Fundamentals and Applications of Biofilms Analysis, Structure and Physiology of Bacterial Biofilms Ching-Tsan Huang ( 黃慶璨 ) Office: Agronomy

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1 1 Fundamentals and Applications of Biofilms Analysis, Structure and Physiology of Bacterial Biofilms Ching-Tsan Huang ( 黃慶璨 ) Office: Agronomy Building, Room 111 Tel: (02) cthuang@ntu.edu.tw

2 2 Introduction Characteristics of biofilms Heterogeneous Biologically-active Dynamic Why study biofilm structure and physiology? Biofilm structure and physiology will be modified by microenvironmental changes such as the concentration or depletion of nutrients or inhibitors extracellular enzyme activity gas availability ph macromolecules

3 3 Introduction Quantitative v.s. Qualitative Direct v.s. Indirect Invasive v.s. Non-invasive Destructive v.s. Non-destructive

4 4 Qualitative Analysis Cryoembedding and cryosectioning Substratum Biofilm Side View OCT Compound Fluorescent Staining Annular Reactor Dry Ice Dry Ice Cryoembedding 5 m Thickness Cryosectioning

5 5 Fluorescent Process Excited-state lifetime Excitation Emission Source: Molecular Probes, Inc., Eugene, OR, USA

6 Excitation and Emission 6

7 7 Fluorescent Microscopy Source: Molecular Probes, Inc., Eugene, OR, USA

8 Confocal Laser Scanning Microscopy (CLSM) 8 Source: Molecular Probes, Inc., Eugene, OR, USA Source: Leica Inc.

9 9 Fluorescent Staining Viability: Respiratory activity CTC/DAPI Respiratory CTC CTC-formazan activity (colorless) (red crystal) Counterstain with DAPI Respiring (live) cells: red Nonrespiring (dead) cells: green CTC : 5-cyano-2,3-ditolyl tetrazolium chloride DAPI: 4,6-diamidino-2-phenylindole

10 10 Fluorescent Staining Viability: LIVE/DEAD BacLight bacterial viability kit Viability: Membrane potential Rhodamine Growth rate RNA/DNA Acridine Orange: single strand RNA fluores red double strand DNA fluores green

11 11 Fluorescent Staining Species identification Immunofluorescent staining (Antigen-Antibody) Fluorescent in-situ hybridization (FISH) Structure identification Nucleic acid (cells): Propidium iodide or Ethidium bromide Polysaccharides (EPS): Calcofluor white Enzyme activity -galactosidase: Fluorescein di- -D-galactopyranoside phosphatase: Enzyme-label-fluorescence

12 12 Quantitative Analysis Direct measurement Biofilm mass Biofilm thickness by microscopy by micrometer Biofilm density: Cell enumeration Viable cells: plate count Total cells: acridine orange, DAPI Indirect measurement Polysaccharides Phenol-H 2 SO 4 method Proteins Crude cell extract Microbial activities Respiratory activity Enzyme activity Reporter gene expression Chemical gradients ph Dissolve oxygen Chloride Calcium

13 13 Image Analysis: Basic concepts Digitization An image is a spatial representation of an object. Picture elements or pixels The values of f(x,y) are called gray levels If n bits per pixel are available, the number of possible gray levels is 2 n (2 8 = 256; 0 ~ 255) Analog-to-digital conversion Digital imaging Image out Data out Image in Image processing Image analysis Data in Computer graphics Data analysis

14 14 Image Analysis System CCD (charge-coupled device) cameras CCD camera is a semiconductor device that acts as a transducer between incoming light and electrical charge. Video CCD camera Scientific CCD cameras Color CCD camera: (a) using a single CCD chip in which each pixel is covered by a single color filter; (b) using photon sorting to separate incoming color image into three color channels: red, green and blue (RGB camera) Image analysis software Computer

15 15 A new approach Combination of fluorescent staining, cryoembedding, cryosectioning and image analysis Biofilm cultivation Fluorescent staining Epifluorescent microscopy Cryoembedding Image analysis (Digitization) Cryosectioning Data analysis

16 16 Image analysis Substratum Fluorescence Intensity Fluorescein Tetramethyl Rhodamine Biofilm Thickness ( m)

17 a d 17 b e c f Gradients in gene expression

18 h h Fluorescence Intensity h 4 h h 24 h Biofilm Thickness ( m)

19 Factors Affecting Structure and Physiology 19 Trophic state Nutrients presented in biofilm systems are heterogeneously distributed in both time and space Oligotrophic environment Oligotrophic environment means the carbon flux less than 1 g-c m -3 day -1 Oligotrophic = nutrient-limited Attached bacteria can grow in conditions where the nutrient concentration is below the level required by most organisms Eutrophic (copiotrophic) environment Nutrient flux is at least 50-fold higher than that found in oligotrophic environment and the available carbon did not fall to zero for a prolonged period.

20 20 Response to Adverse Environments Under starvation, physiological and morphological adaptation is evoked to maintain viability Miniaturization in cell size Reduce endogeneous respiration Increase surface to volume ratio Increase levels of certain catabolic enzymes and decrease levels of some anabolic enzymes Starvation gene expression Carbon starvation in E. coli may induce more than 40 starvation genes Phosphorus starvation may lead to the induction of phosphatase

21 21 Biofilm Structure Heterogeneous structure Substratum 7 Mushroom-like conceptual model more

22 22 Biofilm Structure Extracellular production G B Pseudomonas aeruginosa PAO1 Biofilm cryosection stained with Calcofluor white (EPS) and Ethidium bromide (cells) 50 U

23 Biofilm Physiology 23 Species distribution Microbial Ecol. 33: 2-10, Cell Concentration (cfu ml-1) Biofilm Density (cfu cm -2 ) P. aeruginosa K. pneumoniae Time (h) P. aeruginosa K. pneumoniae Time (h) 30 35

24 24 Biofilm Physiology Growth Biotechnol. Prog. 12: , Intensity Ratio Growth Rate (h -1)

25 25 Biofilm Physiology Gradients in respiratory activity Gradients in gene expression Appl. Environ. Microbiol. 64: , Appl. Environ. Microbiol. 61: , 1995.

26 P. aeruginosa Biofilms 12 h Nutrients, O 2 Substratum 24 h 36 h

27 P. aeruginosa Biofilms Growth and Starvation AO ELF Nutrients, O 2 Substratum

28 P. aeruginosa Biofilms Growth and Starvation AO ELF Nutrients, O 2 Substratum

29 P. aeruginosa Biofilms Pulse-Chase-Pulse High P 48 h Low P 12 h High P 48 h Low P 12 h Nutrients, O 2 Substratum

30 K. pneumoniae Biofilms In response to phosphate starvation 8 h 24 h