Explanation for the area distribution shape given the uneven surface of the insert.

<p>A) Scanning electron microscope image showing the uneven surface of a sterile insert; B) Schematic of the uneven insert surface with biofilm growth, HDPE roughness height is commonly taken in modelling to be 20 μm, 80 μm was the greatest depth measured in Day 28 biofilms. Broken red lines indicate parts of an example area distribution curve and the corresponding cut through position of the biofilm.</p

Fluorophores (fluorescent stains) evaluated for their use in visualising cells, proteins or carbohydrates within drinking water biofilms.

<p>Fluorophores (fluorescent stains) evaluated for their use in visualising cells, proteins or carbohydrates within drinking water biofilms.</p

Drinking Water Distribution System (DWDS) simulation pipe facility and Pennine Water Group (PWG) Coupon.

<p>A) Test facility, total volume (tank and loops) 4.5 m³, tank volume 1.53 m³, MV = Manual valves used to separate the three loops, 1a, 2a and 3a indicate the 5^th coil of each loop into which PWG coupons were inserted, 1b, 2b and 3b indicate the 50 mm internal diameter pipes containing flow meters (FM) and control valves; B) Detail of loop arrangement, T = turbidity meter, CV = control valve, other annotations as for A); C) Coupons secured in the apertures; D) HDPE PWG coupon and rubber gasket, to ensure a watertight fit; E) PWG coupon components (insert for microscopy and outer coupon for molecular analyses) and dimensions.</p

Cluster analysis of similarity using fingerprint profiles to show the similarity between A) bacterial, B) archaeal and C) fungal drinking water biofilm communities.

<p>Relative abundance data was derived from T-RFLP or ARISA analysis, sample identification numbers are shown and clusters are indicated with a bracket and number. Red lines indicate profiles not significantly dissimilar according to SIMPROF analysis.</p

Oligonucleotide primer pairs used to amplify 16S rRNA genes and ITS regions, PCR cycling conditions used in each case are indicated.

<p>Oligonucleotide primer pairs used to amplify 16S rRNA genes and ITS regions, PCR cycling conditions used in each case are indicated.</p

An example of the arrangement, volume and distribution of cells, carbohydrates and proteins of drinking water biofilms.

<p>A) 3D projection of example Day 0 and Day 28 biofilms, plotting region shown is 420 μm × 420 μm × 30.6 μm and 420 μm × 420 μm × 94.4 μm, respectively; B) Volume (log) of biofilm components at Day 28, relative to the thresholds used in digital image analysis, each data point (n = 25) represents a FOV, box and whisker plots show the range, interquartile range and median—indicated by the solid black line; C) Day 28 area distribution plot, each line (n = 25) indicates a FOV, note the different x-axis scales between components. Area coverage fraction refers to the proportion of each XY image of the Z-stack covered by the particular component, blue dashed line at “0” indicates the cell peak location; peak location is the aligned slice number at which the maximum area fraction occurs. Area fractions for carbohydrates and proteins are plotted relative to cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115824#sec002" target="_blank">Materials and Methods</a> for details).</p

Emission spectra (fingerprints) of the triple stain combination targeting cells (Syto 63), carbohydrates (Con A Rho) and proteins (FITC), at each excitation wavelength.

<p>Note that plastic/biofilm autofluorescence is also shown and that the whole spectrum in each case was used in linear unmixing. A) Emission at 488 nm excitation, image settings for FITC; B) Emission at 543 nm excitation, image settings for Con A Rho; C) Emission at 633 nm excitation, image settings for SYTO 63.</p