Biofilm investigations in washing machines An overviewaboutavailablemethodstoassessbiofilmremovalin washing processes Caroline Amberg, Daniel Faeh Swissatest testmaterials ag, St. Gallen, Schweiz Düsseldorf, 19th of May, IDC 2015 1
Biofilms: A fascinating micro-world P. Dirckx, Center for Biofilm Engineering, Montana State University, Bozeman Biofilm = Community of microorganisms embedded in a extracellular polymer matrix(eps) Bacteria, fungi, algae, protozoa EPS = exopolymere substance consist of polysacharides, proteins, lipids, nucleic acids 2
Biofilms Wastewater treatment Bioremediation Copper leaching Symbiosis with plants Production of certain biochemicals Bacterial infections like Otitis media, Cystic fibrosis, legionella disease Implants Hospital aquired infections (catheters) Resistant to antibiotics Diverse problems and expenses of industries 3
Biofilm challenge in washing machines Mild washing conditions: Low temperature cycles + Detergent without bleach + Short washing cycle Biofilm formation and Soap scum/ soil residues Malodor of washing machine and washed textiles Reservoir of potentially pathogenic germs 4
Biofilm Standards ASTM E2562_12: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with High Shear and Continuous Flow using CDC Biofilm Reactor Growth and sample of standardized P. aeruginosa biofilm for different applications. Later on also a biofilm removal treatment could be simulated in the reactor or in an external treatment High shear environment, firmly attached biofilm Determination of microbial counts ASTM_E2871_13: Standard Test Method for Evaluating Disinfectant Efficacy Against Pseudomonas aeruginosa Biofilm Grown in CDC Biofilm Reactor Using Single Tube Method Growth of P. aeruginosa biofilm according to E2562_12. Disinfectant test in a single 50 ml tubes Determination of microbial counts 5
Biofilm Standards ASTM E2647_13: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown Using Drip Flow Biofilm Reactor with Low Shear and Continuous Flow Growth and sample of standardized P. aeruginosa biofilm for different applications. Later on also a biofilm removal treatment could be simulated in the reactor or as an external treatment Low shear environment, loosely attached biofilm Determination of microbial counts ASTM E 2196_12: Standard Test Method for Quantification of Pseudomonas aeruginosa Biofilm Grown with Medium Shear and Continuous Flow Using Rotating Disk Reactor Growth and sample of standardized P. aeruginosa biofilm for different applications. Later on also a biofilm removal treatment could be simulated in the reactor or as an external treatment medium shear environment, medium attached biofilm Determination of microbial counts 6
Biofilm Standards ASTM E2799_12: Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay Disinfectant testing against P. aeruginosa biofilm Only screening test Determination of microbial counts(quantitative) or optical density(qualitative) 7
Labscale testing method (Impact on biofilm formation) 1) Growth of a young biofilm 24 h incubation at 30 C in growth medium Mixed biofilm Pseudomona s aeruginosa E. coli Staphylcoccus aureus Microbial growth in the solution OD 595 2) Simulated washing test(s) Simulated washing machine cleaning test Quantification of growth in the suspension Microbial growth in the solution OD 595 4 h Incubation at 30 C in medium 3) Biofilm quantification Quantification of biofilm amount Rinsing to remove planctonic cells Crystal violet assay
Biofilm reduction test in a washing machine Microbial counts of mixed biofilms before washing cycle Microbial counts of mixedbiofilmson carrier after washing cycle 24 h incubationat 30 C in growth medium Biofilm coupon is analysed Pseudomonas aeruginosa E. coli Staphylococcus aureus Koch-/Buntwäsche 30 C Nodetergent, liquid detergent, IEC-A* Sterile cotton or Polyester swatches Microbial counts of formersterile textile swatches Textile swatches areanalysedafter washing cycle 9
Comparison of labscale test and in situ test Lab scale test Mixed 24-hours old biofilm with E. coli, Staphylococcus aureus, Pseudomonas aeruginosa biofilm in a 96- well microplate Simulatedwashingtest30minmain wash,2rinsingcyclesà10minutes Biofilm analysis via microbial plate counts Temperatur: 60 C Detergent / cleaner: liquid detergent, IEC-A*, WM cleaner 1, WM cleaner 2, WM cleaner 3 InSitutestinthewashingmachine Mixed 24-hours old biofilm with E. coli, Staphylococcus aureus, Pseudomonas aeruginosa biofilm on polypropylene supports 60 Ccycle,shortprogram,1h18min, 35to37L waterconsumption Biofilm analysis by microbial plate counts Sterile laundry load of cotton or Polyester Detergent / cleaner: liquid detergent, IEC-A*, WM cleaner 1, WM cleaner 2, WM cleaner 3 10
Comparison of labscale test and in situ test Lab scale test: better differentiation between the three washing machine cleaners Less remaining biofilm in the washing machine Resultingtrendsareequalin thelab scaletestandin thein situ test in the washing machine Mechanicalactionhasa remarkable impact on biofilm removal The lab scaletestisableto represent the situation in the washing machine With the mechanical action of thein situ test, morebiofilmis removed and some products have a better efficacy against biofilm In thelab scaletestthe differention between products is more clear 11
In situ test in the washing machine More complexdatafromthein situ testin thewashingmachine Test microorganisms behave differently 12
Summary Biofilms can be either beneficial or harmful A potential health risk and/or odor formation is a result of biofilm in washing machines Several biofilm standards exist where the production of reproducible biofilms is described. Pseudomonas aeruginosa biofilms are used in this standards. One standard (ASTM E2799_12: ) for testing disinfectants efficiency against Pseudomonas aeruginosa biofilms Standard test procedure to test washing processes / laundry products and additives were developed by Swissatest Lab scale test method for systemic investigations In situ test method for more consumer relevant test
Thank you for your attention 14