Bacterial Source Tracking How One Small Environmental Lab

Bacterial Source Tracking How One Small Environmental Lab

Bacterial Source Tracking How One Small Environmental Lab Developed an Impactful Molecular Biology Program Overall Goal of Bacterial Source Tracking Improve the water quality of the San Antonio River basin Identify the source of the bacteria in order to decrease the levels in the basin to the TCEQ /EPA recreational levels Reduce outsourcing of samples Added revenue stream for the lab 2 3

What is BST? Bacterial source tracking (BST) is a set of techniques used to determine the sources of fecal indicator bacteria in the environment. BST techniques attempt to determine whether fecal bacteria are being introduced into water bodies through human, wildlife, or domestic animal sources. 4

What is PCR? 5 SARAs Method Filtration DNA extraction qPCR Data analysis 6 Filtration Collection similar to MPN Hold time 8 hours Filter each sample

Analysis stopping point # 1 7 DNA Extraction Addition of lysis buffer to lyse cells Series of washes through a column Analysis stopping point # 2 8 qPCR Analysis Set up appropriate run on the instrument

Prep samples for analysis Load wells according to your run Set it and Forget it 45min 9 Designate a clean room Logistics Research and purchase equipment Lab set up Implementation of analysis

10 Clean Room Specific to BST analysis 3 separated rooms or areas Unidirectional workflow Designated supplies 11 Research and Purchase Equipment

Low, Medium, or High throughput? Industry standards Consider the space youre working with 12 Lab Set Up Install equipment

Order consumables Calibrate equipment 13 Implementation of Analysis QA/QC Measures DOCs Validation of paperwork Calibration & standardization SOPs and Bench sheets Training of analysts

Financials fee schedule 14 Training Life Technologies Principals of Quantitative PCR and Data Analysis 15 Lessons Learned Unusual equipment unusually long and or difficult delivery Always be on the lookout for training and conferences Helps to have a team member

who is familiar with molecular biology No standardized method be ready to improvise, Very detailed protocol 16 Resource allocation staff planning, research and development Expect the unexpected staff changes, projects, sample loads Networking with other labs Current Testing

SARA LAB E. coli OUTSOURCE LAB MPN/100 mL Human BST Human Bird Dog

Ruminant SAR @ Myrtle 2/9/2016 14:39 46 Present NA NA NA

NA SAR @ Myrtle 4/5/2016 14:39 55 Absent Absent Present (Trace)

Absent Not Detected SAR @ Pecan 2/9/2016 14:49 210 Present Present Not

Detected Not Detected Not Detected SAR @ Pecan 4/5/2016 14:20 230 Present

Present Present (Trace) Present (Trace) Not Detected SAMPLE 17

SAR Draining Results Sample # Sample Site MPN value (MPN/100mL) Present/Absent Human DNA AA99318-A Seep at Little Rhein

14 Absent AA99319-A Storm drain @ Commerce St. 77 Present AA99320-A

Storm drain @ Pecan St. >24000 Present AA99260-A Salatrillo influent N/A Present

18 How Can We Use BST For Stormwater Management? In pipe sampling Pre/post BMP water quality testing 19 Questions 20

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