PowerPoint Presentation - … · 27.02.2018 · Introduction to the ... •Education and training ... Experimental system used to model the development of dairy biofilms on SS surfaces.
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▪ The goal when designing or selecting a laboratory system for estimating real world observations is to find the proper balance between field relevancy and practicality while achieving the statistical specifications required of a standard method.
The Goal
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Grow AnalyzeTreat SampleASTM Biofilm Methods
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ASTM Biofilm Methods ASTM Standard Method E2196
▪ Continuous stirred tank reactor
▪ Grows biofilm under moderate shear
Rotating Disk Reactor
Pseudomonas aeruginosa
Polycarbonate coupons
Tryptic soy broth: 300 mg/L INB & BNB,
30 mg/L CFNB – All 24 hr
200 rpm
6.7 mL/min
20oC
ASTM Standard Method E2647
▪ Continuous stirred tank reactor
▪ Grows P.a. biofilm under high shearBacterial air vent
Reactor top
Berzelius Pyrex
beaker
Polypropylene rod
Coupon
Baffled stir bar
Effluent spout
Media inlet/inoculation port
Set screws
L. Lorenz, S. Goeres 2007
CDC Biofilm Reactor
Pseudomonas aeruginosa
Polycarbonate coupons
Tryptic soy broth: 300 mg/L INB & BNB,
100 mg/L CFNB – All 24 hr
125 rpm
11.7 mL/min
20oC
ASTM Standard Method E2647
▪ Plug flow reactor
▪ Grows biofilm under low shear
▪ Air/liquid interfaceDrip Flow Reactor
Pseudomonas aeruginosa
Glass microscope slides
Tryptic soy broth: 3000 mg/L INB – 24 hr
3000 mg/L BNB – 6 hr
270 mg/L CFNB – 48 hr
10° angle
0.8 mL/min/channel
20oC
ASTM Standard Method E2799
▪ Batch reactor with gentle mixing
▪ Grows biofilm under very low shear
▪ Easily implemented in a standard
microbiology laboratory
▪ Useful when only a small volume of the test
compound is available
▪ Suitable for screening multiple bugs and/or
antimicrobials at a range of concentrations
MBEC Assay
P. aeruginosa
Tryptic soy broth:
Full strength INB – 24 hr
Dilute for growth
110 rpm
36oC
GrowTreat
MBEC Assay
1 2 3 4 5 6 7 8 9 10 11 12
A 100 100 100 100 100 50:N N UC SC
B 50 50 50 50 50 50:N N UC SC
C 25 25 25 25 25 50:N N UC SC
D 12.5 12.5 12.5 12.5 12.5 50:N N UC PC
E 6.25 6.25 6.25 6.25 6.25 50:N N UC PC
F 3.125 3.125 3.125 3.125 3.125 50:N N UC PC
G 1.5625 1.5625 1.5625 1.5625 1.5625 50:N N UC PC
H 0.7813 0.7813 0.7813 0.7813 0.7813 50:N N UC PC
▪ # = % Antimicrobial
▪ N = Neutralizer
▪ UC = Untreated controls
▪ SC = Sterility controls
▪ PC = Peg controls
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ASTM Standard Method E2871 ASTM Standard Method E2871
Single Tube Method
P. aeruginosa ATCC 15442
Borosilicate glass coupons
Sonicate/vortex vs
Scrape/homogenize
Methods…with benefits
▪ Teaching tools
▪ Communication
▪ Comparison
▪ Product registration
REGULATORY
INDUSTRYACADEMIA
Industrial
Associates
Collaboration
▪ Reduces/controls biofilm bacteria
(low level of efficacy)
▪ Kills biofilm bacteria (high level of efficacy)
▪ Prevents bacterial biofilm
▪ Removes bacterial biofilm
Biofilm Label Claims Center for Biofilm Engineering
30 min residence time 30 min residence time 30 min residence time
Room Temperature 36oC Room Temperature
300 mg/L INB300 mg/L BNB100 mg/L CFNB
10% INB10% BNB1% CFNB
30 g/L INB30 g/L BNB3 g/L CFNB
ASTM E2562 Modifications
Future modifications for method optimization:
▪ Growth temperature: 36oC
▪ Duration: 48-72 hr
▪ Supplement milk: TSB
▪ Condition system with milk: grow in TSB
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Listeria biofilms
ASTM Standard
Method E2562Modifications
Pseudomonas aeruginosa Listeria monocytogenes
300 mg TSB/L (Inoculum &
Batch)2 g TSB/L (Inoculum & Batch)
100 mg TSB/L (CF) 2 g TSB/L (CF)
11.7 mL/min 5.35 mL/min
125 rpm 60 rpm
24 hour CF 72 hour CF25 x 25 x25 x
Sometimes the reactor you may want to use or
already own is not the best choice for the bacteria
you are trying to grow.
Fluid shear is important
Kelli Buckingham-Meyer
ASTM E2647 Modifications
ASTM Standard
Method E2647Modifications
Pseudomonas aeruginosa Listeria monocytogenes
Polycarbonate coupons Stainless steel coupons
TSB TSBYE or BHI or ?*
48 hr 72 hr?
10o angle 5o angle?
0.8 mL/min/channel 0.4 mL/min/channel?
Room temperature Optimal range 30-37oC
1:100 INB1:100 BNB1:300 CFNB
1:100 INB1:100 BNB1:300 CFNB
*Jarvis et al. (2016) Food Control 66:256-269
Ben Klayman
200 mm
1 mm
Green is gfp P. aeruginosa PA01 Red is dsRed E.coli 0157:H7
T = 72 hrs
Klayman BJ, Volden PA, Stewart PS, Camper AK, "Escherichia coli O157:H7 requires colonizing partner to adhere and persist in a capillary flow cell," Environ Sci Technol 2009; 43(6):2105–2111
Pathogen Survival in Biofilm
Modifications for mixed species including L.p.
Bacterial air vent
Reactor top
Berzelius Pyrex
beaker
Polypropylene rod
Coupon
Baffled stir bar
Effluent spout
Media inlet/inoculation port
Set screws
L. Lorenz, S. Goeres 2007
CDC Biofilm Reactor
Pseudomonas aeruginosa
Klebsiella pneumoniae
Flavobacterium spp.
Legionella pneumophila
Hartmannella vermiformis
Stainless steel coupons
TSB
24 hr (base biofilm)/4 d after L.p. addition
125 rpm
11.7 mL/min
30oC
▪ Selective media
PIAR2A w/ & w/o chloramphenicolBCYE w/GPAV
Biofilms in Beer Draught Lines
L. Lorenz, CBE 2017
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Long Draw Draught System: Clean-In-Place Schematic Overview
Dispense line & faucet
Keg coupler
Beer manifolds
Gas manifolds
CO2 tank & regulator
Dispense line coupler
Kegerator
Credit: Brewers Association & New Belgium Brewing
Beer Lines: 3 Tubing Types
Credit: Brewers Association & New Belgium Brewing
Coupler/Simulated Keg
5/16” vinyl
tubing (5 ft.)
5/16” PET
tubing (15 ft.)
3/16” vinyl
tubing (3 ft.)
Faucet
A B C
Shank and faucet
Pre-conditioning the Draught System
Cleaning canister used to
pre-condition lines with
3% NaOH.
Lines were rinsed with tap
water, then beer.
Draught System: Inoculation Pouring Beer
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Draught System: Sampling Draught System: Sampling
Bacteria enumerated on Universal Beer Agar with
cycloheximide.
Yeast enumerated on WL Nutrient Agar.
▪ Biofilms impact many industries▪ Challenging to eliminate▪ Lab biofilms are engineered▪ Standard biofilm methods help industry and