COCK RESEARCH GROUP 2017

EXTRACT PREPARATION

…When plant material arrives in unprocessed form (tree cuttings etc.) …

1. Dry materials if required (‘wet’ or sticky materials will result in loss of desired compound and logistical issues
during the extract preparation process).
2. Grind materials to a powder using a coffee/ spice grinder. Ensure it is cleaned thoroughly with water and
ethanol (80%) between different materials.

…When plant materials are powdered…

3. ‘Tare’ a 50ml tube (red cap) on a 4 point balance. Weigh out 1g of plant material powder into a 50ml tube
per extract.
 This works out to 1g per 10ml; essentially, 1 extract (i.e. Acacia in water) = 1g which translates to
10ml of extract
4. Fill the tube to the max line with desired solvent (water, methanol, ethanol…etc.)
5. Shake thoroughly until mixed. Place in 4C storage overnight. Ensure correct labelling (EXTRACT NAME,
initials, date, solvent type)
6. Vaccuum Filter:
 Set up vacuum filtration apparatus (sketch when shown). Filter into 50ml falcon tubes (red cap) –
use a new filter paper per extract.
7. Move extracts to ‘drying’ step:
 Organic solvents (i.e. ethanol, methanol, hexane etc.) need to be moved to the incubator where they
need to be left open to the air to dry over 1 – 3 days. A resin will form on the bottom of the tube –
this is the extract concentrate.
 Water dissolved extracts will need to be freeze-dried: move to -80C freezer, and freeze dry using
correct apparatus under supervision (this will take several hours)
8. Once dry:
 Label 1 10ml falcon tube per extract; weigh said empty tube without the lid & record weight
 Transfer extracts into respective tubes: wash with solvent (approx. 3 – 9 ml). allow 10ml tubes to
dry
9. Once FINAL dry step complete:
 record final weight (tube + extract)
 Add 5ml de-I. water (& DMSO ?)
 Sonicate
10. Syringe filter (0.22um & 0.45um)
11. Top up with de-I. water to 10ml extract per tube.
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ANTIBIOTIC PREPARATION

…Need to use a 0.01mg/ml concentration of antibiotic made with sterilised (autoclaved) water ONLY. Antibiotics are
usually available in powdered form; follow protocol below. If in liquid form, make appropriate dilution….

1. Weigh out 10mg antibiotic into 10ml falcon tube. Top up to 10ml with autoclaved water. Label
appropriately… This is your stock antibiotic of 1mg/ml concentration, to be store at 4°C with parafilm
covering when unused.
2. Pipette 100ul of 1mg/ml Ab into a sterile Eppendorf tube. Add 900ul water. Vortex & label appropriately
(0.1mg/ml) … This is your intermediate Ab. Which you will use to make your working Ab solution.
3. Pipette 100ul of 0.1mg/ml (intermediate Ab solution) into another sterile Eppendorf tube & add 900ul
autoclaved water. This is your working Ab solution (0.01mg/ml), to be used in assays.

PATHOGEN PREPARATION

…You are provided with a bacterial culture – the following protocol will explain how to prepare a 1:100 McFarland
solution. Note that this is per bacteria; if working with X number of bacteria, then X number of McFarland solutions
need to be prepared. Note also that when working with more than one bacteria, there is to be strictly one bacterial
culture per plate for the purposes of liquid dilution assays (to avoid cross contamination). Use aseptic technique …

1. Label three schott bottles & fill each with 40ml autoclaved nutrient broth:
A – Primary culture: this is your personal culture of bacteria, used to make standards etc.
B – Standard: this contains a standard amount of bacteria to then make a 1:100 dilution
C – 1:100 McFarland: working bacterial solution (1:100) to be used in assays

2. Take enough of the lab cultured bacteria (approximately 1ml) & inoculate in schott bottle A until slightly
cloudy. Incubate for 2 days – this is your primary culture to be prepped once.

3. Take enough of A (primary culture), and inoculate into B until slightly cloudy– this is your standard solution
to be prepped per assay.

4. Take 400ul of B and inoculate in 40ml nutrient broth to produce C – your 1:100 McFarland culture or
‘working bacterial solution’

INT PREPARATION

…Make a solution of 0.08g INT powder in 200ml autoclaved water. Store at 4°C …
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LIQUID DILUTION ASSAY

…Label the sides of each plate prior to use. Find attached plate maps – fill these out prior to starting an assay for
your own ease. Perform assays in duplicate. Use aseptic technique …

1. Using a multichannel pipette, add 100ul of nutrient broth to all wells of the plate. Note that you will likely
be using a number of plates – all controls go on the last plate ON THEIR OWN. Controls are single shot
assays. This should essentially be the layout of your plate map:

1 2 3 4 5 6 7 8 9 10 11 12

A X1 X1 X1:Ab1 X1:Ab1 X1:Ab2 X1:Ab2 X2 X2 … CC +ve -ve

CC = Culture Control X1 = extract 1 (extract only)

-ve = Negative control (contains water) X1:Ab1 = extract1 / antibiotic 1 combination
+ve – Positive control (contains pure Ab)

2. To wells of row 1 as per your map:

i. Extracts: 100ul of mapped extract to all extract only rows, 50ul of mapped extract to all
combination rows
ii. Antibiotics: 50ul of mapped antibiotic to all combination rows, 100ul of mapped antibiotic per
mapped antibiotic (positive) control
iii. Controls: add 100ul of nutrient broth to culture control well as mapped, add 100ul autoclaved
water to negative control well as mapped, ensure that all positive controls are completed.
3. Using a multichannel pipette, serially dilute the rows down the plate by taking 100ul from row A and adding
it to row B, pipette mix, and dilute to row C and so on. Discard the last 100ul and change tips before
performing the next serial dilution.
There are 12 columns so for each plate perform the first with 8 tips and the second with four tips.
4. Pour your 1:100 McFarland solution into a petri dish within the aseptic zone. Using a multichannel pipette,
add 100ul of working bacterial solution C into each well of each plate – there is no need to change tips
provided you do not touch the solutions of any wells.
5. Incubate at 37°C for 24 hours
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6. Results: add 40ul INT solution to each well using a multichannel pipette & wait for colour development (5
minutes)
pink = live bacteria, no inhibition
clear = dead bacteria, inhibition
Note down which wells have turned pink by marking an X on your map
COCK RESEARCH GROUP 2017