate a hexane:isopropanol extract into its component
lipid classes, the following thin-layer chromatography methods
are used.

1. The extract, dissolved in chloroform is spotted on a
single spot 2 cm from the bottom and 2 cm from the right edge on
a thin layer chromatography plate (Silica Gel G).
2. To separate the neutral lipids, the plate is developed in
petroleum ether: diethyl ether: acetic acid (70:30:1).
Development should take about 45 minutes.
3. The plate is removed from the first solvent tank, turned 90
degrees so that the right hand edge is at the bottom and then
developed in the second direction which will separate polar
lipids in CHCl3:CH3OH:CH3COOH:H2O (85:15:10:3.5).

4. After development in the second direction for about 1 hour, the plate
is removed from the tank, air dried, sprayed with fluorescein to
fluorescently label the lipid spots and analysed under UV light.

Spots are identified by comparison with the two dimensional
TLC of lipid standards. Compare the different extracts
qualitatively for presence of particular lipid classes and
variation in amounts of the classes.

II PART TWO: BIOASSAY

BIOASSAY FOR PHOTOACTIVATED ANTIMICROBIAL COMPOUNDS IN PLANT
EXTRACTS

PROTOCOL:

A. Materials
4 nutrient agar plates.
4 clean Petri dishes
Paper disks (6.5 mm ID)
Pipetman (20 or 100 ul)
Glass bar and spinner
95% ETOH
Bacillus cereus and E. coli cultures.
1 ml glass pipet and rubber bulb.

The following chemicals and crude extracts in Eppendorf tubes:
A. Chemicals:
        Psoralen (Ps),
        5,7-Dimethoxycoumarin(5,7 DMC),
        8-Methoxypsoralen(8 MOP),
        5-Methoxypsoralen(5 MOP).
B. Crude extracts (MEOH extracts):
        Fl. (Flaveria),
        Bi. (Bidens).
        Bottlebrush
        lime or lemon
        spinach
        grass

METHODS
A. Procedures for crude extracts.

1. Each group will extract one sample and share their extract
with all other groups for the bioassay.
2. weigh 20 g plant material,
3. cut with scissors into small pieces.
4. add to 50ml centrifuge tube and add 20ml MeOH.
4. grind with scalpel for 1 min.
5. centrifuge and remove supernatant to a 15 ml centrifuge tube.

Use this as crude extract in the following bioassay.

B. Procedures for the bioassay.

0. Light your Bunsen burner.

1. There are 5 different chemical compounds and some crude extracts to be
tested. Number your compounds and extracts.

2. In a clean Petri dish labeled with the number of the individual compound or extract place four (4) filter disks/group of each compound or extract (so prepare as many petri dishes as needed to cover all the groups)

3. To the disks in the clean Petri dish labelled 1, transfer 20
ul of compound 1 to each disk. Do the same for the rest of the
compounds to be tested.

4. Let the paper disks containing the chemicals or extracts air dry
around the Bunsen burner for 30 min. to evaporate the solvent.

B. Bacterial culture

5. WEAR GLOVES. Each groups gets 4 nutrient agar plates. Prepare 2 nutrient agar plates with B. cereus and 2 with E. coli. Label E.coli dark, E.coli UVA, B. cereus dark, B. cereus UVA.

6. Put one nutrient agar plate on the spinner and transfer two
drops of bacteria culture (E. coli, B. cereus) to the plate and spread with a glass
bar while spinning the spinner.

7. Transfer one filter disk of each compound and extracts to the nutrient
agar plate where you spread the bacteria and put over a mark where you placed the first filter disk. Place the second through tenth disks on the plate in a
clockwise fashion with even spacing between the disks. Repeat for all nutrient agar plates.

8. Incubate the nutrient agar plates with the paper disks in an
incubator at 37 0C for 30 min.

7. For each set of plates for E. coli and B. cereus, expose
one plate to UVa light for 30 min.,and one in the dark as control.

8. After exposure to UV light, the plates will be incubated in
the dark at 37 C for 8 hours (B. cereus) and 16 hours (E. coli).

9. After incubation, the plates will be removed from the
incubator and stored in the fridge until the next lab session.

WEEK TWO:

Observe and measure the inhibition zones. Determine which
compounds have antimicrobial activity and which compounds have
photoactivated antimicrobial activity by comparing the dark plates to the light plates.

Tabulate the results.