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AGR-WATE71-05 · Lesson 7 · 15 clock hours · lab

Microbiological Testing: Total Coliform and E. coli Presence/Absence

Objective. Students will inoculate presence/absence broth or a membrane filtration system with drinking water samples using aseptic technique, incubate cultures at the required temperature and time, and correctly read and record results according to the method.

⚠ Pending professional review

This lesson, its worked examples and its practice questions has not yet been verified by a licensed professional in the field or by a certification or approval body. It is a study draft and may contain errors. Do not use it as instructions for work on a job site or on live equipment. Always follow your instructor, your supervising professional, and current official references and regulations.

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Why Microbiological Testing Matters

Drinking water regulations in the United States require public water systems to monitor for microbial contamination on a routine basis. The Total Coliform Rule (TCR), administered by the EPA under the Safe Drinking Water Act, requires systems to test for total coliform bacteria as an indicator of potential fecal contamination and pathogen risk. A positive total coliform result triggers follow-up testing for Escherichia coli (E. coli), which is a direct indicator of fecal contamination. These results drive public notification requirements and regulatory action, making accurate sampling and testing critical skills for any licensed operator.

What We Are Testing For

Total coliforms are a broad group of gram-negative, rod-shaped bacteria that ferment lactose and are used as general indicators of water quality. Their presence does not automatically mean the water is unsafe, but it signals that the system may have a problem — a breach in the distribution system, inadequate disinfection, or cross-connection.

E. coli is a subset of total coliforms found specifically in the intestines of warm-blooded animals. A confirmed E. coli positive in a routine distribution sample indicates fecal contamination and is considered a more serious violation requiring immediate action.

Two Approved Methods

Presence/Absence (P/A) Broth Method

The P/A broth method does not count bacteria — it only tells you whether total coliforms and E. coli are present or absent in the volume tested (typically 100 mL). The broth contains lactose and a pH indicator. Coliform bacteria ferment lactose, producing acid and gas, which causes the broth to change color (usually from purple to yellow) and may produce gas bubbles. This color change is a positive result. Under UV light, some formulations also fluoresce to confirm E. coli.

Membrane Filtration (MF) Method

In membrane filtration, 100 mL of sample is drawn through a sterile 0.45-micron membrane filter that traps bacteria on its surface. The membrane is then placed on a selective growth medium such as m-ColiBlue24® or equivalent. After incubation at the required temperature and time, colonies are counted. Blue colonies typically indicate total coliforms; red/magenta colonies may indicate E. coli, depending on the medium. The MF method can provide both presence/absence and quantitative (count) data.

Aseptic Technique: The Non-Negotiable Foundation

Aseptic technique prevents contamination of your sample with environmental bacteria, which would produce false positives. Any contamination event invalidates results and can trigger unnecessary regulatory actions or, worse, mask a real problem.

Key aseptic practices:

Sample Collection Requirements

Samples must be collected in sterile bottles pre-dosed with sodium thiosulfate to neutralize residual chlorine. Chlorine in an un-neutralized sample will continue killing bacteria after collection, causing false negatives. Samples must be kept cold (typically at or below 10 °C) and analyzed within the holding time specified by the approved method — generally within 24 to 30 hours of collection. Chain-of-custody documentation must accompany every sample.

Incubation and Reading Results

Both the P/A and MF methods require incubation at a specified temperature (consult the current approved method or your state's approved laboratory procedures for the exact temperature and time). Incubator temperature must be checked and logged before and during use. After the required incubation period:

Always read and record controls first. Your positive control must read positive and your negative control must read negative. If either control fails, the entire run is invalid and must be repeated.

Recording and Reporting Results

Record results immediately in your lab notebook: date, sample ID, sample source, incubation start and end time, temperature, and result (positive/negative or colony count). Never record from memory hours later.

Under the TCR, a routine positive total coliform sample requires the system to collect a set of repeat samples within a defined timeframe to determine whether the contamination is widespread. If repeat samples confirm E. coli, the system must issue a Tier 1 public notification within 24 hours. State primacy agencies have specific thresholds for the number of positive routine samples that trigger a Tier 2 violation requiring public notice within 30 days. Operators must know their state's specific requirements and reporting deadlines.

Safety in the Microbiology Lab

Key terms10

Total Coliform Rule (TCR)An EPA regulation under the Safe Drinking Water Act that establishes monitoring, reporting, and public notification requirements for total coliform bacteria in public drinking water systems.
Total coliformA broad group of gram-negative, rod-shaped bacteria used as an indicator of potential contamination in drinking water; their presence triggers follow-up testing for E. coli.
E. coli (Escherichia coli)A species of coliform bacteria found in the feces of warm-blooded animals; its detection in a water sample is a direct indicator of fecal contamination.
Presence/Absence (P/A) broth methodAn approved microbiological test method that determines whether total coliforms or E. coli are present or absent in a 100 mL sample by detecting color change or fluorescence caused by bacterial fermentation.
Membrane filtration (MF) methodAn approved method in which water is passed through a 0.45-micron sterile membrane that traps bacteria; the membrane is incubated on selective media and colonies are counted or their presence is noted.
Aseptic techniqueA set of laboratory practices designed to prevent the introduction of external microorganisms into a sample or culture, ensuring that test results reflect only what was in the original sample.
Sodium thiosulfateA chemical pre-added to sterile sample bottles to neutralize residual chlorine in drinking water samples, preventing continued disinfection that would kill bacteria after collection and cause false-negative results.
Positive controlA sample known to contain the target organism, run alongside test samples to verify that the growth medium and method are functioning correctly; must read positive for results to be valid.
Tier 1 public notificationThe highest-urgency public notice required under the Safe Drinking Water Act, issued within 24 hours when a water quality violation poses an acute risk to human health, such as a confirmed E. coli positive.
Holding timeThe maximum elapsed time between sample collection and analysis, as specified by the approved method, beyond which the sample is considered invalid and results cannot be reported.

Worked examples4

1. A community water system serves 4,200 people and is required to collect 10 routine distribution samples per month. During a given month, 2 of the 10 routine samples test positive for total coliform. What percentage of routine samples are positive, and does this percentage exceed the TCR acute violation threshold of greater than 5% positive routine samples in a month (for systems collecting 40 or more samples/month)?
  1. Step 1: Identify the number of positive samples and total routine samples. Positive = 2; Total = 10.
  2. Step 2: Calculate the percentage positive: (2 ÷ 10) × 100 = 20%.
  3. Step 3: Determine which TCR threshold applies. This system collects fewer than 40 samples per month, so the percentage-based threshold does NOT apply to determine a monthly violation for small systems. For systems collecting fewer than 40 samples/month, any single positive routine sample triggers repeat sampling requirements. A violation occurs if any repeat sample is also positive.
  4. Step 4: State the finding. 20% of routine samples tested positive. Regardless of the monthly percentage threshold, the positive results require the system to collect repeat samples within the required timeframe per state procedures.
Answer: 20% of routine samples were positive. For this system size (fewer than 40 samples/month), the percentage-based threshold does not apply; any positive routine sample triggers repeat sampling, and the operator must follow state-specific TCR repeat sampling protocols.
2. A student incubates 3 P/A broth tubes — a positive control, a negative control, and a blind sample — for the required time. At reading, the positive control has turned yellow with gas production, the negative control remains purple, and the blind sample has turned yellow. How should the student record and interpret these results?
  1. Step 1: Check the positive control. It turned yellow with gas — this is the expected positive result. The method is functioning correctly.
  2. Step 2: Check the negative control. It remained purple — this is the expected negative result, confirming no contamination was introduced during inoculation.
  3. Step 3: Both controls performed as expected, so the run is valid.
  4. Step 4: Read the blind sample. It turned yellow, which matches the positive color-change criterion. Record the result as POSITIVE for total coliform.
  5. Step 5: Document in the lab notebook: sample ID, date/time of inoculation and reading, incubation temperature and duration, control results, and the blind sample result of POSITIVE.
Answer: The run is valid (both controls correct). The blind sample is recorded as POSITIVE for total coliform. This result, if from a routine distribution sample, would trigger required repeat sampling per the TCR.
3. A student is collecting a drinking water sample for microbiological analysis. The tap water has a measured free chlorine residual of 0.8 mg/L. The sterile sample bottle holds 120 mL and is pre-dosed with sodium thiosulfate sufficient to neutralize up to 5 mg/L of chlorine in a 120 mL sample. Is the pre-dosed bottle adequate for this sample, and what must the student verify before capping the bottle?
  1. Step 1: The bottle's sodium thiosulfate is rated to neutralize up to 5 mg/L chlorine in 120 mL. The sample has 0.8 mg/L free chlorine residual.
  2. Step 2: Since 0.8 mg/L is well below the 5 mg/L capacity, the pre-dosed bottle is adequate to neutralize residual chlorine in this sample.
  3. Step 3: Before capping, the student must verify: (a) the sample volume is correct (do not fill to the very top — leave air space as directed, or fill to the marked line so the pre-dosed chemical is in the correct ratio); (b) the sample bottle label is complete (sample ID, location, date, time, collector initials); (c) the bottle was not rinsed before filling, as rinsing would wash out the sodium thiosulfate.
  4. Step 4: Cap immediately after filling, invert gently to mix, place on ice, and transport to the lab within the method holding time.
Answer: Yes, the pre-dosed bottle is adequate. The student must not rinse the bottle, must fill to the correct volume mark, complete the label, cap immediately, and place the sample on ice for transport within the required holding time.
4. After a routine positive total coliform result is confirmed (with no E. coli detected), the operator needs to inform the public. What tier of public notification is required, and within what general timeframe must it be issued under the Safe Drinking Water Act framework?
  1. Step 1: Identify the type of violation. A routine positive for total coliform with NO E. coli is a potential monitoring or treatment technique violation — it is not an acute health risk violation.
  2. Step 2: Under the SDWA public notification rule, violations that do not present an acute risk to human health require Tier 2 notification.
  3. Step 3: Tier 2 notification must be issued as soon as practical but no later than 30 days after the system learns of the violation.
  4. Step 4: If instead E. coli had been confirmed, that would escalate to a Tier 1 (acute) notification required within 24 hours.
Answer: A confirmed total coliform positive without E. coli requires a Tier 2 public notification, issued no later than 30 days after the system learns of the violation. An E. coli-confirmed positive would require Tier 1 notification within 24 hours.

The math in this lesson1

Learners compute percent coliform-positive samples, 2 of 10 = 20%, for Total Coliform Rule checks.
6.RP.3
In class: Instructor demonstrates the P/A broth method and the membrane filtration method side by side, narrating aseptic technique at each step; students identify the critical aseptic steps on a written checklist during the demonstration. · Each student inoculates three P/A broth tubes with assigned samples (including a known positive control, a known negative control, and one blind sample), labels correctly, and places in incubator set to the method-required temperature; after incubation (results read following day), students record positive/negative result and color change. · Students calculate the percentage of samples that would be in violation of the Total Coliform Rule if the blind positive result came from a routine distribution sample; write a brief summary of required repeat sampling and public notification triggers using the TCR framework.
Materials: P/A broth tubes or m-ColiBlue24 or equivalent membrane filtration media, sterile membrane filtration apparatus, incubator set to method temperature, sterile sample bottles with sodium thiosulfate, positive and negative controls, aseptic technique supplies (alcohol lamp or biosafety cabinet), lab notebooks, EPA Total Coliform Rule summary reference
Check: Aseptic technique checklist (no contamination events observed); correct reading of positive and negative controls; accurate result recording and compliant interpretation statement; pass = all controls read correctly and no observed aseptic technique breaks.

Practice check8

Answer each question, then check it. Nothing is recorded; this is your own practice.

Laboratory Procedures and Sample Analysis
1. In the Presence/Absence broth method for total coliform testing, what color change indicates a POSITIVE result?
Sampling and Monitoring Procedures
2. Why must sterile sample bottles for microbiological analysis be pre-dosed with sodium thiosulfate?
Laboratory Quality Control
3. During a microbiological test run, the positive control tube shows NO color change after incubation. What is the correct action?
Regulations and Compliance — Total Coliform Rule
4. A confirmed E. coli positive result in a routine distribution sample triggers which level of public notification under the Safe Drinking Water Act?
Laboratory Safety and Aseptic Technique
5. Which of the following is a critical aseptic technique requirement when inoculating P/A broth tubes?
Laboratory Procedures and Sample Analysis
6. In the membrane filtration method, which membrane pore size is used to trap bacteria on the filter surface?
Regulations and Compliance — Total Coliform Rule
7. After a routine positive total coliform result (with E. coli absent), what immediate action is required of the operator under the Total Coliform Rule?
Laboratory Safety and Hazardous Waste Handling
8. A student is preparing to discard used P/A broth tubes and membrane filter materials after testing. What is the correct disposal procedure for these microbiological waste materials?
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