16.1 Drinking Water Microbiological Testing: Coliform, E. coli & RTCR

8.1 Drinking Water Microbiological Testing: Coliform, E. coli & RTCR

Waterborne pathogenic microorganisms—including bacterial agents such as Salmonella enterica, Shigella dysenteriae, and Vibrio cholerae, enteric viruses such as norovirus and hepatitis A, and protozoan parasites such as Cryptosporidium parvum and Giardia duodenalis—pose immediate acute risks to human health when present in public drinking water supplies. However, directly isolating and enumerating every specific pathogen in daily compliance monitoring is practically impossible due to low ambient pathogen concentrations, protracted incubation times, high analytical costs, and specialized biosafety requirements.

To safeguard public water systems, environmental public health standards rely on indicator organisms. An ideal microbiological indicator is consistently present in high numbers when fecal contamination occurs, behaves similarly to enteric pathogens during water treatment and disinfection, persists longer than vegetative pathogens in distribution networks, is non-pathogenic to laboratory analysts, and can be detected quickly using standardized, reproducible procedures.

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Indicator Organisms & The Coliform Hierarchy

The coliform bacterial group serves as the primary microbiological indicator for drinking water treatment efficacy and distribution system sanitary integrity. Coliform bacteria are taxonomically organized into three nested tiers:

 ┌────────────────────────────────────────────────────────┐
 │ Total Coliform Group                                   │
 │ (Escherichia, Klebsiella, Citrobacter, Enterobacter)   │
 │ ┌────────────────────────────────────────────────────┐ │
 │ │ Fecal / Thermotolerant Coliforms                   │ │
 │ │ (Thermotolerant lactose fermenters at 44.5°C)      │ │
 │ │ ┌────────────────────────────────────────────────┐ │ │
 │ │ │ Escherichia coli (E. coli)                     │ │ │
 │ │ │ (Definitive indicator of mammalian waste)     │ │ │
 │ │ └────────────────────────────────────────────────┘ │ │
 │ └────────────────────────────────────────────────────┘ │
 └────────────────────────────────────────────────────────┘

1. Total Coliform Group

Total coliforms are defined biochemically as all aerobic and facultatively anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria (bacilli) that ferment lactose with gas and organic acid production within 48 hours at $35.0 \pm 0.5^\circ\text{C}$. This group includes members of the family Enterobacteriaceae belonging to genera such as Escherichia, Klebsiella, Enterobacter, Citrobacter, and Serratia.

While some coliform genera inhabit the gastrointestinal tracts of warm-blooded animals, many species are ubiquitous in nature, thriving naturally in soil, decaying vegetation, surface runoff, and distribution system biofilms. Consequently, the presence of total coliforms does not definitively prove fecal pollution; instead, it indicates a breakdown in sanitary barriers, treatment plant filtration failure, cross-connections, backflow, or bacterial regrowth within the distribution network.

2. Fecal Coliforms (Thermotolerant Coliforms)

Fecal coliforms are a specialized subset of the total coliform group capable of fermenting lactose with gas and acid production at an elevated incubation temperature of $44.5 \pm 0.2^\circ\text{C}$. This subgroup consists predominantly of Escherichia coli and thermo-adapted strains of Klebsiella pneumoniae. Their thermotolerance reflects adaptation to the warm intestinal environments of birds and mammals, making them a more specific indicator of animal or human waste than total coliforms.

3. Escherichia coli (E. coli)

Escherichia coli is the definitive species of fecal coliform that originates almost exclusively in the gastrointestinal tracts of humans and warm-blooded animals. Unlike other coliforms, E. coli rarely multiplies in ambient temperate distribution networks without a biological host. The detection of E. coli in finished potable water is conclusive evidence of recent fecal contamination, signaling an imminent public health emergency and the probable presence of co-occurring waterborne enteric pathogens.

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The Revised Total Coliform Rule (RTCR) & OAR 333-061

In 2013, the United States Environmental Protection Agency (EPA) finalized the Revised Total Coliform Rule (RTCR) under the Safe Drinking Water Act (SDWA), which Oregon adopted in OAR 333-061 (monitoring in OAR 333-061-0036; Oregon calls the RTCR assessments coliform investigations, in OAR 333-061-0078). The RTCR established a major regulatory paradigm shift:

1. Elimination of the Monthly Total Coliform MCL: Under the previous 1989 rule, having two or more positive total coliform samples in a month resulted in a non-acute Maximum Contaminant Level (MCL) violation accompanied by mandatory public notice. The RTCR eliminated this numeric MCL, recognizing that total coliforms are operational indicators rather than direct toxicological hazards.
2. The "Find-and-Fix" Approach: Total coliform detections now function as Treatment Technique Triggers. Instead of issuing administrative penalties or confusing public notices for minor biofilm regrowth, the rule requires utilities to conduct structured investigations (Level 1 or Level 2 Assessments) to identify and correct sanitary defects before disease outbreaks occur.
3. Acute E. coli MCL Maintained: An acute E. coli MCL violation occurs when any of the following compliance scenarios arise:
   • An E. coli-positive routine sample is followed by a total coliform-positive repeat sample.
   • An E. coli-positive routine sample is followed by an E. coli-positive repeat sample.
   • A total coliform-positive routine sample is followed by an E. coli-positive repeat sample.
   • An operator fails to collect all required repeat samples following an initial E. coli-positive routine sample.
   • An operator fails to test for E. coli after a repeat sample tests positive for total coliforms.

Routine Microbiological Monitoring Requirements

Public water systems must monitor for total coliform and E. coli according to a written Sample Siting Plan approved by the Oregon Health Authority (OHA) Drinking Water Services. Routine sampling frequency is determined strictly by the population served under OAR 333-061-0036:

• Small systems serving $25 \text{ to } 1,000$ individuals: Minimum 1 sample per month (or 1 per quarter for eligible groundwater systems).
• Systems serving $1,001 \text{ to } 2,500$ individuals: 2 samples per month.
• Systems serving $2,501 \text{ to } 3,300$ individuals: 3 samples per month.
• Large municipal systems serving 100,000 residents: 100 samples per month (increasing with population).

Samples must be distributed evenly throughout the monitoring period and taken from representative consumer taps reflecting all pressure zones, storage tank draw zones, and dead ends.

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Compliance Sample Collection Protocol

Bacteriological compliance results are extremely vulnerable to false positives caused by improper sample collection technique. A single environmental bacterium from an operator's fingertip, a dirty spigot threads, or an aerator gasket can trigger a false positive, causing regulatory panic, mandatory repeat testing, and costly assessments. Certified operators must execute strict sampling hygiene:

1. Tap Selection: Select an appropriate cold-water service tap directly connected to the distribution main. Avoid:
   • Swivel faucets, single-lever mixing faucets, or pull-out sprayers (internal O-rings harbor bacteria).
   • Leaking faucet stems or packings (water running down the outside of the spout will contaminate the sample).
   • Faucets with internal or external hose threads.
   • Taps located downstream of domestic point-of-use treatment units (water softeners, carbon filters, reverse osmosis units).
   • Frost-proof yard hydrants with underground drain ports (weep holes can siphon contaminated groundwater into the valve barrel).
2. Aerator and Attachment Removal: Unscrew all aerators, screens, sediment filters, and rubber hoses from the spigot. These components accumulate sediment, organic debris, and thriving bacterial biofilms.
3. Tap Disinfection: Clean and sanitize the exterior and interior orifice of the faucet spout. For metal faucets, sanitize by flaming the spout with a propane torch until dry and hot, or swab thoroughly with a freshly prepared sodium hypochlorite solution ($100 \text{ mg/L}$) or 70% isopropyl alcohol. Allow chemical contact time for at least 2 minutes.
4. Flushing: Open the cold-water tap to a smooth, non-splattering, laminar stream (approximately $0.5 \text{ to } 1.0 \text{ gpm}$). Flush the line continuously for 2 to 3 minutes (or until the water temperature stabilizes as measured by a thermometer). Thermal stabilization proves that stagnant service line water has been cleared and fresh water is entering directly from the water main.
5. Bottle Handling & Preservative: Use only state-certified, sterile, non-toxic containers (typically $120 \text{ mL}$ polypropylene or polystyrene bottles). Containers are dosed prior to autoclaving with sodium thiosulfate ($\text{Na}_2\text{S}_2\text{O}_3$, approximately $10 \text{ mg}$ of powder or liquid equivalent), which instantly neutralizes up to $15 \text{ mg/L}$ of free or combined chlorine residual. This stops chlorine from continuing to kill bacteria during transit.
   • Never rinse the container before filling (rinsing washes out the sodium thiosulfate preservative).
   • Grasp the bottle by the base, remove the cap without touching the inside rim or threads, hold the cap face down, and do not lay the cap on any surface.
6. Filling & Headspace: Fill the container to the $100 \text{ mL}$ fill line. Never fill the bottle to the brim: leave at least $1 \text{ inch (25 mm)}$ of air headspace below the cap. This headspace is essential for the laboratory analyst to invert and vortex-mix the sample before testing.
7. Transport and Holding Time: Tightly seal the cap, label with chain of custody data, and store immediately in an insulated cooler chilled with wet ice to $<10^\circ\text{C}$ (ideally $1 \text{ to } 6^\circ\text{C}$ without freezing). Under federal SDWA and OAR 333-061 rules, the maximum allowable holding time between collection and laboratory incubation is 30 hours (commercial laboratories strongly recommend $<24 \text{ hours}$). Samples exceeding 30 hours must be rejected.

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Standard Analytical Testing Methodologies

Water utilities and commercial laboratories utilize three primary analytical methodologies approved by the EPA (codified in Standard Methods for the Examination of Water and Wastewater) for compliance coliform enumeration.

  Compliance Sample (100 mL potable water)
             │
   ┌─────────┼────────────────────────┐
   ▼         ▼                        ▼
Membrane  Multiple Tube           Defined Substrate
Filtration Fermentation (MTF)     Technology (Colilert)
(0.45 µm)  (Lauryl Tryptose LTB)   (ONPG & MUG reagents)
   │         │                        │
m-Endo    Durham gas tube          Yellow = Total Coliform
24 hr     BGBB confirm             UV Blue Fluo = E. coli
Sheen     EC-MUG fecal

1. Membrane Filtration (MF) Method (Standard Methods 9222B / 9222D)

A $100 \text{ mL}$ drinking water sample is drawn through a sterile, gridded, porous membrane filter ($47 \text{ mm}$ diameter, $0.45 \ \mu\text{m}$ nominal pore diameter) using a vacuum filtration flask. Bacteria larger than $0.45 \ \mu\text{m}$ are retained on the filter surface.

• The membrane is placed grid-side up onto an absorbent cellulose pad saturated with m-Endo broth or onto an m-Endo agar Petri dish.
• The plate is inverted and incubated at $35.0 \pm 0.5^\circ\text{C}$ for $24 \pm 2 \text{ hours}$.
• Coliform colonies ferment lactose, producing acetaldehyde and intermediate aldehydes that react with sodium sulfite and basic fuchsin dye. Positive coliform colonies develop a distinctive golden-green metallic sheen.
• Colonies exhibiting sheen are enumerated directly. For fecal coliform confirmation, filters can be incubated on m-FC medium in an airtight water bath at $44.5 \pm 0.2^\circ\text{C}$ for 24 hours, where fecal coliforms form distinct blue colonies.

2. Multiple Tube Fermentation (MTF) / Most Probable Number (MPN) (Standard Methods 9221)

The MTF technique is a statistical assay using multi-tube dilution series (ten $10 \text{ mL}$ tubes or five $20 \text{ mL}$ tubes) to determine the Most Probable Number (MPN) of coliforms per $100 \text{ mL}$:

1. Presumptive Phase: Sample aliquots are inoculated into Lauryl Tryptose Broth (LTB) containing lactose, sodium lauryl sulfate (which suppresses non-coliform bacteria), and an inverted glass Durham tube. Tubes are incubated at $35.0 \pm 0.5^\circ\text{C}$ for $24 \text{ to } 48 \text{ hours}$. Turbidity accompanied by gas bubbles trapped in the Durham tube constitutes a positive presumptive reaction.
2. Confirmed Phase: Inocula from positive LTB tubes are transferred using a sterile wire loop into Brilliant Green Bile Broth (BGBB 2%) and incubated at $35.0 \pm 0.5^\circ\text{C}$ for up to 48 hours. Gas production confirms total coliform presence.
3. Fecal / E. coli Phase: Inocula from positive presumptive tubes are transferred into EC-MUG broth and incubated in a circulating water bath at $44.5 \pm 0.2^\circ\text{C}$ for 24 hours. Gas production confirms fecal coliforms, while bright blue fluorescence under long-wave ultraviolet illumination confirms E. coli.

3. Defined Substrate / Enzyme Substrate Technology (Standard Methods 9223B - Colilert)

Enzyme substrate technology (such as IDEXX Colilert® or Colisure®) is the most widely adopted regulatory testing method for drinking water compliance. The test utilizes two specific nutrient-indicators:

• ONPG (ortho-nitrophenyl-$\beta$-D-galactopyranoside): The total coliform group produces the specific constitutive intracellular enzyme $\beta$-galactosidase. When coliforms metabolize ONPG, the enzyme hydrolyzes the colorless substrate into galactose and ortho-nitrophenol, imparting a distinct yellow color to the solution.
• MUG (4-methylumbelliferyl-$\beta$-D-glucuronide): Escherichia coli uniquely produces the enzyme $\beta$-glucuronidase (present in approximately $97\%$ of E. coli strains). When E. coli metabolizes MUG, the enzyme cleaves the molecule to produce 4-methylumbelliferone, which produces intense bright blue fluorescence when viewed under a $365 \text{ nm}$ long-wave ultraviolet (UV) lamp.

Reagents are dissolved directly into a $100 \text{ mL}$ sample and incubated at $35.0 \pm 0.5^\circ\text{C}$ for 24 hours (or 18 hours for Colilert-18). The method can be configured as a simple Presence/Absence (P/A) bottle test or quantified as an MPN using sealed multi-well Quanti-Trays.

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RTCR Repeat Sampling Protocols & Assessment Triggers

When any routine monthly compliance sample tests positive for total coliform (TC+), the water utility must trigger immediate mandatory follow-up actions under OAR 333-061-0036.

24-Hour Repeat Sampling Protocol

The utility must collect a set of at least three repeat samples within 24 hours of being notified of the positive result:

1. Original Tap: One repeat sample from the exact same faucet where the positive routine sample was drawn.
2. Upstream Tap: One repeat sample from an active service connection located within 5 service connections upstream of the original positive tap.
3. Downstream Tap: One repeat sample from an active service connection located within 5 service connections downstream of the original positive tap.

(Note: For groundwater systems subject to the federal Groundwater Rule, the utility must also collect triggered source water samples from each operating wellhead before chemical disinfection to determine if the aquifer itself is contaminated).

 ───────┬──────────────┬──────────────┬──────────────┬───────
        │              │              │              │
    [Tap -3]       [Tap -1]       [Tap 0]        [Tap +2]
                   UPSTREAM       ORIGINAL      DOWNSTREAM
                  (within 5)     (Positive)     (within 5)
                   Repeat #2      Repeat #1      Repeat #3

Coliform Investigations (Oregon's Level 1 and Level 2 Assessments)

Level 1 Coliform Investigation

A Level 1 investigation is an evaluation conducted by the water supplier or its representative to identify sanitary defects, distribution pressure losses, storage tank roof damage, recent main breaks, or sampling errors. It is triggered when:

• For systems taking $\ge 40$ samples per month: Greater than $5.0\%$ of routine and repeat samples test positive for total coliform.
• For systems taking $< 40$ samples per month: 2 or more routine and repeat samples test positive for total coliform.
• The utility fails to collect all three required repeat samples after a single positive routine sample.

The completed Level 1 investigation report is due to OHA within 30 days of learning of the trigger.

Level 2 Coliform Investigation

A Level 2 investigation is a more comprehensive examination conducted by OHA or a party OHA approves; the water supplier communicates with OHA so it is completed within 30 days. It involves rigorous engineering review of treatment barriers, pressure management, reservoir security, cross-connection programs, and distribution maintenance. It is triggered when:

• An acute E. coli MCL violation occurs.
• The water system triggers a second Level 1 investigation within any rolling 12-month period, unless OHA had already determined the likely cause of the first and confirmed it was corrected.

Public Notification Mandates

• Tier 1 Public Notice (Boil Water Advisory): Issued within 24 hours for any acute E. coli MCL violation. Utilities must notify the public via broadcast media, emergency website postings, social media, reverse-911 calls, and hand-delivered door hangers. Consumers are advised to boil all tap water vigorously for at least 1 full minute (or 3 minutes at elevations above 6,500 ft) before drinking, cooking, brushing teeth, or making ice.
• Tier 2 Public Notice: Issued within 30 days for treatment technique violations, such as failing to perform a required Level 1 or Level 2 coliform investigation or failing to correct an identified sanitary defect within the state-approved timeframe.