Laboratory Products


Achieving Asepsis - Selecting a sterilisation procedure Gareth West, Astell Scientifi c Ltd


The sterilisation of microbial life and biological material is a prerequisite of both experimental and environmental control in laboratories across myriad disciplines.


Whether ensuring aseptic conditions in a culture plate or maintaining an area of biocontainment, sterilisation’s goal is always the eradication of unwanted biological agents. This aim can be reached through a variety of procedural pathways, each with their own benefi ts and shortcomings.


The ideal sterilisation process needs to work quickly and effectively; deactivating microbiology and unwanted biological material such as prions. It should also ensure the minimal toxicity, health risks to operators, and change to sterilised objects, while providing maximum adaptability - accommodating different materials. The procedure should also be able to overcome any physical resistance to sterilisation that is demonstrated by the materials being sterilised. All of this should ideally be achieved in a cost-effi cient way, which also allows for consistent monitoring [1, 2].


Meeting all of these conditions, however, is frequently impractical. Consequently, incorporating many of these factors is often a secondary concern based on situation and necessity, with the primary goal being achieving asepsis in a functional end product. What follows is an assessment of commonly available sterilisation methods, their positives, and negatives.


Sterile Filtration


If a fl uid material is to be sterilised, fi ltration forms an option for consideration. A liquid or gas can be passed through a sterilising fi lter membrane, which forms a mechanical barrier to all particles of a larger diameter than the pores in the membrane. Microorganisms bigger than the pores are trapped behind the fi lter, assuring they cannot enter the fi ltrate.


Heat, radiation and chemical sterilants work by changing the physical structures of molecules and organelle within microorganisms - a process which can also change the structural components of more sensitive substances. Filtration has no such effects, only removing particles over a certain size. As such, fi ltration forms a plausible choice for more unstable and reactive fl uids.


Unlike other sterilisation methods, fi ltration does not deactivate microbiological entities. As such, other sterilisation procedures - often heat or radiation - are required to sterilise the fi lter and residue post-processing.


Close attention must also be paid to the size of pores in the fi lter. Frequently, a pore size of 0.2µm is used - and with the smallest mycoplasma measuring around 0.3µm - this is suffi cient for fi ltering out most bacteria. Yet ultramicobacteria can measure less than 0.1 µm [3, 4, 5], while viruses and prions are often smaller still. Reducing pore sizes to 0.001µm ensures fewer microbiological entities can enter the fi ltrate, although this becomes increasingly prohibitive to fl uids that can be fi ltered. A decrease in pore size also increases processing time - a 0.1µm pore fi lter will have around 40% of the fl ow rate of a 0.2µm pore fi lter.


Radiation


As a sterilant, radiation takes multiple forms. Take, for example, ionising radiation sterilants; including x-rays, gamma rays, and electron beams. These technologies sterilise by electromagnetically exciting particles in the area being sterilised, causing them to release free radicals. These radicals combine with the double bonds in biomolecules, (such as DNA, RNA, and enzymes,) changing their form, stopping their function, and leading to the invalidity of the microorganisms containing them. Non-ionising radiation sterilants work by causing new bonds - pyrimidine dimers - to form between nucleobases in DNA, which - like with ionising radiation - changes the macromolecule’s structure and stops it functioning.


Due to their functionality, radiation sterilants can degrade organic chemicals such as plastics [6, 7] and biological material. Unfortunately, their degradative effects do not extend to prions which remain largely too stable to be affected by radiation sterilants [8]. However, it is worth noting that some studies show a possibility of prion denaturation via gamma radiation [9].


A further important consideration is that the penetrative ability and processing times of radiation sterilants vary signifi cantly. Ultraviolet Light (UV) is only able to penetrate transparent materials, which makes it a viable sterilant for air and purifi ed reverse osmosis water, but it is limited to a surface sterilant for opaque substances. UV’s processing time can also run into hours, as is the case with X-rays and gamma. However, these latter two methods bring their own advantages.


Gamma and X-rays are highly penetrative, meaning they can be used to sterilise objects held within otherwise impermeable containers. Electron beam sterilisation’s party piece is to sterilise near-instantaneously. Often it is used as a surface sterilant due to the electron particle’s poor penetrative abilities - however given enough energy it too can deeply permeate matter.


The running costs for X-ray, e-beam, and UV can be relatively low, with effi cient electricity use an attribute of each. X-ray, e-beam, and gamma hardware have a high initial capital cost [10]. Gamma sterilisation has an ongoing cost associated with replacement of its radiation source, commonly the colbolt-60 isotope. It should, however, be recognised that while operators should always be shielded from the damaging effects of all radiation sterilants, the physical presence of radioactive material in gamma sterilisers mean extra precautions should be made during maintenance of the device.


Chemical


Chemical sterilants encapsulate a wide range of liquids and gases with one unifying feature - their ability to destroy microbial life. Due to these characteristics they are often harmful to humans, if not deadly. The most commonly used chemical sterilant - ethylene oxide - can cause headaches, nausea, vomiting, diarrhoea, shortness of breath, respiratory irritation, lung injury, and cyanosis in the short term, while long-term exposure is associated with occurrences of cancer, mutagenic changes, neurotoxicity, and sensitisation [11].


A selection of autoclave steam sterilisers


INTERNATIONAL LABMATE - FEBRUARY 2021


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