4.2 Microbiology & Coliform Testing

For water treatment operators, microbiological testing is the most critical analysis performed. Biological contaminants can cause immediate, acute illness in a community. Unlike chemical hazards with long-term chronic risks, a single pathogen (a disease-causing bacterium, virus, or protozoan) can spark a widespread outbreak within days. Consequently, operators must master coliform sampling, understand regulatory rules, and comprehend detection methods.

Indicator Organisms and the Coliform Group

Testing drinking water for every individual pathogen is impractical, so regulatory monitoring relies on an indicator organism: the coliform bacteria group. Coliforms are ideal because they are present in warm-blooded animal intestines in large numbers, survive longer in water than most pathogens, and are easy to detect.

The coliform group is categorized hierarchically for testing and regulatory action:

• Total Coliform: Aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas and acid production within 48 hours at 35°C. They occur naturally in soil, vegetation, surface water, and animal intestines.
• Fecal Coliform: A subgroup of total coliforms that specifically grow and ferment lactose at an elevated temperature of 44.5°C, indicating recent fecal contamination.
• Escherichia coli (E. coli): A specific species within the fecal group. Its presence is a direct indicator of fecal contamination from humans or animals, presenting a high risk of pathogenic contamination.

Regulatory Monitoring: TCR and RTCR

Microbiological water quality is regulated under the EPA Total Coliform Rule (TCR), updated to the Revised Total Coliform Rule (RTCR). The RTCR aims to reduce fecal pathogens in distribution systems. Under the RTCR, the EPA establishes a Maximum Contaminant Level Goal (MCLG) of zero for E. coli. If a routine sample is positive for total coliforms, the laboratory must test that sample for E. coli. Additionally, the operator must collect repeat samples within 24 hours of notification.

Sampling Protocols and Transport

Proper sample collection is paramount; poor technique can introduce environmental bacteria, resulting in false-positive compliance tests. A coliform sample must be collected in a specialized sterile sample bottle, typically made of clear plastic with a 100 mL or 125 mL capacity. The bottle must contain a small amount of sodium thiosulfate. Sodium thiosulfate acts as a dechlorinating agent, immediately neutralizing chlorine residual in the sample. If chlorine is not neutralized, it will continue to disinfect the sample during transit, potentially killing target bacteria and yielding a false-negative result.

When collecting a coliform sample, operators must follow a strict protocol:

1. Select a clean tap, avoiding swivel faucets, drinking fountains, or taps with aerators or attachments where bacteria can colonize.
2. Remove any aerators, screens, or hoses from the tap.
3. Open the tap and flush the line for 2 to 5 minutes to clear stagnant water.
4. Do not rinse the sample bottle. Rinsing would wash out the sodium thiosulfate reagent.
5. Carefully open the bottle without touching the inside of the cap or the rim.
6. Fill the bottle directly to the fill line, leaving at least 1 inch of headspace at the top. Headspace is necessary to allow the sample to be thoroughly mixed by shaking prior to analysis.
7. Secure the cap immediately and label the bottle with the date, time, location, and collector's name.

Once collected, the sample must be kept cool (under 10°C, but not frozen) during transport. The maximum holding time from collection to the start of analysis is 30 hours. If a sample exceeds this limit, the laboratory must reject it, and the operator must collect a new sample.

Analytical Testing Methods

Three primary analytical methods are used in laboratories to detect and quantify coliform bacteria in drinking water samples:

1. Presence-Absence Test (P-A): A qualitative method that determines only whether coliforms are present or absent in a 100 mL sample. It uses a nutrient medium containing color-changing indicators. If coliforms are present, they ferment the nutrients and cause a color change (e.g., from clear to yellow). E. coli is detected in the same bottle by observing fluorescence under ultraviolet (UV) light.
2. Membrane Filtration (MF): A quantitative method where a 100 mL water sample is filtered through a sterile membrane filter with a pore size of 0.45 microns. The filter, which traps the bacteria, is placed on an M-Endo agar medium in a petri dish and incubated at 35°C for 24 hours. Coliform colonies grow on the membrane and develop a characteristic golden-green metallic sheen.
3. Multiple-Tube Fermentation (MTF): A quantitative method that uses a series of tubes containing lactose broth. If coliforms are present, they ferment lactose, producing acid and gas (trapped in small inverted tubes). The number of positive tubes is compared to a statistical table to calculate the Most Probable Number (MPN) of coliforms per 100 mL.

Table 4.2: Microbiological Testing Methods Comparison