What is Microbial Source Tracking?

Traditional water quality testing tells you that fecal contamination is present (typically via E. coli or Enterococcus). It does not tell you where it came from. Microbial Source Tracking (MST) closes that gap by detecting host-associated microbial DNA markers that are highly associated with humans, cattle, dogs, birds, horses, and other animal sources. With MST, water managers can distinguish a failing sewer lateral from cattle grazing impacts, gull roosting from urban runoff, or septic failures from pet waste, and direct remediation accordingly.

H₂O Molecular performs MST by digital PCR, which delivers absolute quantification of each marker without standard curves and provides superior sensitivity in inhibitor-rich environmental samples.

The CSI-MST Methodology

H₂O Molecular’s team pioneered the Collection System Investigation Microbial Source Tracking (CSI-MST) approach during a decade of work inside a wastewater utility. CSI-MST applies human-associated MST markers strategically across stormwater conveyance networks to identify compromised sewer infrastructure such as cracked pipes, lateral leaks, illicit cross-connections, and failing manholes that contribute to dry-weather and wet-weather contamination.

CSI-MST is uniquely powerful because it moves MST from a passive characterization tool into an active infrastructure-assessment tool. Rather than telling a utility there is “human fecal contamination in the watershed,” CSI-MST tells them which segments of their collection system are leaking.

Human-Associated Fecal Markers

Our human-associated MST assays detect Bacteroides and other host-associated microbial markers shed in human feces. Targets we offer include:

• HF183: the most widely validated and US EPA-evaluated human-associated Bacteroides marker
• HumM2: a supplementary human marker for confirmation and triangulation
• crAssphage: a human gut-specific bacteriophage marker, useful as a complementary indicator of recent contamination
• PMMoV (Pepper Mild Mottle Virus): a human dietary virus marker with extraordinary persistence and high concentration in sewage

Using two or more human markers in combination significantly strengthens the evidentiary value of MST results, particularly for regulatory or enforcement applications.

Animal-Source Fecal Markers

Our animal-source assays are customizable to the species of concern in your watershed. Available targets include:

• Bovine / cattle: CowM2, CowM3
• Dog / canine: DG3, DG37
• Bird / avian: GFD, AV43, LA35
• Horse / equine: HoF597
• Swine: Pig2Bac
• Ruminant: Rum2Bac
• Universal Bacteroides: GenBac3

Custom panels can be assembled for any combination of host species. Contact us to design a panel for your specific watershed.

Viral Tracer Panel

For applications where high-specificity tracking is required, particularly in complex urban watersheds, H₂O Molecular offers viral tracer assays that complement bacterial markers. These viral markers are shed by the same human and animal host sources but offer complementary specificity and persistence characteristics.

• PMMoV
• crAssphage
• Tomato brown rugose fruit virus (ToBRFV)

Viral tracers are particularly valuable for distinguishing recent contamination from legacy contamination and for source attribution in mixed-source watersheds.

See Resources for our full list of MST assays

Applications & Use Cases

H₂O Molecular’s MST services support a range of regulatory and management programs:

• MS4 stormwater compliance and IDDE programs (Illicit Discharge Detection and Elimination)
• Total Maximum Daily Load (TMDL) development and implementation under the Clean Water Act
• Beach water quality and recreational water management
• Drinking water source water protection (Surface Water Treatment Rule, source water assessments)
• Shellfish growing area sanitation surveys under the NSSP Model Ordinance
• Septic system failure investigations
• Sewer infrastructure assessment via CSI-MST
• Onsite Wastewater Treatment System (OWTS) impact studies
• Watershed assessment and restoration monitoring

Why Digital PCR for MST?

Digital PCR has become the preferred platform for MST for several reasons:

• Absolute quantification without standard curves means inter-laboratory and longitudinal comparisons are direct and defensible, important for litigation, enforcement, and TMDL reporting.
• Inhibitor resistance is critical for stormwater, sediment, and sewage matrices, where qPCR is often compromised.
• Improved sensitivity at low marker concentrations matters in finished or partially-treated waters.
• Reproducibility across laboratories is significantly better for dPCR than qPCR, making results stronger for regulatory and legal use.

Read more about digital PCR for environmental samples

Sample Types We Analyze for MST

Stormwater · Dry-weather urban runoff · Recreational waters (beach, lake, river) · Shellfish growing waters · Source water · Wastewater · Sewer infrastructure samples · Soil and sediment · Septic effluent

Who We Work With

Municipalities and stormwater agencies · Water and wastewater utilities · State and federal environmental agencies · Watershed associations and conservation groups · Public health departments · Engineering and environmental consultants · Academic and research institutions