Ag Sop

MATERIALS
Material Quantity
Gingko biloba bark and leaves
Rotary evaporator 2
Scalpel blade 1
Cotton swabs 10
Test tubes 50
Petri dishes 30
Inoculating loop 1
Incubator 1
Calliper/ measuring ruler 1
Bijou/ McCartney bottles 10
10ml measuring cylinder 4
200ml beaker 2
Filter paper
Autoclave 1
Droppers 3
1ml micropipette 1
Micropipette tips 1 case
Bunsen burner 1
Tripod stand with wire gauze 1
Match sticks Box
Crucible tongs/ test tube holder 1
Weighing balance 1
Spatula 1
Cold room/ refrigerator 1
Mortar and pestle 1
Strainer 1
REAGENTS
Chemical Quantity
Methanol 1500ml
Distilled water 2500ml
Fehling’s A 5ml
Fehling’s B 5ml
Aqueous copper (II) sulphate/ cupric
sulphate
Sulphuric acid 25ml
Benzene 25ml
10% ammonia solution 15ml
Acetic anhydride 5ml
Concentrated sulphuric acid 15ml
chloroform 10ml
Sodium hydroxide 2ml
Olive oil 1ml
1% aqueous hydrochloric acid 15ml
Dilute hydrochloric acid 2ml
Dragendorff’s reagent (potassium 1ml
bismuth iodide)
Mayer’s reagent (potassium mercuric 1ml
iodide)
Glacial acetic acid 5ml
Ferric chloride (0.1%) 1ml
Saline solution (0.9%) 50ml
0.5 McFarland turbidity standard 1
Muller – Hinton Agar 500ml
Nutrient broth 60ml
Gram staining chemicals (gram’s
iodine, safranin, crystal violet,
alcohol/ acetone)
Hydrogen peroxide 2ml
Rabbit plasma 1ml
METHODS
1. PLANT EXTRACT PREPARATION
o Collect plant samples (leaves) and wash under running water to remove
any dirt.
o Let it air dry for a few minutes.
o Grind the leaves and roots using pestle and mortar.
o Add 500ml of methanol to plant extract.
o Leave soaked for 48 hours, shaking mixtures periodically.
o Filter the extracts through a Whatman paper.
o Concentrate the extracts in a rotary evaporator.
o Store the extracts in airtight glass containers at 4oC until use.
2. SCREENING FOR PHYTOCHEMICALS
 Fehling’s test for reducing sugars

 Dissolve 0.5g of extract in 5ml of distilled water and filter.
 Mix 1ml of Fehling’s A and 1ml of Fehling’s B and bring it to a boil.
 Add 2ml of the extract filtrate to the boiling Fehling’s solution
 A brick red precipitate indicates the presence of reducing sugars.
 Salkowski test for terpenoids

 Dissolve 10mg of extracts to 2ml of chloroform.
 Add 1ml of acetic anhydride followed by 3ml of concentrated
sulphuric acid.
 A colour change from pink to violet shows the presence of terpenoids.
 Sodium hydroxide test for flavonoids

 Dissolve 0.2g of plant extract in 200ml of distilled water and filter.
 Add 3-5 drops of sodium hydroxide solution.
 A yellow colour that disappears on standing indicates the presence of
flavonoids
 Borntrager’s test for anthraquinones

 Add 0.2g of plant extract to 10ml of benzene.
 Shake and filter the mixture.
 Add 5ml of 10% ammonia solution to the filtrate and shake well.
 A pink red or violet colour indicates the presence of anthraquinones.
 Liebermann- Buchard’s test for steroids
 Add 100mg of plant extract to 2ml of chloroform. Dissolve and filter.
 Add 2ml of sulphuric acid.
 A reddish- brown indicates the presence of steroids.
 Foam test of saponins

 Dissolve 0.5g of plant extract in 5ml of distilled water.
 Shake mixture vigorously and observe a stable froth.
 Add 3 drops of olive oil, shake vigorously.
 An emulsion indicates the presence of saponins.
 Dragendorff’s test for alkaloids

 0.5g of extract was diluted to 10ml with acidified alcohol, boiled,
filtered and allowed to cool.
 To 5ml of the resulting filtrate, 2ml of dilute ammonia was added.
 Followed by addition of 5ml chloroform and shaken gently to extract
the alkaloidal base.
 The chloroform layer was extracted with 10ml acetic acid.
 Extract dispensed into two equal portions, thus Mayer’s reagent was
added to one while Dragendorff’s reagent is regarded positive for the
presence of alkaloids.
 Keller- Killians test for cardiac glycosides

 Dissolve 0.5g of crude extract in 5ml distilled water.
 Add 2ml of glacial acetic acid and a drop of ferric chloride solution.
 Add 1ml of concentrated sulphuric acid.
 A brown ring in the junction indicates a positive result.
 Ferric chloride test for tannins

 Boil 0.5g of plant extract in 10ml of distilled water and filter.
 Add a few drops of 0.1% ferric chloride.
 A brownish green or blue-black colour indicates the presence of
tannins.
3. EVALUATION OF ANTIBACTERIAL
4. PREPARATION OF BACTERIAL INOCULUM

 Measure 3ml of 0.9% saline solution and place in each bijou bottle.
 Using a loop, pick about 3 bacterial colonies (one at a time) from a
pure culture and inoculate it into 3ml saline solution.
 Compare the turgidity with that of the 0.5 McFarland standard
solution until they are the same.
 Repeat for all five bacterial test organisms.
 Use these bacterial suspensions within 15 minutes.
5. PREPARATION OF PLANT EXTRACT CONCENTRATIONS
 Prepare concentrations of 25mg/ml, 50mg/ml, 100mg/ml,

200mg/ml and 400mg/ml by diluting the plant extracts in distilled
water.
 25mg/ml = 0.025g plant extract + 1ml distilled water. Repeat
process to prepare concentrations of 50mg/ml, 100mg/ml,
200mg/ml and 400mg/ml.
 These plant extract concentrations will be impregnated in the
Whatman filter paper disks (approximately 6mm diameter) for the
antibacterial susceptibility test (AST).
6. PERFOMING THE ANTIMICROBIAL SUSCEPTIBILITY TEST

 Prepare Mueller- Hinton Agar (MHA) as described by
manufacturer.
 Pour MHA in petri dishes, let it set and label.
 Dip a cotton swab in the E. coli inoculum and press tip against
bottle to remove any excess. Repeat for the rest of the bacterial
suspensions.
 Swab the whole agar surface and rotating the plate to as to prepare
a lawn culture.
 Dip (holding with forceps) the prepared paper disks in respective
prepared plant extract concentrations.
 Carefully and aseptically place the paper disks onto the pre-
inoculated MHA plate. Press them down lightly with the forceps.
 Prepare a negative control by inoculating 1 or 2 paper disks with
distilled water and place on the pre-inoculated MHA plate.
 Prepare a positive control by placing these antibiotic disks:
penicillin g and tetracycline. This can be done by impregnating
these antibiotic solutions on the paper disks and place on pre-
inoculated MHA plate.
 Cover and incubate the plates inverted at 37oC for 24 hours.
 Measure and record the zones of inhibition in millimetres (mm) if
any.
 NB this test will be carried out in triplicates.
7. DETERMINATION OF MINIMUM INHIBITORY

CONCENTRARION (MIC)
 For both methanol and water extracts, prepare dilutions of 6.25
mg/ml, 12mg/ml, 25mg/ml, 50mg/ml, 100mg/ml, 200mg/ml and
400mg/ml.
 Dissolve 6.25mg (0.00625) of plant extract in 1ml of distilled
water to make a concentration of 6.25mg/ml.
 Repeat the process to prepare the rest of the extract concentrations.
 Place 0.5g of the plant extracts each in a different test tube.
 Add 1ml of nutrient broth and 0.5ml (standardized bacterial
suspension in saline as described above 4) of the test organisms in
each test tube containing the plant extract (each bacterium into a
different test tube).
 Thus on the negative control, do not add plant extract.
 Swirl slightly to mix and incubate aerobically at 37oC for 24 hours.
 After 24 hours, check the test tube (for all bacteria) with the lowest
concentration in which bacterial growth is absent. This will serve
as the MIC.

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MATERIALS

2. SCREENING FOR PHYTOCHEMICALS

 Fehling’s test for reducing sugars

 Salkowski test for terpenoids

 Sodium hydroxide test for flavonoids

 Borntrager’s test for anthraquinones

 Foam test of saponins

 Dragendorff’s test for alkaloids

 Keller- Killians test for cardiac glycosides

 Ferric chloride test for tannins

4. PREPARATION OF BACTERIAL INOCULUM

5. PREPARATION OF PLANT EXTRACT CONCENTRATIONS

 Prepare concentrations of 25mg/ml, 50mg/ml, 100mg/ml,

6. PERFOMING THE ANTIMICROBIAL SUSCEPTIBILITY TEST

7. DETERMINATION OF MINIMUM INHIBITORY

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