And now to something not so medical.... …but with the same biofilm techniques

1. And now to something not so medical.... …but with the same biofilm techniques

2. Diversity in Pseudomonas putida biofilm Biofilm as an industrial capacity Exercise 4 Anne-Mette Juel Christensen PhD Student, Infection Microbiology group August 2014

3. Biofilm as the bad guy…

4. Biofilm as the bad guy… • Surfaces – a space to be occupied! • Localizing cells in close proximity • Communication • Exhange of genetic material -> establish niche growth • Protection from environmental challenges e.g. antibiotics, host immune response, toxic compounds • Extracellular polymeric substances (EPS) forms a barrier • Creation of dorment cells • Differentiation of cells into subpopulations e.g. non-motile -> heterogeneity -> Robust community of cells that causes problems for the host

5. Biofilm as the good guy… -> Robust community of cells that can be… • Resilient to variety of environmental stress • Potent biocatalyst of toxic and insoluble products In industry: • Microbial subpopulations in industrial fermentations • Undesirable bi-products • Alter substrate uptake • Growth reduction • Alter metabolic activities

6. Exercise 4: Diversity in P. putida biofilm • Industrial relevant production organism – Pseudomonas putida • Non-pathogenic • Biofilm capability • Produce compounds interesting for biofuel production -> isobutanol • Isobutanol toxic to the cell • Pseudomonas sp. VLB120 • originally isolated from forest soil • has shown to be able to grow on a wide range of carbon sources • grow in the presence of different solvents e.g. toluene and styrene • GFP tagged

7. Aim of the exercise • Study diversification and change of biofilm structure of Pseudomonas sp. VLB120 biofilm when exposed to 2.5 % isobutanol during growth in FB medium with citrate as carbon source in the flow chamber system.

8. Experimental procedure Assembly of the biofilm system, preparation of medium +/- isobutanol Sterilization and washing. Preparation of cultures Inoculation of flow chamber channels CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Done for you  COMSTAT and Imaris analysis Phenotypic characterization: Stability, growth, CV staining of biofilm, motility Select morphology variants for phenotypic characterization

9. Biofilm set up FB medium + 1 mM citrate FB medium + 1 mM citrate + 2.5 % isobutanol • Each team has one flow cell with. • 2 channels treated with FB medium + citrate + 2.5 % isobutanol and 1 channel treated with FB medium + citrate • OR • - 1 channel treated with FB medium + citrate + 2.5 % isobutanol and 2 channels treated with FB medium + citrate

10. Confocal laser scanning microscopy

11. Identification of variants • Sample from effluent tubes • Plating on selective media

12. Day 2 – Monday 11th august • Morning: • CLSM • Prepare LB PIA plates • Afternoon: • CLSM cont. • Collect effluent in epp. tubes – keep sterile! • Make dilutions in 0.9 % NaCl and plate on LB PIA plates • Incubate ON at 30 °C Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

13. Slight change of experimental set-up • Flow cell 1 • VLB120 wt • VLB120 wt • VLB120 + isobutanol from t=0 • Flow cell 2 • VLB120 wt • VLB120 + isobutanol from t=0 • VLB120 + isobutanol from t=0 • Flow cell 3 • VLB120 wt • VLB120 wt • VLB120 + isobutanol from t=0 • Flow cell 4 • VLB120 wt + isobutanol from day 4 • VLB120 + isobutanol from t=0 • VLB120 + isobutanol from t=0 • Flow cell 5 • VLB120 wt + isobutanol from day 4 • VLB120 wt + isobutanol from day 4 • VLB120 + isobutanol from t=0 Only microscopy and effluent collection on VLB120 wt and VLB120 wt + isobutanol from day 4

14. Day 4 (day 3 in manual) • Team 1-3 -> Pseudomonas sp. VLB120 wt • Team 4-5 -> Pseudomonas sp. VLB120 treated with 2.5 % isobutanol at day 4 • Look for variants on plates • Take pictures (1.st floor, lab 101 – Leica) • Select 4 variants and re-streak on LB PIA plates • Incubate ON at 30 °C • Keep original plates at room temperature Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

15. Day 5 (day 4 in the manual) Stability: • Compare original plates (pictures) with re-streak. Stable? • Leave plates at room temperature (to be used for motility) Growth study and crystal violet biofilm staining (CV staining) • If stable inoculate single colony in 10 mL LB • Incubate ON at 30 °C • Prepare FB medium + 1mM NaCitrate Motility (Swimming, swarming & twitching): • Prepare 5 plates for each motility type Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

16. Day 6 (day 5 in the manual) Growth study: • 50 mL LB medium in 100 mL flasks • Measure OD600 on ON culture -> inoculate to a conc. Of OD600=0.01 • Measure OD600 at appropriate intervals e.g. every hour • Calculate doubling time and compare with wt Crystal violet staining: • Dispense 140 uL in all wells. • Add 10 uL ON cultures • Remember blanks Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

17. Day 6 (day 5 in the manual) Motility: • Twitching (bottom) Swarming (top) • Swimming (middle) • Incubate ON at 30 °C Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

18. Day 7 (day 6 in the manual) Crystal violet staining: • Wash away planktonic cells • Stain with 0.1 % CV • Wash wells • Dissolve stained biofilm with 96 % EtOH • Measure OD Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis

19. Day 7 (day 6 in the manual) Day 2 Day 3 Day 4 – 6 Day 7 CLSM image acquisition (Images for COMSTAT and Imaris), plating of effluent cells Select morphology variants for phenotypic characterization Phenotypic characterization: Stability, growth, CV staining of biofilm, motility COMSTAT and Imaris analysis