Tuesday, June 22, 2010
Monday, May 31, 2010
2) Sterility check Day 2 for RPMI-1640 basal at 4/June/2010 @ 13:00hrs. Venue: Bioreactor Lab.
3) Sterility check day 5 for RPMI-1640 basal at 7/June/2010 @ 13:00hrs. Venue: Bioreactor Lab.
5) Prepartion of RPMI-1640 Complete Medium at 9/June/2010 @ 10:00hrs. Venue: Bioreactor Lab...
updated @ 13:06hrs
Basal RPMI-1640 medium
Foetal Calf Serum
200mM L-Glutamine Sterile stock
Penicillin/Streptomycin 100x stock
500mL Duran-Schott bottle steriled
1. Thaw a bottle of Foetal Calf Serum ( FCS ) by placing it in 56 degree celcius water bath for 30 minutes.
2. Complete 400mL ( x10 ) of the RPMI-1640 medium by adding the components as follows:
- Basal RPMI-1640 312mL
- Foetal Calf Serum 80mL
- 200mM L-glutamine strile stock 4mL
- Penicillin/Streptomycin 100x stock 4mL
- TOTAL VOLUME 400mL
NOTE: Glutamine is unstable and should be kept frozen. Half-life in medium at 4 degree celcius is anout 3 weeks. Can be added to medium to be stored at -2o degree celcius ( i.e All complete medium with glutamine added to be stored at -20 degree celcius freezer ). Only the working medium bottle to be stored at 4 degree celcius for use.
Updated @ 12:58hrs
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
Sunday, May 23, 2010
1) 16/June/2010 after 13:00hrs for a diagnosis test at TTSH at 14:30hrs.
2) 23/June/2010 after 14:00hrs for specialist doctor appointment at TTSH at 15:25hrs.
A reminder of this notice will be posted on a later date.
Saturday, May 22, 2010
- Stock Taking : Tuesday 25/May/2010 ( 11:00 hrs - 12:00hrs ).
- Introductory Meeting with Mr Suresh: Wednesday 26/May/2010 ( 16:00 hrs - 18:00 hrs ).
Venue: Level 4 research lab.
Updated @ 22:30hrs
Friday, May 21, 2010
( i ) non-specific ( innate )
( ii ) Specific ( acquired immunity )
The innate immunity of the chicken refers to the natural or inherited ability to resist disease by non-specific mechanisms and this response is systemic.
Examples of innate immune cells: intraepithelial leukocytes, including macrophages, dendritic cells, heterophils, natural killer cells and T-lymphocytes ( Jeurissen and Janse, 1996 ). The innate immune cells have several functions, including the recognition and control of invading pathogens as a first line of immunological response as well as antigen presentation and the activation of the mechanism of acquired immunity.
Innate response is non-specific, the immune cells are not targeted against specific antigen, but instead, it recognises generalised and conserved molecules across many pathogens through pathogen molecular pattern ( PAMP; Humphrey and Klasing, 2004 ). Because of this non-specificity, the innate response is generally faster than the acquired response ( Merlino and Marsh, 2004 ).
Acquired immunity is exquisitely target-specific which has immunological memory.
uploaded by Jasmine @ 21:37hrs