Alcohol fixation of protein to surfaces

The phenomenon of protein fixation by alcohol has been well documented, with HTM 01-05 advising caution in the use of alcohol as a cleaner and disinfectant. The objective of decontamination procedures is to reduce the risk of cross-infection to patients and staff, accomplished by a combination of cleaning and disinfection operations.

Cleaning means removal of any soiling from surfaces. Within soiling matter, pathogens and proteinaceous material are likely to be present. Disinfection means deactivation of pathogens. Disinfectants work best in clean conditions, so disinfecting without cleaning can be limited in effectiveness if the disinfectant used is unable to provide an effective cleaning action for all the soil types present.

Alcohol solutions act rapidly without leaving any surface residue, providing effective inactivation of a wide range of microorganisms. Their fast-drying property is seen as an advantage in the fast turnaround of patients, and the availability of a wide range of alcohol-based preparations has led to widespread adoption of these products in dental decontamination.

Why is protein fixation problematic?

On clean surfaces requiring disinfection, there is little soiling matter present and inactivating any remaining microorganisms is the main focus. Where extensive soiling matter is present, its removal is important if complete disinfection is to be achieved. If proteins are present and an alcoholic solution used as a disinfectant, proteinaceous material can be fixed to the surface, possibly trapping microorganisms and providing additional surface roughness to aid adhesion of further soiling. The potential for fixation of protein that is potentially contaminated with prions should be considered as a significant risk factor in decontamination.

Demonstrating the effects of alcohol and protein fixation

Protein can be fixed onto a wide range of solid surfaces, including metals, plastics and coated materials commonly found in dental surgeries. Our study used stainless steel (grade 316), dental handpieces, ABS plastic and melamine-coated particleboard to demonstrate fixation.

Stainless steel plates were cleaned using a detergent solution, rinsed and treated to remove any traces of surfactant, oil or grease. Bovine serum albumin solution was evenly applied to the surface of each plate and allowed to dry.

Handpieces were cleaned using a detergent solution, rinsed and treated to remove any traces of surfactant, oil or grease. They were dipped in a BSA solution and allowed to dry.

Application of disinfectants to metal surfaces

Wet wipes: The surface was uniformly wiped once, then again with a fresh wipe. Wiped surfaces were allowed to dry followed by a cold-water rinse process in a washer-disinfector.

Solutions: Test pieces were immersed in the relevant alcohol solution for the required contact time, rinsed and left to dry. A cold-water rinse process was then carried out in a washer-disinfector.

Quantification of residual protein

Residual protein was assessed using the ProReveal system. ProReveal reagent is sprayed over the item’s surface which is then placed inside the ProReveal Viewer where specially configured lighting causes the spray to fluoresce where it has reacted with any residual protein. An image is displayed on screen which shows fluorescence on the instrument, indicating any remaining protein. Software assesses the fluorescence and hence the amount of protein remaining on the instrument*.

Preparation of plastic and coated materials

Melamine-coated particleboard and ABS white plates were cleaned, degreased and dried. The protein was prepared using fresh egg white with addition of the appropriate marker dye quantity.

Disinfectants on plastic surfaces

Disinfectants were applied via wet wipes in a standard pattern and with a standardised wiping technique (fig 6a).

Residual protein was assessed using the depth of remaining marker dye colour as an indicator. Application of a non-alcohol wipe across the previously untreated and alcohol treated areas demonstrates the presence of fixed protein.

Results

Fig 1 shows the effect of protein fixation on stainless steel by different alcohols with different contact times. The degree of fixation was similar for 30% and 70% solutions. Contact times of 30 seconds and 5 minutes produced fixation to the same extent.

Fig 2 illustrates the results obtained using dental handpieces. Significant fixation is seen using an alcohol-based wipe compared with a water-based preparation. In fig 3, similar results are seen when stainless steel plates are used as the substrate for protein fixation.

Fig 4 (b and c) give results for fixation of protein to melamine-coated particleboard and ABS surfaces. Again, protein is strongly fixed to these surfaces by alcohol and the fixed material is resistant to a subsequent detergent treatment. Fig 4a illustrates the experimental procedure that was used.

How can protein fixation be avoided?

To avoid protein fixation, disinfection using alcohol-based preparations or disinfectants with other fixatives can be carried out after thorough cleaning with a water-based formulation in a two-stage process. Alternatively, a faster, single-stage cleaner and disinfectant based on chemistry that does not fix proteins can be used.

Conclusion

Alcohol rapidly fixes proteins to hard surfaces. The degree of fixation is largely independent of the alcohol type, or contact time with protein, but is influenced by formulation specifics. Fixation of proteins is not observed when water-based formulations without alcohols or other fixatives are used.

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