UA Biophysics:Protocols:Plate turbidostat

MEDIA

• To LB media add Ampicillin at 100 mg/mL. For growing E.coli cells with plasmid pUC19-COLE1.

• Ampicillin, 1000X (100 mg/mL)

Dissolve 5 g ampicillin (sodium salt) in 50 mL MQH2O. Filter sterilize using syringe filters into 15 ml conical tubes. Label and keep at -20 °C. Leave one tube at 4 °C for immediate use.

• To LB media add Kanamycin at 20 mg/mL. For growing E.coli cells with plasmid pGWB5-R1.

• Kanamycin, 1000X (20 mg/mL)

Dissolve 1 g kanamycin in 50 mL MQH2O. Filter sterilize using syringe filters into 15 ml conical tubes. Label and keep at -20 °C. Leave one tube at 4 °C for immediate use.

PLATING E. COLI CELLS

• 1. Place plates on bench top to let them come to room temperature.
• 2. Using an inoculating loop (either metal burner-sterilized or plastic disposable), lightly scratch the surface of the frozen glycerol stock. DO NOT ALLOW STOCKS TO THAW.
• 3. Streak the loop across the plate.
• 4. Return the stock to the freezer immediately.
• 5. Incubate LB plates with AB at 37 °C overnight.
• 6. Store plates in refrigerator for no more than one week. Wrap plates in Parafilm to keep them from drying out.

GROWTH PROCEDURE

• 1. Using sterile serological pipet, add 5 mL LB plus appropriate antibiotics to a loose-capped culture tubes.
• 2. Inoculate the tubes with a single colony using a disposable loop, sterile toothpick, or pipette tip.
• 3. Incubate with 250 rpm shaking overnight at 37 °C. Do not tighten the lid of the tube; bacteria need oxygen to grow.

MORBIDOSTAT USE

• 1. Check the connections between the motherboard and the mechanical parts (fluid pumps, UV lamp and plates structure) then plug in the electrical source.
• 2. Turn On the computer and open the LabView program called Turbidiostato. The UV light should turn on, so try to place the UV lined filtered incubator on the turbidostato before turning on the computer.
• 3. Connect the hole pump system with silliconed hoses. Besides the ray emisor on the plate side is a slot to fit the hose using a U-shaped guide. The entrance hose must be quite separately from the culture and the outing hose just in contact to maintain enough media volume.
• 4. For eight-plate experiments use about 3L of the appropriate media (Aprox. 400mL by plate). After connecting the media to the pumps system, fix the plate media volume using the pump controls in the software, for putting in place clean media use between 10 to 20 seconds depending on the length of your hoses.
• 5. Set the conditions for your UV light during your experiment, i.e. 25 min ON and 40min OFF. Run your experiment (check time interval for data acquisition and number of data you want to average in every take, the software would ask you to turn on the mechanical device) for around an hour and then inoculate your growth culture in every plate using a burner.
• 6. Turn on the incubator a set the appropriate temperature according to your need.

General suggestions

One of the most important matters to take into acount before starting your experiment is trying to guarantee the conditions to not comiting errors, this is critical because you usually run long term experiments (about months) and any mistake in the begining could make you waste lots of time and even mess up your hole assay. For this reason it is important to always check media pumps work out and review the settings for your UV lamp. It is also important to check the time interval for data acquisition and amount of data to take in every measure, the program does a mean of this sampling and report one datum. When need to sample your culture, take 100 mL of clean media dilute it on your plate and then take 100mL of culture (Remember always using the burner and checking the UV light not be ON, during this procedure).