1. Bacterial Source Tracking
How One Small Environmental Lab Developed an Impactful Molecular Biology Program

2. 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

3. Look to determine where we need to start when it comes to impairments in our water bodies

4. 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.

5. What is PCR?
-Human Associated fecal indicator bacteria

6. SARA’s Method
Filtration
DNA extraction
qPCR
Data analysis

7. Filtration Collection similar to MPN Hold time 8 hours
Filter each sample
Analysis stopping point # 1

8. DNA Extraction Addition of lysis buffer to lyse cells
Series of washes through a column
Analysis stopping point # 2

9. 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

10. Logistics Designate a clean room Research and purchase equipment
Lab set up
Implementation of analysis

11. Clean Room Specific to BST analysis 3 separated rooms or areas
Unidirectional workflow
Designated supplies

12. Research and Purchase Equipment
Low, Medium, or High throughput?
Industry standards
Consider the space you’re working with

13. Lab Set Up
Install equipment
Order consumables
Calibrate equipment

14. Implementation of Analysis
QA/QC Measures
DOCs
Validation of paperwork
Calibration & standardization
SOPs and Bench sheets
Training of analysts
Financials – fee schedule

15. Training
Life Technologies – Principals of Quantitative PCR and Data Analysis

16. 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
Resource allocation – staff planning, research and development
Expect the unexpected – staff changes, projects, sample loads
Networking with other labs

17. Current Testing SAMPLE SARA LAB OUTSOURCE LAB E. coli Human Bird Dog
MPN/100 mL
Human BST
Human
Bird
Dog
Ruminant
Myrtle
2/9/ :39 46
Present NA
4/5/ :39 55
Absent
Present (Trace)
Not Detected
Pecan
2/9/ :49
210
4/5/ :20
230

18. 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 Commerce St. 77
Present
AA99320-A
Storm Pecan St.
>24000
AA99260-A
Salatrillo influent
N/A

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

20. Questions