Bacterial propagation

Stock culture of Staphylococcus epidermidis

1. Prepare media 10 mL according to manufacturer’s protocol. Once media is fully dissolved in HPLC water autoclave for 30 min at 121 °C. Let media cool to room temperature.
2. Open vial. Withdraw 0.5-1.0 mL of media and rehydrate entire pellet.
3. Aseptically transfer aliquot back to broth tube. Mix well.
4. Incubate at 37 °C for 24 hr.
5. Add glycerol to achieve a final concentration of 20% v/v.
   1. Prepare 40% glycerol solution and sterilize in autoclave. Let cool to room temperature.
   2. Add a volume of sterile 40 % glycerol to equal volume of culture broth to give a final glycerol concentration of 20% v/v.
6. Aliquot 1 mL into sterile cryogenic vials.
7. Place cryogenic vials in refrigerator for 30 min to permit equilibration between cells and suspending medium.
8. Store in -80 °C freezer until needed.

Working culture of S. epidermidis

1. Thaw a vial of stock S. epidermidis. On an already prepared media plate, streak plate to get an isolated colony. Culture overnight at 37 °C.
2. This is the working culture and should be sealed with Parafilm then stored upside down at 4 °C. It will keep for a few weeks.
3. Subculture by selecting a single colony and inoculating 6-10 mL sterile media. Culture overnight.

Preparation up to start of assay
S. epidermidis standard curve

1. Create a subculture of S. epidermidis.
2. Make a series of dilutions. Measure absorbance at 490 nm.
3. For each dilution, mass a 1 mL microfuge vial, add 1 mL of dilution solution and re-mass.
4. Dry in vacuum oven then remass.

Preparation up to start of assay

Silica surface preparation

1. Piranha wash: Place wafers in large, deep Petri dish. Securely place Petri dish in hot bath (80°C). Cover wafers with 5 H₂O: 1 NH₄: 1 H₂O₂ base solution. Let sit in hot bath for 10 min. Remove Petri dish from hot bath, cool 15 min. Remove base solution and rinse wafers with water (in Petri dish). Remove water from Petri dish. Place back in hot bath. Add 5 H₂O: 1 HCl: 1 H₂O₂ acid solution to Petri dish. Let sit 10 min. Remove from hot bath, cool 15 min. Rinse with water thoroughly.
2. OSCAR Silanization: Rehydrate silica gel trap. Make sure there is enough nitrogen. Blow dry each wafer individually than place each in OSCAR set up. Seal OSCAR and let nitrogen flow through system (valve set to “bypass”) for approximately 1 h to equilibrate the system and remove any excess surface water. Draw up approximately 0.5 mL of TCVS into a plastic syringe with a pipette tip (should fill the tip and part of the syringe). Inject into reactant chamber through the septum. Leave the syringe there. Switch valve to “vapor.” Let react for 4 h. Set to “bypass” for a short time before turning off (removes excess anything).
3. PEO-PPO-PEO covalent attachment: Prepare a sterile solution of 0.5 mg/mL F108 in PBS. Remove wafers from OSCAR and rinse with water. Place wafers in sterile Petri dish. Add solution to Petri dish, enough to cover. Orbital rotate for 4 h or more. Send to radiation center for 0.3 Mrad of gamma irradiation.
4. Nisin entrapment: Prepare a sterile solution of 0.5 mg/mL nisin in PBS. Rinse wafers with water. Place wafers in a new sterile Petri dish. Add nisin solution, enough to cover. Orbital rotate for 4 h or more. Rinse with water before use.

Catheter segment surface preparation

1. Segmenting and cleaning: Using a razor blade cut the catheter into 3 segments per catheter. The segment should be long enough to stick out of the agar plate… when the time comes for that. The catheters are already sterile in packaging and should be mostly clean. Rinse micropipette tips in ethanol. Stick catheter segments on the end of the micropipette tips. Rinse catheters with ethanol then HPLC water.
2. PEO-PBD-PEO covalent attachment: The PBD already has a double bond that can be broken and covalently bound to the surface via gamma irradiation. Prepare a 0.5 mg/mL solution of Hillmeyer triblocks in PBS. Place catheter segments in microfuge vials. Add solution, enough to cover catheter segment. Rotate (orbital or rotisserie?) for 4 h or more. Send to radiation center.
3. Nisin entrapment: Prepare a sterile solution of 0.5 mg/mL nisin in PBS. Rinse catheter segments with water. Place catheter segments in new sterile microfuge vials. Add solution, enough to cover segments. Rotate 4 h or more. Rinse with HPLC water before use.

Incubator set up

1. Place silica wafers in sterile 24-well plates. Place catheter segments in sterile cryogenic vials. See the diagrams at the end for set up/arrangement.
2. Add blood plasma to each sample well. See blood plasma preparation.
3. Place the orbital shaker in the incubator, and the well plates on top of the orbital shaker. Keep it on low RPM throughout the test.

Sample testing during the assay

Sample testing during the assay

Blood plasma preparation

1. Get blood sample from Hemostat. Distribute into wells. Volumes/well TBD.
2. Every _______ days, remove plasma and replace with new plasma.

Media preparation

1. Dissolve 8 g in 1 L of purified water. Completely dissolve (use gentle heat) before autoclaving. Autoclave media for 15 min at 121 °C to sterilize.

Overnight culture

1. Prepare 20 mL of sterile media. Place 6 mL into 3 culture tubes.
2. Aseptically inoculate each tube with an isolated colony from the working culture.

Plate preparation

1. Prepare 1% (10 mg/mL) agar solution in nutrient broth.
2. Sterilize at 121 °C for 15 min.
3. Add volume of liquid culture to nutrient media to achieve 0.1% inoculation (based on standard curve). Do this when media is cooled enough to be warm to the touch or room temperature.
4. In the laminar flow hood, distribute incolulated media to sterile Petri dishes. For the medium sized Petri dishes use 3.2 mL per plate to achieve a 5 mm depth.
5. Let set in the refrigerator.
6. Place one wafer upside-down in the center of the Petri plate.
7. Place covered plate in 37 °C incubator overnight (or for 2 days, depending on growth).

Data collection

1. Measure kill zone: average diameter, average ‘radius’ from edge of the wafer.
2. Take pictures.