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1. Cisco Certified Network Associate: 640-802
2. CCIE Routing and Switching Written Exam: 350-001
3. BSCI – Building Scaleable Cisco Internetworks: 642-901
4. MCDBA Managing and Maintaining a Microsoft Windows Server 2003 Environment: 70-290
5. BCMSN – Building Converged Cisco Multilayer Switched Networks: 642-812
6. ISCW – Implementing Secure Converged Wide Area Networks: 642-825
7. Designing for Cisco Internetwork Solutions: 640-863
8. MCSA 2003 Implementing Managing and Maintaining a Microsoft Windows Server 2003 Network Infrastructure: 70-291
9. ONT – Optimizing Converged Cisco Networks: 642-845
10. IP Communications Quality of Service (QoS): 642-642
11. TS: Upgrading MCSE on Wndws Serv 2003 to Wndws Serv 2008: 70-649
12. Composite: 642-892
13. Microsoft SQL Server 2005 Implementation & Maintenance: 70-431
14. MCSE 2003 Security Planning Implementing and Maintaining a Microsoft Windows Server 2003 AD Infrastructure: 70-294
15. SECURITY+ CERTIFICATION EXAM: sy0-101
16. Network+ Certification Exam : n10-003
17. VMware Certified Professional on VI3: VCP-310
18. MCSE 2003 Security Planning and Maintaining a Microsoft Windows Server 2003 Network Infrastructure: 70-293
19. Interconnecting Cisco Networking Devices Part 1: 640-822
20. TS: Exchange Server 2007, Configuring: 70-236
21. MCSE Installing Configuring and Administering Microsoft Windows XP Professional: 70-270
22. Troubleshooting Unified Communications (TUC): 642-426
23. MCSE Managing and Maintaining a Microsoft Windows Server 2003 Environment for a W2K MCSA: 70-292
24. TS: Microsoft Office SharePoint Server 2007, Configuring: 70-630
25. CompTIA A+ Essentials Exam: 220-601
26. IP Communications Cisco Voice Over IP: 642-432
27. Interconnecting Cisco Networking Devices Part 2: 640-816
28. Gateway Gatekeeper(GWGK): 642-453
29. MCSE 2003 Planning Implementing and Maintaining a Microsoft Windows Server 2003 Environment for a W2K MCSE: 70-296
30. CCIE Voice Written: 350-030

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A schematic diagram of “Resuscitation of Frozen Cell Lines”

Materials

• Media– pre-warmed to the appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and size of flask to resuscitation into.)
• 70% ethanol in water
• DMSO

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Glass Bottom Dishes
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre labeled flasks
• Marker Pen
• Pipettes
• ELISA plates
• Ampule Rack
• Tissue

Procedure

1. Read Technical data sheet to establish specific requirements for your cell line.
2. Prepare the flasks as appropriate (information on technical data sheet). Label with cell line name, passage number and date.
3. Collect ampule of cells from liquid nitrogen storage wearing appropriate protective equipment and transfer to laboratory in a sealed container.
4. Still wearing protective clothing, remove ampule from container and place in a waterbath at an appropriate temperature for your cell line e.g. 37oC for mammalian cells. Submerge only the lower half of the ampule. Allow to thaw until a small amount of ice remains in the vial – usually 1-2 minutes. Transfer to class II safety cabinet.
5. Wipe the outside of the ampule with a tissue moistened (not excessively) with 70% alcohol hold tissue over ampule to loosen lid.
6. Slowly, dropwise, pipette cells into pre-warmed growth medium to dilute out the DMSO (cell culture flasks prepared in Step 2).
7. Incubate at the appropriate temperature for species and appropriate concentration of CO2 in atmosphere.
8. Examine cells microscopically (phase contrast) after 24 hours and sub-culture as necessary.

Key Points

1. Most text books recommend washing the thawed cells in media to remove the cryoprotectant. This is only necessary if the cryoprotectant is known to have an adverse effect on the cells. In such cases the cells should be washed in media before being added to their final culture flasks. See Protocol 7 for further details.
2. Do not use an incubator to thaw cell cultures since the rate of thawing achieved is too slow resulting in a loss of viability.
3. If a CO2 incubator is not available gas the flasks for 1-2 minutes with 5% CO2 in 95% air filtered through a 0.25m filter.
4. For some cultures it is necessary to subculture before confluence is reached in order to maintain their characteristics e.g. the contact inhibition of NIH 3T3 (Prod. No. 93061524) cells is lost if they are allowed to reach confluence repeatedly.

Scouce: ECACC Handbook Protocol 2

　　”Because I’m a woman,” she told him.

　　”I don’t understand,” he said.

　　His mum just hugged him and said, “and you never will.”

　　Later the little boy asked his father, “why does mother seem to cry for no reason?”

　　”All women cry for no reason,” was all his dad could say.

　　The little boy grew up and became a man, still wondering why women cry.

　　Finally he put in a call to god; and when god got on the phone, he asked, “god, why do women cry so easily?”

　　God said: “when I made the woman she had to be special. I made her shoulders strong enough to carry the weight of the world; yet, gentle enough to give comfort.”

　　”I gave her an inner strength to endure childbirth and the rejection that many times comes from her children.”

　　”I gave her a hardness that allows her to keep going when everyone else gives up, and take care of her family through sickness and fatigue without complaining. “

　　”I gave her the sensitivity to love her children under any and all circumstances, even when her child has hurt her very badly.”

　　”I gave her strength to carry her husband through his faults and fashioned her from his rib to protect his heart.”

　　”I gave her wisdom to know that a good husband never hurts his wife, but sometimes tests her strengths and her resolve to stand beside him unfalteringly.”

　　”And finally, I gave her a tear to shed. This is hers exclusively to use whenever it is needed.”

　　”You see: the beauty of a woman is not in the clothes she wears, the figure that she carries, or the way she combs her hair.”

　　”The beauty of a woman must be seen in her eyes, because that is the doorway to her heart and the place where love resides.”