Frequently asked questions

1. Why is vapour phase LN₂ recommended rather than liquid phase LN₂?

2. What Quality Control (QC) tests do ECACC cell lines undergo?

3. Can I use cell lines obtained from colleagues?

4. How can I make sure that I keep my cell line safe in a shared laboratory?

5. What does seeding density mean?

6. I do not have a CO₂ fed incubator – can I grow the cells without it?

7. The subculture routine states that CO₂ is not required.  However, I only have access to a shared incubator that has a CO₂ supply – can I use this anyway?

8. Why are my cells not adhering to the culture flask?

9. Can I use antibiotics in the cell culture medium?

10. How do I culture serum free cell lines?

11. How will my DNA order be supplied and how do I handle it upon receipt?

12. What’s the best way to keep my laboratory clean?

13. What biosafety level are ECACC cell lines? Why?

14. I have received my order and there is no data sheet or MSDS documentation included. How can I get this information?

 

1. Why is vapour phase LN₂ recommended rather than liquid phase LN₂?

If vials are immersed in liquid phase LN₂ there is a risk of LN₂ seeping into the vial; this could result in cross-contamination and an increased risk of the vial exploding when thawed.

 

2. What Quality Control (QC) tests do ECACC cell lines undergo?

The following tests are carried out:

• cell count, viability and plating efficiency

• sterility testing for the detection of bacteria and fungi

• mycoplasma testing (using a minimum of two out the following three different methods: PCR, Indirect Hoechst DNA stain, culture isolation)

• cell line authentication

• human cell lines: cells will undergo STR profiling.

• animal cell lines: cells were previously authenticated using isoenzyme analysis (for speciation) and DNA fingerprinting, but due to technological advances they now undergo DNA barcoding.

• hybridoma cell lines: cells will undergo antibody isotyping.

A Certificate of Analysis, showing the results of these tests, is available for each lot number of cells on the product pages. 

 

3. Can I use cell lines obtained from colleagues?

Although it can be tempting to source cell lines from colleagues they may inadvertently supply you with contaminated or misidentified cell lines. If they can provide you with confirmation that the cells are mycoplasma free and the DNA profile has been confirmed to be as expected you can have more confidence.  If not, is it worth taking the risk? 

 

4. How can I make sure that I keep my cell line safe in a shared laboratory?

Effective segregation of cell lines is essential to safeguard the credibility of your work especially in shared laboratories. In order to do this we would recommend the following steps:

• make sure the microbiological safety cabinet is clutter free and cleaned before you begin working on your cells. This can be done with 70% IPA on a daily basis.

• only work with one cell line at a time in the cabinet. Once you have finished working on the cell line spray down the cabinet with 70% IPA, allow it to settle and then set up the cabinet for the next cell line. This reduces the chances of cross contamination.

• keep separate and clearly labelled bottles of media and all other reagents for each cell line

• aliquot media and reagents from stock bottles for use in the cabinet where possible. Discard any unused media and reagents; never return this to the stock bottles

• practice good aseptic techniques

• if possible, keep flasks segregated in the incubators

• keep accurate and detailed records

If you suspect that your cells may have become cross contaminated, take a look at our cell line authentication services.

 

5. What does seeding density mean?

Seeding density refers to the number of cells per cm² (for adherent cell lines) or per ml (for suspension cell lines) seeded into a flask when setting up a culture.  Check the subculture routine information available on the product detail page on our website and the data sheet included with your order.

 

6. I do not have a CO₂ fed incubator – can I grow the cells without it?

If the subculture routine specifies CO₂ is required then it is necessary to provide a CO₂ supply.  If you do not have a CO₂ fed incubator you can manually gas the flasks using gas cylinders with the appropriate mix of air and CO₂. The requirement for CO₂ is linked closely to the type of culture medium used.  Some culture media, such as L15, do not require a supply of CO₂ 

 

7. The subculture routine states that CO₂ is not required.  However, I only have access to a shared incubator that has a CO₂ supply – can I use this anyway?

You can use the incubator to incubate at the correct temperature but you must use sealed flasks (rather than flasks with vented caps) or put the flask in a sealed plastic box and then into the incubator to exclude the CO_2.

 

8. Why are my cells not adhering to the culture flask?

There may be several reasons why. Firstly, check the expected growth mode of the cell line i.e. is it an adherent cell line or a suspension cell line? This information is available on the product detail pages on our website and the data sheet included with your order. 

If the cell line is an adherent line there may be a problem with the culture.  The majority of adherent cell lines will adhere to the substrate within a few hours after seeding.  If the cells have not adhered 24 hours after resuscitation this would indicate that the culture may be non-viable.  However, a few adherent cell lines can take some time to adhere, particularly immediately post resuscitation e.g. the HEK 293 cell line can take up to seven days to adhere post resuscitation. 

Secondly, consider whether you are using the correct type of cell culture flask; there are several types of cell culture plastic available. Cell culture treated plastic for use with adherent lines and ultra-low attachment flasks, which enables cells to be cultured in spheroid culture.  Non-treated flasks are ideal for cell lines that grow in suspension. It is not always obvious which type of flask is which; make sure that you are using the one best suited to the cells you are culturing.

To determine if the cells are dead – do not rely solely on the visual appearance of the cells observed using an inverted microscope.  In addition, perform a viable cell count e.g. using trypan blue stain, a haemocytometer and an inverted microscope or an equivalent viable cell counting method.

 

9. Can I use antibiotics in the cell culture medium?

We do not generally recommend the use of antibiotics in cell culture work as they can mask a low level contamination and poor aseptic technique. The ampoule of cells which you received either directly from us or via one of our distributors will not have been cultured using antibiotics. However, we understand that many laboratories do use antibiotics although this is not considered to be best practice. 

Here is a table of common antibiotics used in cell culture (Information taken from Culture of Animal Cells: A manual of basic technique. Fifth Edition by R. Ian Freshney).

Antibiotic	Working Concentration	Activity Against
Amphotericin B	2.5 ug/ml	Fungi, yeasts
Ampicillin	2.5 ug/ml	Bacteria, gram negative and gram positive
Ciprofloxacin	100 ug/ml	Mycoplasma
Neomycin	50 ug/ml	Bacteria, gram negative and gram positive
Penicillin	100 U/ml	Bacteria, gram positive
Streptomycin	100 ug/ml	Bacteria, gram negative and gram positive
Tetracycline	10 ug/ml	Bacteria, gram negative and gram positive

 

A combination of Penicillin (100U/ml), Steptomycin (100ug/ml), and Amphotericin (2.5ug/ml) is effective against the most common forms of cell culture contamination; bacteria (gram positive and gram negative), yeast, and fungi; when used at the concentrations stated.

Please note: Antibiotics should not be added to freeze media as during the freezing of the cells the antibiotics will become concentrated and result in a toxic effect on the cells

 

10. How do I culture serum free cell lines?

Cell lines adapted to serum free medium can be more difficult to resuscitate and establish in culture in comparison to cell lines cultured in the presence of serum. For cell lines which have been adapted to serum free or animal component free media, cell viability may be poor on resuscitation and may initially decrease further. It is important to start serum free cultures at a relatively high cell density. Full recovery and establishment of a proliferating cell line may take up to two weeks.

When ordering this type of cell line from us we recommend that you read the subculture routine given in the product detail pages on our website where we recommend a specific protocol for the resuscitation and culture of these cells; this is the protocol which is routinely and successfully used here at ECACC.

 

11. How will my DNA order be supplied and how do I handle it upon receipt?

DNA products are supplied at either +4°C or frozen; consult the data sheet in your delivery. Frozen DNA products should be stored at -20°C upon receipt if not to be used immediately. RNA Transported on dry ice. Handle as for DNA, but RNA should be stored at -80°C

• How to handle DNA on receipt

 

12. What’s the best way to keep my laboratory clean?

In order to maintain a clean and safe working environment tidiness and cleanliness are key. All spills should be dealt with immediately and you should routinely clean all work surfaces both inside and outside of the microbiological safety cabinet, the floors, and all other pieces of equipment i.e. centrifuges. 

Humidified incubators are a particular area for concern due to the potential for fungal and bacterial growth in the water trays. This will create a contamination risk that can only be avoided by regular cleaning. All major pieces of equipment should be regularly maintained and serviced by qualified engineers, for example: 

• Microbiological safety cabinets should be checked every six months to ensure that they are safe to use in terms of product and user protection. These tests confirm that the airflow is correct and that the HEPA filters are functioning properly  

• Incubator temperature should be regularly checked with a NAMAS (National Accreditation of Measurement and Sampling, UK), or equivalent, calibrated thermometer and adjusted as necessary  

• Incubator CO₂ and O₂ levels should also be regularly checked to ensure the levels are being maintained correctly.

 

13. What biosafety level are ECACC cell lines? Why?

ECACC cell lines are routinely handled at Biosafety Level 2 (unless a higher containment level is specified). This is in accordance with the guidelines provided by the UK Advisory Committee on Dangerous Pathogens; any cell line has the ability to harbour an as yet unidentified adventitious agent.

We provide a generic Material Safety Data Sheet (MSDS) which covers the cell lines from our catalogue. The information contained in this document will help you to carry out your own Biological Risk Assessment covering your intended use for your cells in your own laboratory. Once you have assessed the potential risks involved, then, after consultation with your own safety advisors you can decide whether you are happy to reduce the level of containment at which you handle the cells.

We also and run several cell culture training courses throughout the year.

As well as the information available online customers can request a free copy of the Fundamental Techniques in Cell Culture Laboratory Handbook. This compact laboratory handbook, produced in partnership with one of our distributors Sigma Aldrich (now a part of of Merck KGaA, Darmstadt, Germany), provides a wealth of information from the sourcing of cell lines, safety and laboratory design to aspects of cryopreservation and quality control.

14. I have received my order and there is no data sheet or MSDS documentation included. How can I get this information?

For information about how to store, handle or resuscitate the frozen vial of cells you have received, you can use this series of protocols to help you. These are the protocols routinely used by our own cell culturists at ECACC .

For cell line specific information such as recommended seeding densities, culture media etc, please go to the individual product page.

For product safety information a generic Material Safety Data Sheet is available.

 

1. What is a type culture?

2. What is the passage number of my NCTC strain? 

3. Who uses NCTC cultures?

4. How can I use NCTC strains in diagnostic testing?

5. How are researchers using NCTC strains?

6. What do the NCTC cultures look like?

7. How are NCTC cultures dispatched?

8. What is the difference between NCTC and ATCC cultures?

9. Are NCTC cultures reference materials?

 

1. What is a type culture?

A type culture, or type strain, is the strain on which the description of a species is based. For example, NCTC 10332 is the type strain of the species Pseudomonas aeruginosa. Although the type strain is often (but not always) the first strain of a species to be isolated and described as a new species, it is not necessarily the most typical example of that species. An example of this would be the type strain of the species Listeria monocytogenes NCTC 10357, non-motile variant of what is described as a motile species.

When describing new species, the International Committee on Systematics of Prokaryotes stipulates (as outlined in the International Code of Nomenclature of Prokaryotes) that the type strain of a species must be deposited in “at least two accessible culture collection”, such as NCTC. For more information on depositing strains with NCTC, go to: How to deposit strains with NCTC.

 

2. What is the passage number of my NCTC strain? 

NCTC microbial strains produced and distributed in ampoules by the UK Health Security Agency are categorised as Primary Reference Standards and therefore considered “passage zero”, where a passage count is defined as the “transfer of organisms from a viable culture to fresh medium with growth of the microorganisms” at the recipient laboratory. Please note that NCTC cultures are not considered passage zero for the purposes of an import permit.

Additionally maintenance of bacteria in a freeze dried state reduces the need for subculture when creating new batches, and the NCTC QC regime includes testing to ensure that the cultures remain unchanged by passage in house.

 

3. Who uses NCTC cultures?

Many thousands of laboratories all over the world use NCTC cultures. The strains are used in a very wide range of fields including clinical, veterinary, food, water and environmental microbiology. They are used by public and governmental organisations, hospitals, universities and research establishments, private companies and global industries.

 

4. How can I use NCTC strains in diagnostic testing?

NCTC cultures are often stipulated as controls for specific procedures such as the strains recommended by EUCAST* for antimicrobial susceptibility testing.  They are used as positive and negative controls for phenotypic tests and can be used for evaluating and harmonising routine and specialist microbiological tests.

 

5. How are researchers using NCTC strains?

NCTC strains can be used for evaluating the efficacy of potential new drugs and alternative therapies. They are of value in studies to characterise bacterial strains, determining the prevalence of antimicrobial resistance and understanding the mechanisms associated with resistance. NCTC strains are used frequently for evaluating new methods and technologies and for assessing improvements to established methods such as PCR.

 

6. What do the NCTC cultures look like?

The NCTC cultures are provided as freeze-dried (lyophilised) micro-organisms in evacuated glass ampoules. For guidance on opening the ampoules, see: How to open and grow freeze-dried NCTC strains

 

7. How are NCTC cultures dispatched?

Cultures are sent by courier and you would normally receive your cultures within two to five days. Cultures are dispatched as Infectious Substances, Category A (UN 2900 and UN 2814) or Infectious Substances Category B (UN3373), as appropriate. Export restrictions apply for the supply of hazardous pathogens and compliance will impact on the length of the delivery period.

  

8. What is the difference between NCTC and ATCC cultures?

There are over 500 culture collections around the world containing micro-organisms of significance in a wide range of fields. ATCC (American Type Culture Collection) is a general collection based in the US.  NCTC focuses on bacterial strains of biomedical or veterinary importance and there is some overlap between strains available from ATCC and NCTC although many NCTC strains are exclusive to our collection. For guidance on the ATCC strains that are easily available from NCTC go to: NCTC equivalents to ATCC bacterial strains

 

9. Are NCTC cultures reference materials?

Yes. You do not need to provide any validation data for the identity of strains that you have purchased from NCTC.

 

1. I can’t find the virus I’m interested in on your website. Do you have it?

2. Why can’t I pay for virus products by credit card?

3. What cells can I propagate my virus culture in?

4. What containment level should virus RNA and DNA be handled at? Does the hazard group of the live virus impact on this?

5. How should I store the sample I receive?

6. What should I do if the virus fails to grow?

 

1. I can’t find the virus I’m interested in on your website. Do you have it?

Fill in the  and one of our team will get back to you.

 

2. Why can’t I pay for virus products by credit card?

There are security precautions for supplying restricted products. 

 

3. What cells can I propagate my virus culture in?

Permissive cell lines for a particular virus can be found in the published scientific literature. The cell line we use to prepare the virus batch is detailed on the Certificate of Analysis, and linked to on the website catalogue listing under “Related Products”. All cell lines we use can be ordered from our website.

 

4. What containment level should virus RNA and DNA be handled at? Does the hazard group of the live virus impact on this?

Viral nucleic acids are available to be researched on in ACDP Containment Level 2 Laboratories. This is not affected by the hazard group of the live virus benefiting the scientific communities with interests in Containment Level 3 and 4 pathogens. For additional information read our viral nucleic acids news article.

 

5. How should I store the sample I receive?

Most viruses require rapid freezing and storage at -80°C as they are often heat sensitive and suffer damage in temperatures exceeding greater than -60°C. 

1. How will I receive my order?

2. How long will my order take to process?

3. How should I store the sample I receive?

4. How should I open the glass ampoule and resuscitate the freeze dried culture?

5. What agar should I use to culture the strain?

6. Where can I obtain the ampoule openers which appear in the instructions/video?

7. What should I do if the culture fails to grow?

8. How can I find information about the BSL of the strain and the recommended safe handling of the strain in my laboratory?

 

1. How will I receive my order?

NCPF cultures are supplied as either freeze-dried (lyophilised) cultures, sealed under vacuum into glass ampoules; or as growing cultures supplied on agar slopes. Most yeast isolates will be provided as freeze-dried ampoules but many of the moulds will be supplied as growing slopes. The Culture Collections can advise you in which format your order will be supplied.

 

2. How long will my order take to process?

If cultures are available as freeze-dried ampoules then orders can be processed and shipped within a few days. However, if all or part of the order is for an isolate not available as a freeze-dried ampoule then the process involves reviving and growing the isolate on an agar slope and then verifying the identification and checking for purity which may take two weeks or more depending on the growth rate of the organism and its propensity to form spores.

 

3. How should I store the sample I receive?

If you are not ready to begin culturing the strain(s) you receive, then these can be stored at room temperature. Growing slopes should remain viable for several weeks, often much longer provided the slopes do not dry out, whilst freeze-dried ampoules should remain viable for years. Some strains, particularly some dermatophyte species, will not survive in the fridge.

 

4. How should I open the glass ampoule and resuscitate the freeze dried culture?

The ampoule should be resuscitated according to the information available from our website   

How to open an NCPF glass ampoule and reconstitute freeze-dried fungi

 

5. What agar should I use to culture the strain?

The most useful and widely employed mycological agar is glucose-peptone agar (Sabouraud’s agar). There are many commercial suppliers of ready-poured plates, many containing chloramphenicol to prevent bacterial overgrowth and either with or without actidione (chlorheximide) to prevent mould contamination, or powders from which to prepare your own agar. NB The growth of many yeast species and most non-dermatophyte moulds will be inhibited by actidione, such plates are really intended for the primary isolation of dermatophyte species from clinical samples.

Plates should usually be incubated at a maximum of 30°C for dermatophyte and environmental mould isolates and 35° - 37°C for yeast. Many yeast and some of the opportunistic pathogenic moulds will grow faster at 37°C or sometimes even higher depending on the strain and species.

For growth of many Malassezia species a specialist lipid-rich medium will be necessary, or alternatively the surface of the plate should be wiped with a swab dipped in olive oil before inoculation of the organism.

For any of the strains sent as freeze-dried cultures, even if only one or two colonies grow from an ampoule that will be sufficient to further propagate your own cultures of the strain.

 

6. How can I find information about the BSL of the strain and the recommended safe handling of the strain in my laboratory?

Please don’t order strains of fungi if your laboratory is not used to handling them. Information on the Hazard Group classifications can be found on the Advisory Committee on Dangerous Pathogens (ACDP) Approved List of Biological Agents.

 

1. How is cDNA generated at ECACC?

2. What quality tests are carried out?

3. What concentration and quantity of cDNA will I receive? What format will it be in?

4. How long will it take to receive my cDNA?

5. How stable is the DNA and how should it be stored?

6. How much cDNA should I use in my assay?

7. Why should I purchase my cDNA from ECACC rather than generating my own?

8. Do I get a certificate with my cDNA? If so, what information will be on it?

 

 

1. How is cDNA generated at ECACC?

ECACC cDNA can be derived from any human cell lines in the ECACC collection. 1-2 x 10⁶ cells are harvested during the log phase of growth and the RNA extracted before reverse  transcription is carried out to generate single stranded cDNA.

2. What quality tests are carried out?

ECACC checks the extracted RNA for degradation using gel electrophoresis and the presence of genomic DNA is tested for using PCR primers designed to recognise intronic sequences found in genomic DNA.

To check for the presence of cDNA ECACC use PCR using β-actin primers (specific for cDNA) that do not recognise processed pseudo genes (intronless copies of the gene within the genome).

All cell banks that cDNA is generated from are fully authenticated and rigorous in process protocols ensure quality and traceability.

3. What concentration and quantity of cDNA will I receive? What format will it be in?

It is not currently possible to quantify total cDNA so ECACC offer a standard single aliquot comprising 20µl of cDNA in a 2ml tube. Alternatively, ECACC provide cDNA samples in 96 well formats as required. Your cDNA will be dispatched on dry ice to your laboratory via courier. Larger quantities of cDNA are available; please contact us to discuss your requirements.

4. How long will it take to receive my cDNA?

Normally cDNA will be dispatched within 10 working days of receipt of an order. However, on occasion we may need to extend this time line and in those cases you will be contacted by the ECACC customer support team.

5. How stable is the DNA and how should it be stored?

ECACC recommends that cDNA is stored at -80^oC when not in use; repeat thawing should be avoided so we recommend making smaller aliquots of the cDNA after the first thaw.

Some laboratories have reported storing cDNA at -20^oC and +4^oC for extended periods (1-2 months) however there is no stability data for these temperatures currently available. ECACC is currently in the process of carrying out a stability trial, the results of which will be posted on the Culture Collections website as they become available.

We currently recommend using the cDNA within 6 months of manufacture. The date of manufacture can be determined from the lot number. The first two numbers correspond to the year, the second two digits correspond to the month and the last four digits are a consecutive number assigned to the samples we process. So, for example, a sample with lot number 13010001 would have been the first sample processed in January 2013.

6. How much cDNA should I use in my assay?

This will depend on your assay; we recommend checking the literature or contacting the manufacturer of the assay you are using before ordering, a standard single cDNA order consists of 20µl of cDNA. If after this you require further help, the ECACC scientific development team will be happy to advise you.

7. Why should I purchase my cDNA from ECACC rather than generating my own?

Using ECACC cDNA should save you both time and money.  Procuring cDNA from us saves you the time and expense of purchasing cell lines, culturing cells, extracting RNA and generating cDNA only to find the cell line doesn’t contain the gene of interest. ECACC can provide authenticated, quality cDNA in a format to suit you that allows you to progress your research project with confidence.

8. Do I get a certificate with my cDNA? If so, what information will be on it?

ECACC provides certificates of conformity (C of C) on request for cDNA that includes:

• Name of the source cell line

• The ECACC catalogue number of the source cell line

• ECACC Lot (batch) number of the source cell line

• ECACC lot (batch) number of the cDNA