37 degrees celcius incubator
Bottle-top 0.22 micro-meter filtration unit with 45mm outlet
Magnetic stirrer, stir bars ( x4 )
Pyrogen free nanopure water
Beaker 1Litre ( x4 )
Disc filter 0.22 micro-meter
Sterile 15mL tubes ( x8 )
Sterile 500mL Duran-Schott bottles ( x9 )
1N HCl , 1N NaOH
7.5% w/v Sodium Bicarbonate Solution
200mM L-Glutamine solution
9 Sterile universal bottles
9 LB broth culture medium
1. Sterilise the BSC by UV irradiation for 10 to 15 minutes and then clean the working space using 70% ethanol. Aseptic guidelines must be followed everytime while doing sterile work on cells.
2. Measure out 3200mL of clean pyrogen- free water ( nanopure water ) for 4 litres final volume of medium. Split equal volume of 800mL into each 1 litre beaker ( x4 ).
3. Empty the contents of a bottle of RPMI-1640 ( see how much is needed for 4 litres ) and dissolve the powder well with magnetic stirrer. Adjust the pH with 1N HCl to pH 4.0 to completely dissolve the medium before rising it to pH 7.0 with 1N NaOH.
4. Prepare 300 grams of 7.5% w/v Sodium Bicarbonate solution and add 106.8mL for 4 litres of final volume of medium. Stir untill dissolved. Adjust the pH to 7.2 with 1N NaOH and top-up to final volume ( 4 L ) with 693.2mL of nanopure water.
For every 1 litre ( x4 ) beaker of medium, 75 grams of 7.5% w/v Sodium Bicarbonate solution is prepared and 26.7mL was added. Top-up with 173.3mL of nanopure water.
5. Sterilise the medium by using a 0.22 micro-meter filtration unit. Screw the bottle-top filter unit onto a clean sterile 500mL Duran Schott bottle. Dispense 450mL aliquots of basal medium into each 500 ml Duran Schott bottle ( x8 ). The remaining 400mL is filtered into ( #9 ) 500mL Duran-Schott bottle.
( Label the Duran bottles #1 - #9 )
6. STERILITY SCREEN: Aseptically transfer 2mL each of the filtered medium into ( a ) a sterile universal bottle ( label #1 - #9 accordingly ) and ( b ) a LB broth culture medium ( label #1 - #9 accordingly ). Incubate the tubes at 37 degrees celcius for up to 4 days. Store remaining medium at 4 degrees celcius. ( Parafilm the remaining bottles ).
7. Prepare 80mL of 200mM L-Glutamine Soltuion for 4 litres of medium prepared. Filter through 0.22 micro-meter filter disc using the syringe. Dispense 10mL aliquot each into sterile 15mL ( x8 ) screw-capped tubes. Store in -20 degree celcius freezer. ( Parafilm every tubes ).
1. Record results for sterility check for day 2 and 5 respectively.
2. If sterility test passed, continue to make RPMI-1640 complete medium.
updated @ 12:44 hrs