SubscribeYour choice of Laboratory Plastics can have a profound effect on your samples.
Most scientists will carefully choose and evaluate reagents and chemicals used for their sample preparation and analysis to find the purest, highest quality materials that guarantee the best results. But, how many actually consider the sample transfer tools and storage vessels with which all their materials come into contact?
Many may be completely unaware that the quality of the plastic consumables used in their experimental work can have a profound effect on their reactions. There must be maximum confidence in the products used, that they will not interfere with samples or alter experimental results by introducing contamination or degrading samples over time.
Sample preparation and storage should be considered separately, although some issues do apply to both. It is important that plastic wear is evaluated for the individual criteria specific to the required application e.g. accurate sample delivery from pipette tips or no leaching of polymer additives into samples during long term storage.
In this article we review the importance of accurate and precise volume delivery, without contamination when preparing reagents and the effect that the plastics consumables chosen could have on the long term integrity of samples.
Effective sample preparation is paramount in all research projects, getting it wrong at the beginning can have serious downstream implications not only on the findings of the project but also the time, money and resources used. Ensuring the correct equipment and consumables are used is as important as the actual protocol and reagents and can have both short and long term effects on your research.
Have you ever had a PCR reaction just not work? Checked all the variables you can think of, re-optimised and removed all possible sources of contamination and still it doesn't work? Well it may have been caused by one of the most routinely used consumables in your laboratory - Your Pipette Tips! Yes they really can inhibit your PCR reactions and other processes.
Pipette tips vary widely between manufacturers. Different moulding methods, production conditions, grades of polypropylene and combinations of various polymers are used. These can all affect the quality of the resulting tip, the accuracy and precision of sample delivery, risk of contamination or experimental interference.
Some manufacturers incorporate additives into their plastics with the aim to improve performance. During the injection moulding process, additives such as silicon can be combined with the polymer or a coating applied to produce a so called low retention tip for aiding sample recovery. However, this step is often employed simply to alleviate liquid retention caused by inferior moulding or the use of lower grade plastics.
Polypropylene is naturally hydrophobic and so will not retain sample on the inside of a tip manufactured in a high quality mould where no flashes or burrs are produced. Low retention tips can be produced without the need for additives by using 100% pure virgin polypropylene and adhering to strict manufacturing processes and mould maintenance. These will ensure the inside of the tip does not have plastic burrs or inconsistencies that could trap the liquid.
Some manufacturers use a range of chemicals during their tip production processes. Clarifying agents may be added to produce highly transparent tips. Dyes may be included to produce a characteristic coloured pipette tip e.g. blue or yellow. Releasing fluids may be used to ensure that the finished products do not stick to the mould and become damaged once they have been cast. These manufacturing processes are proprietary, often researchers are not aware that the pipette tips they are using contain additives.
Any of these additives may have adverse effects on reaction solutions. The otherwise inert polypropylene polymer can exhibit characteristics such as the inhibition of PCR reactions. The additives which have been mixed with the polypropylene can leach from the plastic into the sample and so cause the inhibition.
These problems are being seen more and more with the increase in the use of very sensitive techniques such as Real Time PCR. With very expensive reagents used in such applications the risk of failure due to plastic additives comes at high cost.
Clean pipette tips are critical in Life Science research. To ensure high quality laboratory results, tips coming into contact with samples must be free of DNase, RNase, DNA, pyrogen and adenosine triphosphate (ATP). Understanding these typical sample contaminants and taking precautionary measures to reduce or eliminate them at their source is vital to protecting sample integrity.
Contamination originates both in and out of the laboratory. Human contact is the primary source of contamination of pipette tips. Poor handling procedures, such as not using protective gloves or inadvertently exposing tips to other contamination sources in the laboratory, can compromise tip cleanliness.
Laboratory personnel typically employ a set of procedures to assure sources of contamination in the laboratory are minimised. But, because sample contaminants can originate outside the laboratory, it is important to evaluate what controls have been adopted during production to assure that risks have been reduced or eliminated before the product arrives in the laboratory.
However, all tips are not created equal. Pipette tips are sometimes produced in environments not designed to reduce or eliminate contamination. Some tip manufacturers produce tips in unclean environments. There are yet some companies who produce so-called "contamination-free" tips, but are often moulding in open manufacturing areas, which may not be free from contamination. This means more risk to the integrity of laboratory samples and less quality assurance.
A clean manufacturing process specially designed to reduce the risks of contamination is the best way to ensure the cleanliness of pipette tips. This involves the implementation of critical procedures and controls at the tip production facility.
- Staff procedures and training
- Clean packaging
- Automated material handling
- Inspection
- Clean Room environment
- Certification
Rainin's Class 100,000 Clean Room - All Rainin tips are manufactured under the same exacting standards and conditions. Look for the bioclean label to ensure your tips are of the highest standard.
Automated material handling and tip racking ensures all Rainin tips are Free from DNase, RNase, DNA, pyrogens & ATP contamination.
All Rainin tips are batch tested to ensure the highest quality possible and undergo both in house & third party microbiology testing for added quality assurance.
Aerosol resistant filter tips are often used for applications where contamination prevention is of primary concern. These tips incorporate individual filters to protect samples and pipetting instruments from contamination that can occur due to aerosol transfer. Filter tips are especially important in critical DNA amplification and microbiology procedures where minute trace carryover can invalidate or interfere with experimental results.
They also protect pipettes and samples when using hazardous materials such as radioactive or corrosive compounds. There are two kinds of filters used in these tips, they are either a strong chemically inert matrix of pure virgin polyethylene, which does not shed or flake and have an average pore size of 20µm preventing aerosol passage without restricting air flow.
Some manufacturer's use a soft filter made from a polyethylene matrix embedded with cellulose gum particles, intended to self-seal on contact with liquid. However, the cellulose gum is not chemically bonded to the filter matrix and can flake or shed particles into the sample being transferred in the tip. These contaminating particles can distort or invalidate experimental results with the potential of inhibiting PCR reactions.
These self-sealing filters can also be problematic if overfilling occurs; samples are absorbed by the filter matrix therefore sample recovery is very limited, if at all possible. With filter tips containing pure virgin polyethylene filters samples are always completely and easily recovered without contamination.
Rainin Aerosol-Resistant Filters - Pure virgin moulded polyethylene filter matrix, won't shed, flake or crumble. (Enlarged photograph of Rainin aerosol-resistant filter. Average pores size 20 microns)
Having minimised the risk to your samples and ensuring accurate and precise volume delivery with high quality tips, it is vital to ensure that sample integrity is not diminished in storage vessels. After all, your reagents spend far longer in their tubes, vials or plates than they do during measurement and transfer.
Have you ever considered what is happening to your samples when they are stored for anything from a few hours to several days, weeks, months or even years? Could your choice of tubes or plates be affecting your samples?
Sample storage is probably one of the most critical areas for scientists in the laboratory but one which is invariably over looked and not taken seriously enough. The Medical Research Council recommend that "Primary research data (and where possible/relevant specimens, samples, questionnaires, audiotapes etc) must be retained in their original form within the research establishment that generated them for a minimum of ten years from completion of the project."1 The integrity of research samples needs to be safe guarded for long periods of time to allow for further experimentation to be undertaken or the re-running of protocols at a later date to confirm experimental results.
Experimental procedures often demand that samples are exposed to extreme diversities in temperature, being analysed on the bench at ambient temperatures, subjected to heating or boiling, then transferred for long term storage in temperatures as low as -190˚C in Liquid Nitrogen.
So, when considering suitable products for short or long term storage they need to be of the highest quality to withstand these extremes. Just as pipette tips vary considerably from manufacturer to manufacturer so can the moulding methods, production conditions, grades of polypropylene and variations of polymers used in storage tubes and plates. All of which could have an effect on your samples.
As with pipette tips some manufacturers will use additives and clarifying agents to improve the quality of the polymer used. However, if these additives can leach into your sample within a matter of seconds from your pipette tip just imagine what is happening during the length of time that your samples are stored.
Leaching of polymer additives could degrade samples and invalidate months of hard work, wasting time recovering or reproducing the materials, with the potential loss of irreplaceable or valuable materials.
Storage tubes/plates must have good chemical resistance, some long term storage requires the use of storage agents which can be highly reactive at extremes of temperatures and so need to be stored in a chemically inert plastic polymer such as pure virgin polypropylene.
The plastic needs to be able to withstand extremely low temperatures for extended periods of time without becoming brittle and cracking. The sealing method used must provide a 100% hermetic seal to ensure there is no leakage or cross contamination of samples, as well as allowing easy sample access and recovery when required.
Manufacturer | Centrifugal Force | Temperature Resistance |
TreffLab – ClickFit, LockFit | 38,000g | -190ºC - +121ºC |
Manufacturer A | 25,000g | Not Available |
Manufacturer B | 20,000g | -90ºC - +120ºC |
Data as specified in company literature | ||
Microtube Specification Comparison - TreffLab Microtubes are the highest specification tubes available.
Anachem provides an extensive range of pipettes, tips, tubes, plates and storage racks manufactured to the highest quality standards by companies such as Gilson, Rainin, TreffLab and SSI. These products ensure your samples are safe and the integrity of your research is protected at all stages of your experimental protocols.
For maximum reagent integrity it is also essential to consider effects on materials due to ambient temperature changes during procedures and manipulations. For example enzymes and PCR reagents may be sensitive to temperate changes and their effectiveness reduced if not stored on the bench top correctly. New innovations are now available that help make this easier and reduce the risk.
SSI has recently developed IsoFreeze™ PCR racks for holding 0.2ml PCR tubes or plates on the bench top or an SBS footprint rack that will fit onto automated liquid handling instrumentation keeping samples at refrigeration temperatures for four hours.
SSI Isofreeze SBS PCR Rack on the Gilson Cyberlab Automated liquid handling workstation.
Remember, it is not only the quality of the reagents used that can contribute to the success of your research. The equipment employed to transfer, prepare and store them can have a profound effect - choose wisely from the vast array of laboratory consumables available. Saving money by buying cheap, low quality plastics could cost you dearly in repeated experimentation and lost or damaged reagents.