BK - Biofilm Generation Methods Flashcards
(25 cards)
What are artefacts?
Artefacts - distortions/inaccuracies in the observed biofilm structure or behavior that are introduced by the experimental methods themselves, rather than representing the true biological reality
What is the trade-off between expediency and relevance when studying biofilms? (2)
- Expedient methods (e.g. high glucose) boost biofilm formation for ease of lab work
- However, such conditions may not reflect in vivo environments.
e.g.
High glucose - Used by labs when generating biofilms to give better/thicker biofilms but don’t really get environments with high glucose
pH - Microbiologists tend to use pH 7 because its convenient and organism grow well. But in many in vivo environments the pH is not 7 e.g. skin pH is 5.5
What factors are important to monitor when modelling biofilms in water systems? (4)
- Physico-chemical parameters: pipe material, roughness, age, corrosion
- Temperature and disinfection residual levels
- Effects of nutrient ingress (e.g. after a cracked pipe)
- Pathogen tracking in projects such as EC FP5 SAFER
Nutrient ingress - the entry or introduction of excess nutrients, such as nitrogen and phosphorus, into a water body.
What are the typical temperature ranges for biofilm formation in water distribution systems? (2)
Mains distribution supply: 4–20ºC
Building plumbing systems: 20–60ºC
What are key design features of on-line and off-line biofilm monitoring systems? (4)
On-Line:
- In situ devices (e.g. Robbins-type devices, side loop pipes, tap “biofilm sensors”)
- Directly measure biofilm formation in the actual system
Off-Line:
- Pilot scale systems (e.g. multi-km rigs like those in Nancy, Kuopio, Kempton) or lab models (flow cells, chemostats, annular reactors)
- Allow controlled experiments away from the actual environment
What microscopy and biochemical techniques are used to assess intact biofilms? (8)
Microscopy approaches:
- SCLM, EDIC for structure
- DAPI or SYTO-9 for total cell count
Viability assays:
- CTC for cell respiration
- Rhodamine 123 for proton motive force
- BacLight for cell integrity
Other analyses:
- Radiolabelled substrates for metabolism (“viability” and function)
- antibodies or rRNA FISH for tracking specific organisms
- Lectins and fluorophores for EPS
How are recovered/homogenised biofilms typically analyzed? (3)
- Microscopy (similar to intact biofilm techniques)
- Cell cytometry for detailed cell counts
- Community analysis (e.g. DGGE) and culture-based methods
What are the benefits (3) and drawbacks (2) of the microtiter plate assay for biofilm formation?
Benefits:
- High throughput screening
- Can incorporate coupons for direct microscopy
- Can screen for anti-biofilm compounds
Drawbacks:
- Direct observation is challenging
- Automated quantification may lack reproducibility
coupons are small, standardized surfaces (often made of stainless steel or other materials) used in labs to study biofilm formation and for monitoring biofilm growth in real-world applications
What is the purpose of the Minimum Biofilm Eradication Concentration (MBEC™) high-throughput assay? (2)
- To screen for anti-biofilm compounds using a standardized 96-well peg lid system
- Designed to avoid gravitational artefacts during biofilm growth
What are the main features of the MBEC peg lid system? (5)
- Contains 96 identical pegs made from plastic or other materials
- Each peg provides a biofilm growth surface averaging 109 mm²
- Designed to work with a corrugated trough that evenly distributes the inoculum
- The entire device is placed on a rocking table inside an incubator -> promotes uniform biofilm growth
- 96-well microtiter plates allows for setting up serial dilutions of antimicrobials to assess biofilm susceptibility
What are the advantages and drawbacks of lab flow cells in biofilm studies? (4)
Advantages:
- Provide flowing medium through transparent channels
- Suitable for direct microscopy and visualization
Drawbacks:
- Not robust; characterized by low shear and slow flow
- Potential for linear gradient effects in the biofilm
Why are transparent substrata important in biofilm microscopy? (2)
- They allow for direct observation of biofilm details such as differentiation and cell death
- Essential for studies requiring high-quality microscopy imaging
What is the function of the ATR-FTIR Ge Crystal flow cell? (3)
- Enables in situ chemical analysis of biofilms via infrared light measurement
- Uses different crystals (e.g. germanium) to grow biofilms
- Requires specialized setup for accurate chemical analysis
Distinguish the Robbins Device and Modified Robbins Device in biofilm research (7)
Robbins Device
- A robust in-line system used to study biofilm formation under controlled flow conditions
- Operates at high pressure, allowing the assessment of biofilms on various materials
Modified Robbins Device
- Functions as a lab model rather than an in-line system
- Operates at lower pressure
- May exhibit initial shear effects and linear gradients
- Not robust
- Offers reproducible conditions for controlled studies
What are the defining features of annular reactor models in biofilm studies? (6)
- Provide a defined environment with controlled physico-chemistry (pH, Eh, nutrients)
- Open flow system that simulates real-world conditions like pipes and tanks
- Reproducible but not as well mixed as chemostat
- Safe, no leaks (suitable for dangerous pathogens)
- Useful for evaluating biofilm structure, pathogen survival, biocide efficiency, and material biofouling
- May face challenges such as eddy current effects and gradients in the x-y directions
What configuration options are available in annular reactors for controlling biofilm formation? (5)
- Flow-through design using tap water as the feed
- Ability to alter influent water quality by adding various feed solutions
- Control over shear stress via the rotational speed of the inner drum
- Independent adjustment of hydraulic retention time through influent flow rate
- Biofilm sampling achieved by removing coupons, which can reveal gradients in biofilm development
How do shear stress and hydraulic retention time factor into biofilm reactor models? (3)
- Devices such as Robbins and annular reactors allow independent control of shear stress and retention time
- These parameters influence biofilm structure, thickness, and detachment
- Critical for simulating real-world flow conditions
What are the defining features of chemostat models
in biofilm studies? (7)
- Provide a defined environment with controlled physico-chemistry (pH, Eh, nutrients)
- Reproducible; well mixed
- Open flow system – model pipes, tanks
- Safe, no leaks (suitable for dangerous pathogens)
- Useful for evaluating biofilm structure, pathogen survival, biocide efficiency, and material biofouling
- Data obtained as found in the field
- Possible disadvantage - Shear forces not completely controlled. but no eddy current effects on biofilms
very similar to defining features of annular reactor models
What are the key components (4) and steps (3) in a chemostat model for biofilm studies?
Components:
- Media reservoir with essential nutrients
- Pump controlling flow rate
- Chemostat vessel where cells grow
- Effluent tube for removing spent medium
Steps:
- Continuous inflow and outflow to maintain a steady state
- Inoculation of microorganisms followed by controlled growth
- Monitoring of cell density and metabolic activity
What types of pathogens can be associated with biofilms in drinking water systems? (5)
- Indicator bacteria: E. coli, coliforms (e.g. Klebsiella)
- Pathogens: E. coli O157, Mycobacterium avium
- Opportunistic pathogens: Aeromonas hydrophila, Pseudomonas aeruginosa
- Microaerophiles: Legionella pneumophila, Campylobacter jejuni, Helicobacter pylori
- Protozoa: Cryptosporidium parvum, Acanthamoeba
What is the principle behind transition analysis in chemostat kinetics? (3)
- Uses the equation Pt/Po = e^(-Dt) to follow the transition between steady states
- Rapid exponential washout indicates turnover of existing biofilm material
- Slower transition may signal the selection of mutant or adapted organisms
How do the CDC Biofilm Reactor (3) and Propella reactor (3) differ in their applications?
CDC Biofilm Reactor:
- Designed for studying biofilm formation under moderate to high shear
- Used to assess sanitizer efficacy
- May have issues with biofilm sharing on coupons
Propella Reactor:
- Combines aspects of chemostat and annular reactor models
- Offers reproducible mixing and independent control over shear stress and retention time
- Can be connected to mains supply but may have leakage concerns
What is the primary application of the drip flow biofilm fermenter? (3)
- Used to study and model dental plaque
- Supports biofilm growth on slides with a drip-wise supply of growth medium (BioSurface Technologies Corp.)
- Emphasizes the role of flow and distribution in biofilm development
What is the purpose of constant depth film fermenters in biofilm research? (2)
- To simulate biofilms of a fixed thickness, particularly for dental plaque and contact lens studies
- Provides a model for biofilm formation on solid substrates with a thin, continuously replenished liquid film
