2nd FYP Meet-up schedule with Mr Suresh

Hi folks.... There will be a scheduled FYP follow-up meeting on the Friday, 25-Jun-2010, at 14oo hrs @ Bioengineering lab...




Jasmine

TimeLine for preparation of media and sterility check

1) Preparation of RPMI-1640 basal medium at 2/June/2010 @ 15:15hrs. Venue: Bioreactor Lab.

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

Protocol for Preparation of RPMI-1640 Medium ( Complete ) from basal medium

MATERIALS NEEDED:

Basal RPMI-1640 medium

Foetal Calf Serum

200mM L-Glutamine Sterile stock

Penicillin/Streptomycin 100x stock

500mL Duran-Schott bottle steriled

METHODS

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

Protocol for Preparation of RPMI-1640 Medium ( Basal )

MATERIALS NEEDED

37 degrees celcius incubator

Powdered RPMI-1640

Bottle-top 0.22 micro-meter filtration unit with 45mm outlet

Magnetic stirrer, stir bars ( x4 )

Pyrogen free nanopure water

Beaker 1Litre ( x4 )

Analytical balance

Disc filter 0.22 micro-meter

Syringe

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


METHODS

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 ).


FOLLOW-UPs

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

Advance notice of absence from FYP laboratory research on June

Please take note of this early notice of absence from lab work on the following dates and timings:

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.


Jasmine
@ 17:46hrs

Confirmed dates for stock taking & FYP introductory meeting

The following dates have been confirmed:

1. Stock Taking : Tuesday 25/May/2010 ( 11:00 hrs - 12:00hrs ).
2. Introductory Meeting with Mr Suresh: Wednesday 26/May/2010 ( 16:00 hrs - 18:00 hrs ).

Venue: Level 4 research lab.

Updated @ 22:30hrs

Past-year FYP report reading on ( The Immune System of Chicken )

There are two types of immune mechanism in birds:
( 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

Literature Search on : IMMUNOMODULATION OF THE AVIAN INNATE IMMUNE RESPONSE TO PREVENT BACTERIAL INFECTION IN POULTRY

Please visit the web page below:
https://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=150334&pf=1


21:22hrs

Tentative proposed stock taking date

The proposed stock taking date will be at 25/May/2010 at 10:45 hrs till 12:00 hrs. Timing yet to be confirmed by Yee Cheng.

Time Line:

10:45 hrs : Look for Yoke Fun to get left over stocks from past year FYP and double confirm quantity to transfer to research lab.

11:00 hrs - 12:00 hrs: stock taking at Bioengineering lab and Bioreactor lab.


updated @ 21:15hrs

FYP Introductory Meeting Postponed.

The meeting have been postponed till next week. Actual date and timing yet to be confirmed.

updated @ 21:10hrs

Stocks currently known to be available.

Location: In -80 degree celcius freezer.

1. Gingseng extract stock root & stem ( left over from 2008 FYP )
2. LingZhi extract in basal medium x 4 ( Left over from 2009 FYP )
3. LingZhi in 1x CM ( 100k ppm ) 10k, 1k & 50ppm ( Left over from 2009 FYP )
4. Penicilin-Streptomycin-Ampicilin antibiotic x 3 ( Left over from 2009 FYP ) supplied by Yoke Fun
5. BSA ( check quantity )
6. 2-Mercaptoethanol
7. 75% Ethanol


updated @ 21:22hrs

Postpone schedule for stock-taking and first experiment

1. The schedule for stock taking will be postponed till next week dued to extreme packed schedule of supervisor and TSO.

2. Wednesday morning lab session will be postpone to friday 21/May/2010 at 1530hrs with Mr Suresh. Wednesday 19/May/2010 morning TSO and Mr Foo would not be available.


Jasmine
21:14hrs

T-cells

For more information on T-cells!
http://www.copewithcytokines.de/cope.cgi?key=T-cells

Have a good weekend!(:

Notice on Leave of Absence

Please take note that I will be unable to be in the laboratory on wednesday 19/May/2010 after 1300hrs due to an approved Leave of Absence ( LOA ) to attend doctor's appointment.

Any delays regretted.


Jasmine
20:14hrs

Tentative dates for Laboratory work

1. Stock taking: Monday 17/May/2010 at 0830hrs.

2. Preparation of media / propagation of cell line : Wednesday 19/May/2010 at 0830hrs till 1130hrs.


Jasmine
20:11hrs

General Experiment Protocol for laboratory work: Thawing of Cryopreserved Mammalian Cells

Acknowledgements: CP2022 /Thawing/FTT/2010-11

INTRODUCTION

Care must be taken when recovering cryopreserved animal cells as animal cells are shear sensitive. Incorret technique in thawing of the sample can lead to cell damage thus causing a low yield of viable cells.
Geral Rule of the Thumb: Slow Freezing, Fast Thawing.
Strict sterility must be observed throughout the process.

PRECAUTIONS TO TAKE

1. When removing cell line from liquid nitrogen, make sure all proper PPEs are worn as liquid nitrogen has a temperature of -190 degree celcius and can cause severe burns.

2. To prevent contamination to cell line, before cell-enumeration step, all procedures must be carried out in the Biosafety Cabinet ( Class II ).

3. Enumeration of cells to be done within 10 minutes to prevet cells from attaching to the plastic vessel.

MATERIALS NEEDED

Frozen adherent cell line in cryovial
Sterile complete RPMI- 1640 media + 10% Foetal Calf Serum ( FCS )
Water-Bath, 37 degree celcius for thawing
Neubauer haemocytometer for cell enumeration
0.2% - 0.4% w/v trypan blue in 0.85% w/v saline
Micropipettes P20, P200 and sterile tips
Sterile pipettes 1mL, 10mL.

EXPERIMENTAL PROCEDURES

1. An ampoule of cryopreserved cells was removed from the liquid nitrogen container and placed immediately into a water-bath pre-set to 37 degree celcius. Take notes of precautions.

2. The contents of the cryovial was thawed by shaking it at the same time keeping the bottom half of the vial submerged in the water-bath. Precaution taken not to let the water touch the cap of the cryovial. Optimal thawing time should be less than 1 minute.

3. As soon as the last trace of ice vanishes, the cryovial was removed quickly from the water-bath and the neck of the cryovial was sprayed with 70% ethanol.

4. The contents of the cryovial was transferred into a tube containing 9-10mL of cold complete RPMI-1640 medium ( RPMI-1640 + 10% FCS + 2mM L-Glutamine ). The contents were mixed gently and spinned at 1,500 rpm for 6 minutes.

5. The supernatant was decanted and cell pellet was resuspended in 5ml of fresh culture medium. A sterile aliquot of about 0.1mL of cell suspension was removed and placed into an Eppendorf tube for cell counting ( enumeration ). Sterility unnescessary after this step.

6. 2 drops of 15 micro-litre of dye was added onto a Nescofilm and the first drop was mixed with the cell suspension before loading the chamber of the Neubauer haemocytometer using a P20. The cells were enumerated and the viability of the recovered cells were accessed.

7. Seed at least 1,000,00 cells/mL in a total volume of 10mL with complete RPMI-1640 medium in a T-25 flask or 60mm petri-dish

8. Cell growth to be examined daily and fresh medium to be replenished every two days as necessary.



Jasmine
14/May/2010
20:06hrs

Phone Conference with Mr Suresh ( 14 / May / 2010 ) 13:15hrs

( A ) THINGS DISCUSSED:

1. Read past-years 2008 & 2009 student FYP reports. Get reports from Mr Foo.

2. Perform stock checking ( take note of complete stocks ). Get help from laboratory technician. Consult Mr Foo about cell line stock.

3. Next wednesday 19-May-2010 start cell line propagation & freeze down cells attained in liquid nitrogen. Take note of the specific concentrations of the cells attained.

4. Start preparing RPMI media.


( B ) FOLLOW-UP TAKEN:

1. Checked with Mr Foo about the cell line. Confirmed in stock and well frozen under crytopreservation using liquid nitrogen.

2. Asked Mr Foo for the past-year reports. Will get it from him once he found it.

3. Initiated tentative stock taking and propagation dates with lab technician by dropping her a short memo as she is not at her desk. Dates yet to be confirmed.


( C ) COMMENTS:

1. Laboratory follow-up needed for section ( A ) point no. 3

2. Confirmed dates for stock taking and propagation will be updated on later date.





Jasmine

Announcement - Phone conference schedule and 1st lab meeting schedule with Mr Suresh

1. The confirmed schedule for the phone conference with Mr Suresh will be on Friday 14-May-2010 at 1315hrs.. Pls meet at library 4th level.

2. The confirmed introductory meeting is schedule on next friday 21-May-2010 at 1530 hrs at level 4 research lab. No lab coat required.

thanks..

Jasmine

Introduction

This blog is meant for the project purposes and no personal stuffs should be posted here.

Day to Day lab work , results, comments and follow-ups will be posted here. This would be an alternative platform to access the log-book in case the person in-charge of the log book is on Medical Certified Leave of Absence.

You can update the literature search in this blog indicating in the title : ' literature search article.......'

If you have comments on any post pls feel free to leave your comments but dun forget to indicate your name below your comments....


Thanks and have a happy FYP... cheers....

Jasmine