Protocol from Matt Ryder 2007-2008 bioactivity assay (Ryder UHC thesis).
Nisin Activity on PS Microspheres
General Schedule
Day Before: Date __________
- Make MRS Broth (change bi-weekly):
Vol __________ Date __________
- Make overnight Pediococcus culture (16 hr incubation):
Time/Date Start __________ Finish __________
Vol __________
OD600 __________
rpm __________
- Incubate microsphres with F108 (16 hr min):
Start __________ Finish __________
Day of Test: Date __________
- Dilute 100x the overnight Pediococcus culture: OD600 __________
- Make MRS agar
Total number samples: __________ MRS Gel Vol: __________ ml
- Make MRS agar tubes
Total number of tubes: __________
- Incubate microspheres with nisin (1 hr) Date __________
Perform Colony Count Assay
Performed Number __________ Date __________
Day 1: __________
Day 7: __________
Day 14: __________
Day 21: __________
Day 28: __________
*Day 35: __________
Inventory Check
- PS microsphers, (Seradyn, Part No: 81002497100290, 1.15 µm, 10% solid. In refrigerator.)
- Pluronic F-108 Solution (5% in refrigerator Lab 300.)
if diluted note here: __________
- South African nisin solution (-80°C freezer)
- 0.01 M phosphate buffer. 0.2 µm filtered
Phosphate buffer is NOT PBS
- Screw-top test tubes
- Pediococcus Stock Solution (FBB 612, In -80°C freezer)
- Lactobacilli MRS Broth, (EMD, Product No: 1.10661.0500)
- Agar, Granulated (Difco, Product No: 214530)
- Petri Dishes, 100 mm in diameter.
Number of dishes: __________
- Serum nedded: __________ ml
Preparation
- Make sure incubator is on 37°C.
- Make sure steam valve for autoclave is open 1 hr before use.
- Wash hands carefully with soap and 70% alcohol. Put on gloves.
- MRS Broth:
Add 52.2g of MRS to every 1 liter of RO water. Heat to dissolve MRS powder, then take to the autoclave at 121°C for 20 min sterilizing and 10 min drying. Run on liquid setting.
Vol made: __________ L
- Overnight Pediococcus Culture:
Take Pediococcus glycerol stock from -80°C freezer. Dip a loop of Pediodoccus in a 50ml Falcon test tube contianing 10ml of MRS broth. Incubate at 37°C for 16hrs. Store at 4°’^C’ immediately after use.
Date/Time Start: __________ Finish: __________ OD600: __________
- 100x Dilution of Pediococcus:
Dilute Peidococcus for neccessary total volume.
Stock Vol: __________ Diluent Vol: _________
Vol made: __________ Date: __________
- MRS Agar Solution:
Add 15g agar to every 1 liter of MRS broth. Heat to dissolve powder, then take to autoclave at 121°C for 20 min sterilizing and 10 min drying. Run on liquid setting.
- Glassware:
Autoclave necessary number of screw-top glass test tubes and flasks with agar solution.
- MRS Agar tubes:
Pipette 12ml of MRS agar solution into each autoclaved screw-top tube. Tubes may be stored at 4°’^C’ for 2 weeks.
Date/Time made: __________
Preparation of Pluronic F-108 Coated PS Microspheres
- Mark 40ml Oakridge tubes to organize for later testing, according to formulation. Add F-108 to beads and incubate overnight — 10% total volume final solution of 5% F108. Date/Time: __________
- After incubation, vortex the tubes for 30 seconds to well-mix the microsphere solution.
- Spin down microspheres at max rpm for 15 min in swing arm centrifuge (if not available, use centrifuge in Gleeson 003).
- Remove supernatant and fill tubes with 0.01M PBS (possibly with NaCl?) up to 30ml.
- Vortex tubes to disperse microspheres again.
- Repeat steps 3-5 twice more.
- Spin down microspheres at max rpm for 15 min, remove supernatant.
- Fill tubes with 0.01M PBS up to 30ml.
Preparation of nisin loaded Microspheres
- Add nisin and other constituents according to experiment, except serum.
- List ingredients and quantities thereof to prevent forgotten material
- Constituents added: _________________________________________________
- Mount samples on rotator. Incubate for 1 hr at room temperature.
1st Washing
- Spin down microspheres at max rpm for 15 min.
- Remove supernatant.
- Fill tube to 30ml with 0.01M PBS.
- Repeat steps 1-3 twice more.
- Let nisin coated microspheres stand for 1 hr.
- Make sure MRS agar solution tubes are standing by in the 40°C water bath. This takes a long time. Start far in advance (approximately 4-5 hours).
- Clean incubator with 70% alcohol before incubation.
2nd Washing
- Spin down microspheres at max rpm for 15 min.
- Remove supernatant.
- Fill tubes to 30ml with 0.01M PBS.
- Repeat steps 1-3 twice more.
- Remove supernatant from the tubes of the previous step. Add buffer and/or serum according to formulation.
- Vortex (1 min) and sonicate (5 min) samples to disperse beads. Put samples on rotator and incubate at 37°C until designated test day.
Date/Time: __________
Assay of Microsphere Acitivty
Plate count Method
- Spin down incubated beads and remove supernatants. Wash, as in previous procedure, microsphere samples three times. Final nisin concentration in worst case: _________
- Add 100x dilute Pediococcus culture up to 1.5ml and disperse the 1ml (perhaps 1.5ml) dead sample by vortexing.
- Add another 1.5ml 100x dilute Pediococcus culture and sonicate for 3 min.
- Pipette solution into 15ml Falcon plastic tubes.
- Add 7ml of 100x Pediococcus culture to reach a 10ml volume.
- Rotate tubes at 37°C for 4 hours.
- Take out 4-hr incubated tube; disperse contents by vortexing.
- Add 1.0ml of of the 4-hr culture to 9ml of MRS broth to achieve 1000x dilution.
- Add 0.5ml of well-mixed 4-hr, 100x diluted samples at the center of Petri dish.
- Obtain a MRS agar tube from water bath and pour on top of the microsphere-containing solution.
- Plate the solution using either the spin or pour plate method (see Plating).
- Count the colonies on the plates. Record data in Excel spreadsheet. (Determine a useful range of colonies?)
Total 100x dilute Pediococcus solution needed = 150ml
Total MRs broth needed = 500ml
Plates Needed = 21
MRS Agar needed = 400ml (15ml/plate)
Experimental treatments and controls: List explicitly so they are consistent and not forgotten
