Aim To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines.

Materials

• Chloros / Presept solution (2.5g/l)
• 1% formaldehyde based disinfectant e.g.Virkon,Tegador
• 70% ethanol in water (Prod. No. R8382)

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Microbiological safety cabinet at appropriate containment level
• Cell Culture Plates(6 well plate, 24 well plate, 96 well plate)

Procedure

1. Sanitize the cabinet using 70% ethanol before commencing work.
2. Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work.
3. Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
4. Whilst working do not contaminate gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding.
5. Discard gloves after handling contaminated cultures and at the end of all cell culture procedures.
6. Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids, cell culture plates) should be wiped with tissue soaked with 70% ethanol prior to use.
7. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
8. Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.
9. After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving.
10. Cell culture discard in chloros (10,000) ppm must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.
11. Periodically clean the cabinet surfaces with a disinfectant such as Presept,Tegador or Virkon or fumigate the cabinet according to the manufacturers instructions. However you must ensure that it is safe to fumigate your own laboratory environment due to the generation of gaseous formaldehyde, consult your on-site Health and Safety Advisor.

    Source: Sigma-Aldrich

Want to become a CCNP? There are six steps to help you becoming a CCNP (Cisco Certified Network Professional) and increasing your chances of capturing the right job and joining network support personnel.

1. Compare professional certification options on the Cisco website. Find out that the CCNP is just one of many professional certifications available. Choose the "Participating in the Program" link to find approved Cisco training facilities and schools in your area whether you've obtained the CCNA elsewhere or prefer to change your current training facility or school.
   
2. Finish curriculum requirements and successfully pass the CCNA exam (and possibly obtain the recommended CCENT training/exam beforehand). Certify your capabilities to install, configure, as well as troubleshoot LANs or WANs and become a CCNP.
   
3. Discover that many traditional colleges offer Cisco networking training; however, some don't have higher level certification courses available. Unearth Cisco-approved training facilities and online courses that typically offer higher level Cisco networking certification training. Evaluate cost, convenience and courses.
   
4. Realize that grasping and memorizing CCNP material and coursework is hard work, and the pace and intensity rivals that of the CCNA training. Set up for taking in new complex information as well as taking on new costs and testing.
   
5. Demonstrate new knowledge and skills acquired in CCNP training for managing routers/switches plus integrated voice, wireless as well as security network applications to become a CCNP. Consume information from the "Building Cisco Multilayer Switched Networks (BCMSN)," "Building Scalable Cisco Internetworks (BSCI)," "Implementing Secure Converged Wide Area Networks (ISCW)" and "Optimizing Converged Cisco Networks (ONT)" training courses to become a CCNP. Make sure programs are taught by certified instructors and cover the Cisco-approved CCNP curriculum.
   
6. Double check course details before signing up for a Cisco certification program and investigate the school or training facility to make sure it is Cisco approved. Become a CCNP once you've successfully mastered the curriculum and completed the certification tests. Pass the combination of the 642-901 BSCI, 642-812 BCMSN, 642-825 ISCW and 642-845 ONT required exams; or pass the combination of the 642-892 Composite, 642-825 ISCW and 642-845 ONT required exams.

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Some Contents from eHow.com

Practice materials for IT professionals looking for certification in CCNA, CCNP, CCDP, MCSE, MCDBA, MCSD, MCSA, MCP, CIW, ORACLE, Security+ and A+.

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Certay Top 30 Certification Exams List:

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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The "Do's and Don'ts" of Cell Culture

Since it was established in 1981, the Cell Culture Facility has made it easier, more economical and more effective to use mammalian cell cultures in research at Fox Chase. Cell culture procedures provide the cellular materials for many of the molecular methods used to study normal and cancer cell proliferation, cellular regulatory mechanisms, and normal and abnormal development. As a result, cell culture has become a core technique in current molecular and cell biological research.

Given below are a few of the essential "do’s and don'ts" of cell culture. Some of these are mandatory e.g. use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas e.g. cell culture plate. However some of them are specific to tissue culture.

The Do’s

1. Use personal protective equipment, (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, full-face visor and splash-proof apron should be worn when handling liquid nitrogen.
2. Always use disposable caps to cover hair.
3. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
4. Keep all work surfaces free of clutter.
5. Correctly label reagents including flasks, medium and ampules with contents and date of preparation.
6. Only handle one cell line at a time. This common-sense point will reduce the possibility of cross contamination by mislabeling etc. It will also reduce the spread of bacteria and mycoplasma by the generation of aerosols across numerous opened media bottles and flasks in the cabinet.
7. Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol) between operations and allow a minimum of 15 minutes between handling different cell lines.
8. Wherever possible maintain separate bottles of media for each cell line in cultivation.
9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially.
10. Quality Control all media and reagents prior to use.
11. Keep cardboard packaging to a minimum in all cell culture areas.
12. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals.
13. Test cells for mycoplasma on a regular basis.

The Don’ts

1. Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of antibiotic resistant strains and may render a cell line useless for commercial purposes.
2. Don’t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators.
3. Don't have too many people in the lab at any one time.
4. Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
5. Avoid keeping cell lines continually in culture without returning to frozen stock.
6. Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet.
7. Do not allow media to go out of date. Shelf life is only 6 weeks at +4oC once glutamine and serum is added.
8. Avoid water baths from becoming dirty by using Sigma Clean (Prod. No.S5525).
9. Don’t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.
10. Anyway, Don't break the Cell Culture Plates(6 well cell culture plate, 24 well cell culture plate, 96 well cell culture plate).

Rings are circles. In all cultures the circle is a symbol of unity and eternity, encompassing all. In early cultures, rings were considered magical or sacred through their association with the sun, moon and gods, thus giving protection to the wearer by binding one with power and energy.

Some of the magical mythology of ancient times still influences how and why we wear rings. Kings, priests and healers wore rings on the index finger because that finger was thought to be especially powerful. Medicines were applied to the body with the index finger to give speed and power to their effect. Even today, wearers of healing gemstones wear the rings on the index finger to take the best advantage of the curing energies.

The strongest magical tradition that still holds fast today is the one about the third or ring finger. The ring finger was thought to contain a nerve that went directly to the heart. Wearing a wedding or engagement ring on the ring finger still symbolizes the binding commitment and union of hearts. The Meaning of Fingers When Wearing Rings:

Right Hand vs. Left Hand

In the dual nature of man, the right and left hands are part of his whole being yet represent his twofold conscious and subconscious mind. The left hand (the subconscious) is a reflection of his deeply buried instinct, beliefs and attitudes. The right hand (the conscious) reflects his logic and his awareness. Wearing rings on your left hand could symbolize your unknowing wish to strengthen the powers associated with the finger that wears the ring. Rings on the right hand reflect your desire to control your life and actions.

The Thumb: Will Power

The thumb represents the self. Set apart from other fingers but working in unison with them to give strength to the hand that the others cannot, the thumb symbolized our self in our world. It is associated with logic and will power which govern our actions. Rings on the thumb symbolize freedom and independence of thought and action.

The Index Finger: Authority & Ambition

This finger reflects our need to enhance our ego. The forefinger, throughout history, has represented authority and power. It is associated with leadership and ambition. Kings, priests and healers wore rings on the index finger because that finger was thought to be especially powerful. Nowadays, people wear rings on the index finger as a show of assertiveness.

The Middle Finger: Identity & Materialism

The middle finger is associated with our role in life. Being in the center of the hand, it symbolizes personal identity and those things that are at central to our world. Those wearing rings on the middle fingers are well balanced yet wish to be at the center of attention.

The Ring Finger: Emotion & Creativity

The third finger is associated with affections because it is the one with a direct connection to the heart. It also represents our creativity and artistic self. A wedding or engagement ring on this finger proclaims to the world that the wearer emotions and creative goals are committed to the giver of the ring. If the ring is acquired by the wearer instead of a mate, it symbolizes self worth and a commitment to one personal skills and talents.

Little Finger: Relationships

The little finger is at the opposite end from the thumb. While the thumb symbolizes the inner self, the little finger is associated with others outside of ourselves. It sits on the outside of our hands, facing the world. It is the relationship finger, expressing our attitudes towards other people, sex, and the material world. Rings on the little finger represent confidence and independence in personal and business relationships. Rings here can also express a person attitude about sexuality and how appealing they consider themselves to be.

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The main aim of risk assessment is to prevent injury, protect property and avoid harm to individuals and the environment. The performance of risk assessment is a legal requirement under the Health and Safety at Work Act, UK. There are other EC directives covering Health and Safety at Work, you can visit the European Agency for Safety and Health at Work website www.europe.osha.eu.int for information on legislation and standards, or you should contact your on-site representative. Consequently risk assessments must be undertaken prior to starting any activity. The assessment consists of 2 elements:

1. Identifying and evaluating the risks.
2. Defining ways of minimizing or avoiding the risk.

For animal cell culture the level of risk is dependent upon the cell line to be used and is based on whether the cell line is likely to cause harm to humans. The different classifications are given below:

Low risk

• Non human/non primate continuous cell lines and some well characterized human diploid lines of finite lifespan (e.g. MRC-5).

Medium risk

• Poorly characterized mammalian cell lines.

High risk

• Cell lines derived from human/primate tissue or blood.
• Cell lines with endogenous pathogens (the precise categorization is dependent upon the pathogen) – refer to ACDP guidelines, 1985, for details.
• Low quality Cell Culture Dishs, Flasks adn Plates.
• Cell lines used following experimental infection where the categorization is dependent upon the infecting agent - refer to ACDP guidelines, 1985, for details*.

*Advisory Committee on Dangerous Pathogens (1985) Categorization of Biological Agents According to Hazard and Categories of Containment, 4th edition, HSE books, Sudbury, UK

A culture collection, such as ECACC will recommend a minimum the containment level required for a given cell line based upon its risk assessment. For most cell lines the appropriate level of containment is Category 2. However, this may need to be increased to Category 3 depending upon the type of manipulations to be carried out and whether large culture volumes are envisaged. For cell lines derived from patients with HIV or HTLV Category 3 containment is required.

Containment is the most obvious means of reducing risk. Other less obvious measures include restricting the movement of staff and equipment into and out of laboratories, especially the Cell Culture Dish(35mm Cell Culture Dish, 60mm Cell Culture Dish, 100mm Cell Culture Dish). Good laboratory practice and good bench techniques such as ensuring work areas are uncluttered, reagents are correctly labeled and stored, are also important for reducing risk and making the laboratory a safe environment in which to work. Staff training and the use of written standard operating procedures and risk assessments will also reduce the potential for harm. Training courses covering the basics of tissue culture safety are offered by ECACC.

Source: ECACC Handbook

Silver jewelry is a special thing in your daily life. You should find the best way to wear it. Maybe you are very puzzled how to choose a silver jewelry for your cloth. Now there are some rules can help you.

1. Professional clothes should come together with simple fine style silver necklace.

2. Leisure clothes should come together with special exaggerate style silver necklace.

3. Lovely clothes can come toether with silver bracelet with a little bell. It will let you very happy and lovely.

4. Silver jewelry with colorful man-made stone should go together with young girl.

5. Tibet silver jewelry must not go together with goldgolden jewelry. But different style silver jewelry can wear at the same time.

6. Tibet silver jewelry must not to together with professional cloth. It will let others think you are not professional person.

7. When you attend party or wear the gorgeous dress, you should come together with silver jewelry with diamond or other gem.

8. Silver jewelry will be oxidized and some part will turn to black. Please don't worry about it will affect you image. It looks like retro style. It's another sense of silver jewelry.

9. Silver jewelry belong to fashion things. Please prepare more styles in order to change to wear.

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David: "My brother is about to face the 642-901. He felt kind of confy two weeks ago, but now he′ve heard about so many people failing, that he′ll be preparing a litte more before diving in.

To prepare he took the Boson training last week with the new courseware and thought it was preety good, however he is still trying to get some decent test simulation software(Boson′s still 801 -oriented and the router simulator simply doesn′t do a good job). He also got the new BSCI book which seems superior to the previous one, at least in the chapters that he reviewed. Anyone knows anything about an updated software? All other comments on the 642-901 exam are welcome too."

Exam Description: The Building Scalable Cisco Internetworks (BSCI 642-901) is a qualifying exam for the Cisco Certified Network Professional CCNP?, Cisco Certified Design Professional CCDP?, and Cisco Certified Internetwork Professional CCIP? certifications. The BSCI 642-901 exam will certify that the successful candidate has important knowledge and skills necessary to use advanced IP addressing and routing in implementing scalability for Cisco ISR routers connected to LANs and WANs. The exam covers topics on Advanced IP Addressing, Routing Principles, Multicast Routing, IPv6, Manipulating Routing Updates, Configuring basic BGP, Configuring EIGRP, OSPF, and IS-IS.

With the new and updated exams its hard to recommend stuff -- the "old" stuff may have been good, but no idea about the "updated stuff."

For the Old CCNP I used the Cisco Press Flash Cards and Practice Exams -- and thought success on the practice questions did indicate possible future success on the real exams. Plus I like the "reference sheets" that were PDF files on the CD (which sound like they may be available separately now from Cisco Press). Not sure if the updated Flash Cards/Practice Exams are available.

Boson does the test engine for the Flash Cards and for the Practice Exams that come with the Cisco Press books. In the past they had different "exam sets" you could buy if you needed more practice (or another evaluation of real exam readiness).

 

 

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Many cultures obtained from a culture collection, such as ECACC, will arrive frozen and in order to use them the cells must be thawed and put into culture. It is vital to thaw cells correctly in order to maintain the viability of the culture and enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic above 4oC therefore it is essential that cultures are thawed quickly and diluted in culture medium to minimize the toxic effects.

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

Tiffany glow with the design of the exquisite dazzling or calm restrained, so that you can feel the culture of Tiffany brand, so the classification is very particular about brands, we can be grading on the main distinction between them and, according to our own demand to choose Tiffany jewelry.

Grade introduced: the hierarchy generally consists TIFFANY: real goods, Genuine; A+, a level; b level; other.

Real Goods is legitimate tiffany store with the sale of the real goods TIFFANY single laboratory;

Genuine refers to the original manufacturer TIFFANY store Availability production mode, and compared the lack of real goods single laboratory report, which is mainly used to export; and real goods Comparatively speaking, the general lack of laboratory report only single, with silver in 92.5 percent above.

A+ international best-selling refers to the TIFFANY products, the price is not high, but production steps, as well as the appearance of the final product are close to authentic.Not necessarily amount of silver up to standard, for the worship of Silver friends not too high, the proposed buy this. Fact to the high volume of silver really unnecessary and people could not see. Suggest that you buy this just general.

A level: bits is a poor place, with an average volume of silver lower number, such as: Will many of the "bread silver" ornaments (this does not ring for the beautiful, But long-time will not change color), in the process of making requests not stringent enough, such as polishing, laser logo, but it is not enough stringent checks, there defective rate is relatively high, the surface may not be entirely smooth, Cheaper prices.

B level refers to some small factories or workshops own family production of the so-called TIFFANY, of course, due to various reasons, the overall quality and quantity of silver have caught up with the front, and some quality really is not bad, but the majority are very difficult to say. If you are luck ,you can and may buy a similar, but in most cases can only feel a loss:)

Genuine: It is now the international market is the most commodity-like tiffany goods, TIFFANY Genuine can buy only a few major cities where the franchise, if scores can buy authentic pieces are deceptive, because not a few hundred pieces of authentic.

A+ goods: This is the best imitation goods, as Tiffany processing requirements for the silver decoration very high, so do the grading here a total of only 30 companies (That we are selling is the one), even as some businesses real goods sold. With its quality is Commodities without distinction, in full accordance with tiffany shape 1:1 manufacturing, materials identical polishing beautifully TIFFANY Lettering, 925 silver signs Fine.

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Serum is a complex mix of albumins, growth factors and growth inhibitors and is probably one of the most important components of cell culture medium. The most commonly used serum is fetal bovine serum. Other types of serum are available including newborn calf serum and horse serum. The quality, type and concentration of serum can all affect the growth of cells and it is therefore important to screen batches of serum for their ability to support the growth of cells(take especial care of 96 well plate). In addition there are other tests that may be used to aid the selection of a batch of serum including cloning efficiency, plating efficiency and the preservation of cell characteristics.

Fetal bovine serum (FBS) has been used to prepare a number of biological and has an excellent record of safety. The recognition of Bovine spongiform encepalopathy (BSE) in 1986 and it’s subsequent spread into continental Europe along side the announcement of the probable link between BSE and a new variant of Creutzfeldt Jacob disease in Humans, stimulated an increased concern about safe sourcing of all bovine materials. In 1993 the Food and Drug Administration (FDA) "recommended against the use of bovine derived materials from cattle which have resided in, or originated from countries where BSE has been diagnosed. The current (European Union) EU guidelines on viral safety focus on sourcing, testing and paying particular attention to the potential risk of cross contamination during slaughtering or collection of the starting tissue. As far as BSE is concerned, the EU guidelines on minimizing the risk of BSE transmission via medicinal products, CPMP/BWP/877/96, recommends the main measures to be implemented in order to establish the safety of bovine material versus the BSE risk. Again, similarly the focus is on geographical origin, the age of the animals, the breeding and slaughtering conditions, the tissue to be used and the conditions of it’s processing.

The use of FBS in production processes of medicinal products is acceptable provided good documentation on sourcing, age of the animals and testing for the absence of adventitious agents is submitted. All responsible suppliers of FBS for bio-pharmaceutical applications will provide such documentation.

Recent regulatory requirements in Europe stress the importance of justifying the use of material of bovine, caprine or ovine origin in the production of pharmaceutical products. Thus, although FBS has been used for many years in the production process of many medicinal products such as viral vaccines , recombinant DNA products and ELISA Plate, at present there is a justified trend to remove all material of animal origin from manufacturing processes. Sigma-Aldrich has recognized this growing trend and works closely with customers to optimize animal free media formulations to meet each customer’s cell culture requirements.

Similarly the FDA has similar guidelines when accepting regulatory submissions. The FDA regulates all medicinal products for Human use, such as therapeutics, vaccines ,diagnostics and Cell Culture Plates, and, usually, the United States Department Agriculture (USDA) are not involved.

More infomation from Sigma-Aldrich

Cisco 642-812 Exam is the associated certifications of CCNP CCIP and CCDP.It's full name is BCMSN - Building Converged Cisco Multilayer Switched Networks : 642-812.

The Building Converged Cisco Multilayer Switched Networks (BCMSN 642-812) is a qualifying exam for the Cisco Certified Network Professional CCNP and the Cisco Certified Design Professional CCDP certifications. The BCMSN 642-812 exam will certify that the successful candidate has important knowledge and skills necessary to implement scalable multilayer switched networks. The exam includes topics on Campus Networks, describing and implementing advanced Spanning Tree concepts, VLANs and Inter-VLAN routing, High Availability, Wireless Client Access, Access Layer Voice concepts, and minimizing service Loss and Data Theft in a Campus Network.

Certay 642-812 is written to coincide with the real test by the experienced IT experts and specialists. With the complete collection of Questions and Answers, Certay 642-812 is high enough to help the candidates to pass this exam easily without any other study materials and no need to attend the expensive training class.

To match the current real test, the technical team from Certay will update the Questions and Answers for any changes in time, and also we are always accepting the feedbacks about this exam from our users, in specialty, we will mend the exam pool with the suggestions from those users who got full scores in this exam, so to perfect Certay 642-812 exam to make it always have the best quality!

We trust Certay!

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Adherent cell lines will grow in vitro until they have covered the surface area available or the medium is depleted of nutrients. At this point the cell lines should be sub-cultured in order to prevent the culture dying. To subculture the cells they need to be brought into suspension. The degree of adhesion varies from cell line to cell line but in the majority of cases proteases, e.g. trypsin, are used to release the cells from the flask. However, this may not be appropriate for some lines where exposure to proteases is harmful or where the enzymes used remove membrane markers/receptors of interest. In these cases cells should be brought into suspension into a small volume of medium mechanically with the aid of cell scrapers.

Schematic diagram of "Subculture of Adherent Cell Lines"

Materials

• Media– pre-warmed to 37oC (refer to the ECACC Cell Line Data Sheet for the correct medium)
• 70% ethanol in water
• PBS without Ca2+/Mg2+
• 0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+
• Trypsin
• ELISA Plate
• Soybean trypsin Inhibitor

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• CO2 incubator
• Pre-labeled flasks
• Cell Culture Plates(96-well plate)
• Marker Pen
• Pipettes
• Ampule Rack
• Tissue

Procedure

1. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants.
2. Remove spent medium.
3. Wash the cell monolayer with PBS without Ca2+/Mg2+ using a volume equivalent to half the volume of culture medium. Repeat this wash step if the cells are known to adhere strongly.
4. Pipette trypsin/EDTA onto the washed cell monolayer using 1ml per 25cm2 of surface area. Rotate flask to cover the monolayer with trypsin. Decant the excess trypsin.
5. Return flask to the incubator and leave for 2-10 minutes.
6. Examine the cells using an inverted microscope to ensure that all the cells are detached and floating. The side of the flasks may be gently tapped to release any remaining attached cells.
7. Resuspend the cells in a small volume of fresh serum-containing medium to inactivate the trypsin. Remove 100-200uL and perform a cell count.
8. Transfer the required number of cells to a new labeled flask containing pre-warmed medium.
9. Incubate as appropriate for the cell line.
10. Repeat this process as demanded by the growth characteristics of the cell line.

Key Points

1. Some cultures whilst growing as attached lines adhere only lightly to the flask and 96 well plate, thus it is important to ensure that the culture medium is retained and the flasks are handled with care to prevent the cells detaching prematurely.
2. Although most cells will detach in the presence of trypsin alone the EDTA is added to enhance the activity of the enzyme.
3. Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+.
4. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged exposure could damage surface receptors.
5. Trypsin should be neutralized with serum prior to seeding cells into new flasks otherwise cells will not attach.
6. Trypsin may also be neutralized by the addition of soybean trypsin inhibitor, where an equal volume of inhibitor at a concentration of 1mg/ml is added to the trypsinised cells. The cells are then centrifuged, resuspended in fresh culture medium and counted as above. This is especially necessary for serum-free cell culture plates.
7. If a CO2 incubator is not available gas the flasks for 1-2min with 5% CO2 in 95% air filtered through a 0.25m filter.

Source: Sigmaaldrich

Disinfection

Methods designed for the disinfection/decontamination of culture waste, work surfaces and equipment represent important means for minimizing the risk of harm.

The major disinfectants fall into four groups and their relative merits can be summarized as follows:

Hypochlorites (e.g. Chloros, Presept)

• Good general purpose disinfectant
• Active against viruses
• Corrosive against metals and therefore should not be used on metal surfaces e.g. centrifuges
• Readily inactivated by organic matter and therefore should be made fresh daily
• Should be used at 1000ppm for general use surface disinfection, 2500ppm in discard waste pots for washing pipettes, and 10,000ppm for tissue culture waste and spillage

Phenolics (e.g. Sudol, Hycolin)

• Not active against viruses
• Remains active in the presence of organic matter

Alcohol (e.g. ethanol, isopropanol)

• Effective concentrations 70% for ethanol, 60-70% for isopropanol
• Their mode of activity is by dehydration and fixation
• Effective against bacteria. Ethanol is effective against most viruses but not nonenveloped viruses
• Isopropanol is not effective against viruses

Aldehydes (e.g. glutaraldehyde, formaldehyde)

• Aldehydes are irritants and their use should be limited due to problems of sensitization
• Glutaraldehyde may be used in situations where the use of hypochlorites is not suitable e.g. cleaning of centrifuge bowls or materials constructed of stainless steel that may be attacked or corroded by using hypochlorite solutions.

Waste Disposal

Any employer has a ‘duty of care’ to dispose of all biological waste safely in accordance with national legislative requirements. Given below is a list of ways in which tissue culture waste can be decontaminated and disposed of safely(especially the solid waste, such as flasks, centrifuge tubes, contaminated golves etc). One of the most important aspects of the management of all laboratory-generated waste is to dispose of waste regularly and not to allow the amounts to build up. The best approach is ‘little and often’. Different forms of waste require different treatment.

• Tissue culture waste (culture medium) - Inactivate overnight in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water
• Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by autoclaving and incineration
• Solid waste, such as flasks, centrifuge tubes(such as 15ml Centrifuge Tube, 50ml Centrifuge Tube), contaminated gloves, tissues etc. should be placed inside heavy duty sacks for contaminated waste and autoclaved prior to incineration. These bags are available from Bibby Sterilin and Greiner.
• If at all possible waste should be incinerated rather than autoclaved

Source: Sigma-Aldrich

Large numbers of cell lines look identical. Cell lines with very different origins and biological characteristics typically cannot be separated on grounds of morphology or culture characteristics. Infection or contamination of a cell line with an adventitious virus or mycoplasma may significantly change the characteristics of the cells but again such contamination will be inapparent. Cell lines will also change with time in culture(even in glass bottom dishes/elisa plate), and to add to all these natural hazards it is all too easy to mis-label or cross-contaminate different cell lines in a busy cell culture laboratory.

The opportunities for inadvertently introducing error into a cell line are limitless and ever present. It is in the nature of the science that, once introduced, an error will be propagated, compounded, consolidated and disseminated.

The integrity and biological characteristics of a cell line have to be actively maintained by a well-organized system of “husbandry” based on systematic cell banking supported by testing regimens in a structured quality assured environment. Such a controlled environment will only prevail in a dedicated professionally organized cell culture laboratory or cell bank. A small research laboratory with a high throughput of short-term research students, a minimum of permanent laboratory staff and no formal quality management program will find it difficult to maintain its cell lines unchanged over many years.

For all these reasons it is strongly recommended that new cell lines should only be acquired from a specialist, reputable culture collection such as ECACC. Moreover, if a laboratory believes it already has a certain cell line in its liquid nitrogen store, the identity and purity of such a cell line should be questioned in the absence of a well-recorded culture history and recent test data. If there is a doubt, it is straightforward and cost effective to replace such cell stocks with authenticated material from a Culture Collection.

When a Cell Culture Collection “accessions” a new cell line it will characterize the cell line using techniques such as isoenzyme analysis and DNA profiling so that the identity of the cell line can subsequently be verified. The Collection will then establish a hierarchy of Master and Working cell banks, cryopreserved in liquid nitrogen, that are demonstrated free from microbial contamination including mycoplasma. Customers are supplied from these authenticated Working Cell Banks (WCB). Replacement WCB's are manufactured from the original Master Cell Bank (MCB) and the new WCB will again be fully tested.

ECACC supplies its cell lines together with advice on how to maintain the line. A Technical Support team will subsequently assist with any difficulties and can often provide additional technical information about the cell line. Culture Collections exist to ensure that animal cell research is conducted using standardized, authenticated material that ensures the work can be reproduced(such as Glass Bottom Cell Culture Dishes, 96 well plate etc). An authenticated cell line of known provenance is the very "bed rock" of any cell based project.

Source: ECACC Handbook-SIGMA

In this article we explain how to choose the right bracelet. Your bracelet could be made in silver or gold, it could be a simple bracelet or be studded with stones and diamonds. Make sure that you know how to evaluate a good bracelet.

Step One

Basic Design: The first thing that you should consider is the basic design of the bracelet. The design should be streamlined without sharp or pointed edges. Sharp edges can not only injure delicate skin, but also get bent or dented easily. The type of mounting used for the diamonds and gemstones in your bracelet will depend on the design. Prongs used to mount gems need to be sturdy and strong. If the gemstones in your bracelet are mounted with thin needle-like prongs, the prongs can get caught in pockets and other places. This causes the prongs to gradually open up, this ofcourse loosens the gemstones which can then drop off. The design dimensions for your bracelet should take the metal weight into consideration. A wide width with low gold or silver weight, will mean that the metal is too thin. This can cause your bracelet to lose shape and get damaged.

Step Two

Metal Weight: The metal weight of the bracelet is another important thing to be checked. Good mounting of the gemstones or diamonds in your bracelet will need sufficient gold or silver weight. There can be no standard weight suitable for all bracelets, much will depend on the design, the number of gems and diamonds, the length and width of the bracelet etc. Looks can be deceptive so pay special attention to the weight of your bracelet. In general you can be sure that, ladies bracelets with a metal weight that is less than 10 grams will not be durable and sturdy. Your jeweller might tell you that, healthy metal weight will make your bracelet appear clumsy and bulky. Stop believing this nonsense, the truth is that the design needs to ensure that the bracelet does not appear bulky. The design for your bracelet should have good metal thickness, this will not be visible from the front of the bracelet. Compromising on the gold or silver weight of your bracelet would also mean that, the links that connect the segments of the bracelet will be weak. This can cause the bracelet to snap, it could then easily fall off your wrist and get lost. So remember that, good metal weight is essential for gold or silver bracelets.

Step Three

Gemstone Options:You should also be given a wide range of gemstone options to select for your bracelet. Many jewellers use whatever few gemstones they have and put a bracelet together. This does not work to your advantage as you are restricted in your choice. You could choose the gemstones in your bracelet based on color, type, birthstone or traditional beliefs. For example, a mother's bracelet could include the birthstone of the mother and all her children. Such family birthstone bracelets can be made only if a wide choice of gems is available. There is ofcourse the issue of budget that you have at your disposal. You might see a gorgeous sapphire bracelet but, the sapphires could drive the bracelet out of your budget. You should be allowed to choose the same bracelet with cheaper gems like, blue topaz, iolite or probably cheaper sapphires. A jeweller with limited access to gems and production facilities will restrict your choice too. There is also the issue of conditions in which your bracelet would be worn. For example, an emerald bracelet needs special care when wearing and storing, this is because of the nature of emeralds. You might pefer the same bracelet design with green tourmaline instead of emeralds. Your jeweller will need to satisfy your requirements and not the other way round.

Step Four

Choose The Metal: The metal for your bracelet will be a component of the price tag, a gold bracelet would add a substantial amount to the price tag. You should therefore have the option to choose gold or silver for your bracelet. Most jewellers are very eager to sell gold bracelets, but will either ignore you are provide you with second grade service if you talk about a silver bracelet. This is sad but true, so look for a jeweller who is willing to make a good quality silver bracelet for you if that is what you want. It is often said and rightly that, a good quality silver bracelet is better than a delicate and light weight gold bracelet. When you choose your silver bracelet remember that, no compromises regarding gemstone choice or craftsmanship need be made. For gold bracelets, you should have the option to select a 14k or 18k bracelet and also choose the gold color. If you are looking for a white gold bracelet, there is no reason why you should be happy with a yellow gold aquamarine bracelet. Jewellers make gold bracelets with low weight, this is done to allow lower price tags. They really don't care what problems these light weight junk will cause you after the payment has been made.

Step Five

Good Craftsmanship: Insist on good craftsmanship for your bracelet, this should be the case for gold or silver bracelets. Bad craftsmanship could also mean loose gemstones and badly connected links. It is true that mass produced bracelets cannot be given individual attention. This is the reason why a custom bracelet could be best for you. Good craftsmanship is essential for ladies and men's bracelets, make sure that your bracelet is well crafted. Good craftsmanship provides more than just good appearance for your bracelet, this will clearly work to your advantage in the longterm.

Tiffany Mall is cheap tiffany jewelry online store. We have added about 200 Tiffany Jewelry to the site for our customers. Our mission is to provide the best products (sunch as tiffany bracelets) and service to our customers at the lowest prices possible.

It was Tiffany & Co. that introduced the engagement ring as we know it today. The celebrated six-prong “Tiffany® Setting” lifts the diamond above the band and into the light. The result is a ring whose beauty has never been equaled.

In 1848 the New York City newspapers dubbed Charles Lewis Tiffany, “The King of Diamonds.” And with good reason. The quality of Tiffany diamonds was then, and remains, exemplary. In the spring of 1887, Tiffany shocked the world by purchasing the French Crown Jewels. From this time on, Tiffany became the world’s authority on the finest diamonds.

Soon Tiffany designers were creating brilliance of their own. From the glittering 1890s on, timeless Tiffany designs graced women from the finest families: the Astors, the Vanderbilts, the Morgans. Celebrities from the theater, sports, and ultimately European royalty and Hollywood stars began to prize Tiffany diamond designs.

Around the world, museums treasure the Tiffany design aesthetic, from the Art Nouveau period to Art Deco to today’s modern classics. Year in, year out, the passion for Tiffany diamonds is clearly demonstrated in the world’s auction houses.

Today, the world-famous 128.54-carat Tiffany Diamond is on permanent display in the New York flagship store—proof positive of Tiffany’s diamond legacy.

List of Tiffany & Co. products:

• Tiffany Necklaces
• Tiffany Pendants
• Tiffany Bracelets
• Tiffany Earrings
• Tiffany Rings

But nowhere is a Tiffany diamond more beautiful or more treasured than in its place of honor, on the hand of a woman.

You can get more infomation from www.tiffany.com.

Tiffany Mall is your best best Tiffany & Co sliver jewelry and diamonds provider. We have added about 200 Tiffany Jewelry and Diamonds to the site for our customers. Our mission is to provide the best products and service to our customers at the lowest prices possible.

The CCDA certification indicates an apprentice knowledge of Cisco network design. Individuals who have earned a CCDA are capable of designing switched or routed networks consisting of LANs, WANs, and various dial services. While a CCNA certification is not required to take the CCDA exam (640-863 DESGN), Cisco recommends being familiar with CCNA material.

This exam indicates a foundation or apprentice knowledge of network design for the Cisco converged networks based on SONA (Cisco's Service-Oriented Network Architecture). CCDA certified professionals can design routed and switched network infrastructures and services involving LAN, WAN, and broadband access for businesses and organizations.

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The CCVP certification recognizes the increased importance placed on IT professionals of today who are responsible for integrating voice technology into underlying network architectures. Individuals who earn a CCVP certification can help create a telephony solution that is transparent, scalable, and manageable. Earning a CCVP certification validates a robust set of skills in implementing, operating, configuring, and troubleshooting a converged IP network. The certification content focuses on Cisco Unified Communications Manager (formerly Unified CallManager), quality of service (QoS), gateways, gatekeepers, IP phones, voice applications, and utilities on Cisco routers and Cisco Catalyst switches.

Exam 642-642: Quality of Service Exam (QOS).

Related Certifications: CCIP, CCVP, IP Telephony Specialists Certifications.

Exam Description

The QOS exam is one of the qualifying exams for the Cisco Certified Internetwork Professional, the Cisco Certified Voice Professional and the Cisco IP Telephony Design Specialist certifications. The QOS 642-642 exam will test materials covered under the Implementing Cisco Quality of Service QOS v2.1 course. The exam will certify that the successful candidate has knowledge and skills necessary to configure and troubleshoot Cisco IOS routers running Quality of Service protocols in Service Provider and Enterprise environments. The exam covers topics on IP QOS, classification and marking Mechanisms, queuing mechanisms, traffic shaping and policing mechanisms, congestion avoidance mechanisms, link efficiency mechanisms, modular QOS command line interface, and QOS Best Practices.

Exam Topics Include:

IP QoS Fundamentals and Best Practices
IP QoS Components
Modular QoS CLI and Auto-QoS
Classification and Marking
Congestion Management and Avoidance Methods
Traffic Policing and Shaping
Link Efficiency Mechanisms
The QOS exam is one of the qualifying exams for the Cisco Certified Internetwork Professional, Cisco Certified Voice Professional, Cisco IP Telephony Design Specialist, Cisco IP Telephony Express Specialist, Cisco IP Telephony Operations Specialist, and Cisco IP Telephony Support Specialist certifications. The exam will certify that the successful candidate has knowledge and skills necessary to configure and troubleshoot Cisco IOS routers running Quality of Service protocols in Service Provider and Enterprise environments. The importance of this exam is evident in the fact that it applies to so many different certifications.

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• Although you may have a favorite designer, there's really no need to stick with just one. In fact, there's no need for the pieces to actually be designer. There are plenty of wonderful pieces on the market that are unsigned but certainly make a fashion statement. Mix and match your jewelry to give it character and a little pizazz!
• Costume Tiffany Jewelry, both signed and unsigned, is an affordable way to complete your look. Costume Jewelry can be bought for a low price and has plenty of life. Heck, we see costume jewelry back to the 1920s still in excellent condition. What's great with costume jewelry is you can use it, get tired of it, put it away or give it away and have still gotten good value for your money.
• Designer pieces tend to cost a little more, last a little longer, and they usually follow more traditional lines so you can wear them for more than one or two seasons.
• Sterling Silver is affordable and very versatile. If you don't want to spend the money for sterling, then purchase some silver plate pieces. Silver is a must have for any wardrobe! You can go from dainty and delicate to large and noticeable. It can be dressed up or down and is acceptable for any event or occasion.
• When purchasing Tiffany Rings, you can have great fun - again the choices are endless from large and overwhelming to dainty and feminine. The length and size of your fingers plays an important role in what will look good. For example, long skinny fingers don't look good with huge oversized rings, yet on a woman that has long thicker fingers they look exquisite. Long nails? Short nails? it all makes a difference. Play and see what you like, and what looks good. Also, determine where you will be wearing these rings. For example, if you're working around fine fabrics, you'll not want any rings that have sharp edges that could snag. Your rings are also part of your total statement.
• When purchasing Tiffany Necklaces, be certain you know your neck size so that you get a good fit. This is very important when purchasing online as you don't get a chance to try it on. If your neck is average in size then a 16" necklace will look great, however if you have a somewhat larger than average neck then a 16" necklace could be too tight. Measure the length of some of your favourite necklaces you already own. This will give you a good indication of what length you prefer. Opera necklaces which are over 30" are terrific. They can be double wrapped, knotted, or whatever else your imagination comes up with. When choosing colored necklaces, make sure to pick a color that complements your skin tone. It may match your outfit fabulously but if it doesn't match or complement your skin tone your look could be lost.
• When purchasing Tiffany Bracelets, you'll need to decide where you're going to wear them, and how noticeable you want them to be. The availability of size and shape is endless - you can go from large bangle bracelets to dainty gem bracelets. If it is a slip-on bracelet, be sure that it will fit over your hand. Make certain to measure your wrist as the fit of a bracelet is very important. Too large and it will look sloppy and may even slide off. Too small and it can constrict your wrist movement.
• Your hands are your expressive area and many of us "talk with our hands." (they are also the smallest part of our body!) Try wearing three bracelets on one wrist - its a sign of power. They don't have to match either so step out and do something different. Don't forget to wear a dainty bracelet with your watch.
• When purchasing Tiffany Earrings, realize that the shapes and styles are endless - from dangles to chandeliers to hoops to studs to buttons. There are materials from enamel to beads to gems to metal to wood. The size of the earrings is very important to the statement you're making. The larger the earring, the more noticeable and the more incorporated they become into your total look. Large earrings aren't for everyone - depending on your face size and shape they may actually detract rather than attract. Experiment and find what looks best on you.
• Your jewelry choices are endless - no matter what your tastes, your likes, your dislikes. You'll be able to complete your look just the way you want it with little effort. So be sure to make your fashion statement!

Source: WikiHow.com

Found the girl of your dreams? And you are going to pop the question? Here are a few steps to help you choose a perfect engagement ring.

1. Take her by some jewelry stores like tiffany jewelry stores, and just browse the engagement ring section. Tell her to feel free to try a few on, and see which ones she really likes.
   
2. Pay close attention to the jewelry she wears everyday. Try to familiarize yourself with the styles she likes.
   
3. Once you've figured all that out, go shopping on your own. Any good jeweler will help to educate you on the four C's of diamonds, Clarity, Color, Cut, and Carat Weight. Figure out what the best diamond is for your budget and pick out a few rings, by the way, tiffany rings is not the bad choise. Tell the jeweler to hold them for you. Most places will hold them for up to a week. Or explain to the jeweler what you plan to do, which will come in the next few steps.
   
4. Schedule an appointment with that jeweler to clean or repair a watch or something of yours. Walk-ins are welcome in a lot of cases. Make sure she comes along. Casually browse over the selection of engagement rings, and get her to randomly try on the ones you picked out for her and judge her reaction. If she shows a strong preference for one ring, then you know which one to get.
   
5. Don't let her talk you down in price. If she is really the one, then she'll appreciate the fact you worked so hard to get the ring.

If you want to choose a tiffany pendant, a pair of tiffany earrings, or some other tiffany accessories, accompany with your engagement ring, just shop online, that can help you save a lot of times and money.

Some tips are from wikihow.com

This world famous New York City store sells high-quality Tiffany jewelry, china and crystal. While many items might cause sticker-shock, there are also a selection of less expensive items (key chains, money clips, etc.) that may make good great New York City souvenirs, but are not the highly sought after Tiffany necklaces, Tiffany pendants, Tiffany earrings, Tiffany accessories.Even if you can't afford to make a purchase, you can make like Holly Golightly from "Breakfast at Tiffanys&qu