Sinton:Protocols/Ferric BG-11 Media preparation

Safety

• Safety goggles
• Nitrile gloves
• Lab coat
• Biosafety cabinet
  

Consumeables

• Weigh boat
• Autoclave tape
• Syringe
• Needle
• Pipette (4)

Equipment

• Clean 1 L bottle
• 10 100mL bottles
• Scoopula or similar device
• Precision Mass Balance

Chemicals needed

• Stock Solution I,II, IV, V
• Ferric ammonium Citrate (check bottle)
• Citric Acid (check bottle)

Protocol

• Clean all Glassware!!

• Fill 1L bottle to 500-750mL with DI water

• Add the Following stock solutions to the DI water in the 1 L bottle

     *10mL Stock I 
     *10mL Stock II
     *40mL Stock IV
     *1mL Stock V

• Add 6mg of Ferric ammonium Citrate
• Add 6mg of Citric Acid

     *THESE STEPS ARE 'difficult' due to the small quantities if you can weight it out well done
     OR
     *Weigh out 30mg in a weight boat using a precision mass balance
     *Add 5mL of of Water to weigh boat, remove 1mL and add to 1L bottle
     NOTE: This would be Stock III anecdotally, this solution was very susceptible to contamination due to Vitamin C presence

• Top up Media bottle (1L) to 1L

• SHAKE IT don't BREAK IT

• Dispense Media into 100mL bottles, filling precisely to either 100,80 or 60, this is to enable easier addition of any antibiotic agents

• Autoclave bottles

• Once cooled, transfer bottles from autoclave to biosafety cabinet

  USE CAUTION IN THE NEXT STEP (SHARPS)

• Extract 6mL of Kanamycin (10mg/mL) and tranfer to weighboat or test tube etc...

• using a pipette add 5microliters/mL to each bottle

• Seal bottles and remove from biosafety cabinet

• label bottle

   *Date
   *FBG11
   *Kanamycin concentration used (5uL/mL)