How do I Define Purity?

Many descriptions of purity exist, but how do they relate to laboratory vessels that come into contact with valuable biological material and which serve a diverse range of experiments? It is crucial that the sample will not be compromised or even destroyed due to possible impurities, and it is equally important to ensure that analytical results will not suffer. Thus, both the sample type and the reaction will determine the most important purity criteria for any given experiment.

Sample material in biological laboratories mainly comprises cells, microorganisms, nucleic acids and proteins that are either processed or analyzed directly. Critical contaminations arising during cell culture are dominated by the faster growing microorganisms (such as bacteria or molds) and infecting mycoplasma, but substances such as endotoxins that are detrimental to cells also play a role. Bacterial cultures, too, can be overgrown with other strains or molds. Nucleic acids and proteins are compromised primarily by molecules; nucleic acids, for example, are threatened by nucleases – enzymes which degrade nucleic acids. This is especially true for RNA, as it is inherently unstable, and RNases are practically ubiquitous. On the other hand, nucleic acids themselves, in particular DNA as the more stable molecule, present a source of contamination. Where sensitive techniques such as PCR are concerned, components of the contaminating DNA are likely to be amplified, thus leading to false positive results. Further to sample-specific factors, it is possible that contaminations may inhibit certain processes, and enzymatic reactions are particularly affected. In the case of PCR, contaminants are considered PCR inhibitors.

The purity criteria applied to vessels containing samples that emerge from the requirements outlined above can be summarized as follows: if I work with live organisms such as cells or microorganisms that will continue to proliferate, sterility is of the essence, and during cell culture, endotoxins must not be allowed to enter the sample. If nucleic acids constitute my sample material, the vessels must be absolutely free from nucleases – especially when working with RNA. If I carry out DNA-amplifications, I must be certain that no foreign DNA will contaminate my sample.

How can I ensure that I am using vessels of a purity grade that corresponds to the requirements of my applications? After answering the question which type of sample material I am working with and which experiments will be performed, the required purity criteria will become evident. The next step will then address the question whether these criteria can be satisfied by vessels that are already on hand in the lab, and whether it is possible for me (if necessary) to remove contaminants myself. Removal of germs can be accomplished via sterilization processes such as autoclaving, which is the standard laboratory method for this purpose.

It is, however, crucial to establish that the vessel material is heat-resistant. The situation is quite different where molecules such as DNA, RNase and endotoxins are concerned. They are very stable and either cannot, or not entirely, be removed or inactivated by autoclaving [1]. The same is true for inhibitors. It is therefore recommended to use plastic consumables that are ready-to-use and that satisfy the required purity criteria. In many cases, products are available in different purity grades that are pre-configured for certain applications, such as the Eppendorf purity grade PCR clean which features a combination of purity criteria which are relevant for amplification techniques. A lot specific test by an independent laboratory is performed on human DNA, DNase, RNase as well as PCR inhibitors and only lots fulfilling all test criteria will be certified.

Figure 1: Lot-specific test-report for Eppendorf PCR Consumables.

(1) G. Elhafi, C. J Naylor, C. E. Savage & R. C. Jones (2004): Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction, Avian Pathology, 33:3, 303-306.