Drinking water quality monitoring is one of the most critical applications of applied microbiology. Of the many methods available for coliform detection, the Presence-Absence (P-A) Broth test stands out for its simplicity, regulatory acceptance, and suitability for routine monitoring programmes — including small water supplies and remote testing sites where more complex methods are impractical.

What is the Presence-Absence Test?

The P-A Broth method is a single-tube presumptive test designed to detect total coliforms and Escherichia coli in a 100 mL drinking water sample. Unlike the multiple-tube fermentation method (MPN), which provides a quantitative estimate of coliform density, the P-A test delivers a binary result: coliforms are either present or absent in the sample volume.

In drinking water compliance monitoring — under regulatory frameworks such as the Australian Drinking Water Guidelines (ADWG) and the US EPA Total Coliform Rule (40 CFR 141) — the acceptable limit for total coliforms is zero in a defined sample volume. A presence/absence result maps directly to this requirement, making the P-A method both scientifically appropriate and operationally efficient.

The method was formally approved under US EPA 40 CFR 141.21 and is recognised as an alternative procedure in APHA Standard Methods 9221 B.

How the Medium Works

P-A Broth is supplied as a dehydrated powder reconstituted at double strength (71.82 g/L). When 100 mL of prepared double-strength broth is combined with 100 mL of water sample, the resulting mixture achieves single-strength nutrient concentrations — a simple and elegant approach to standardising the test volume.

The medium contains four functional components working together:

• Lactose — the fermentable substrate. Coliform bacteria metabolise lactose through mixed-acid fermentation, producing acid and gas. Non-coliform organisms cannot utilise lactose in this way under the test conditions.
• Bromocresol purple — the pH indicator. At neutral pH (6.8) the medium is clear and purple. When coliforms ferment lactose and lower the pH, the indicator shifts to yellow — providing a visible, unambiguous positive signal.
• An inverted Durham tube — placed inside each bottle before autoclaving. Gas produced during fermentation accumulates in this tube. Filling of 10% or more of the tube volume is a positive gas indicator.
• Sodium lauryl sulfate — provides selective inhibition of Gram-positive organisms and non-coliform bacteria, increasing test specificity for coliform detection.

Preparing the Medium Correctly

Correct preparation is critical to test performance. Two common errors — over-autoclaving and failure to neutralise residual chlorine — account for the majority of false results in field settings.

1. Dissolve 71.82 g of dehydrated P-A Broth in 1 litre of purified water with gentle agitation. Heat if necessary to dissolve completely.
2. Place one inverted Durham tube in each 200–250 mL wide-mouth reagent bottle before filling. The tube must be fully submerged.
3. Dispense 100 mL of double-strength broth into each bottle.
4. Autoclave at 121 °C for exactly 12 minutes. This is the most critical step — exceeding 12 minutes degrades both the sodium lauryl sulfate and the lactose, reducing selectivity and productivity respectively.
5. After cooling, inspect each bottle. The broth should be clear and purple. Any turbidity or colour change before sample addition indicates contamination — discard the bottle.
6. Store at 2–8 °C, protected from light. Use within three months of preparation.

Performing the Test

1. Aseptically transfer 100 mL of the prepared water sample into one bottle of double-strength P-A Broth.
2. Mix gently by swirling. Do not invert the bottle — this may dislodge the Durham tube.
3. Incubate at 35 ± 0.5 °C. Temperature control is important: incubation at 37 °C or above can suppress some coliform strains and produce false negatives.
4. Read results at 24 hours and again at 48 hours.

Interpreting Results

P-A Broth vs MPN: Which Method Should You Use?

For routine drinking water compliance monitoring — where the regulatory threshold is simply "coliforms absent in 100 mL" — the P-A method is the more efficient choice. For investigations where the level of contamination needs to be characterised (e.g., a confirmed positive requiring root cause analysis), MPN or membrane filtration enumeration methods are more informative.

Biosafety Considerations

Water samples submitted for microbiological testing may contain pathogenic organisms beyond the target coliforms. All inoculated P-A bottles should be handled as Biosafety Level 2 (BSL-2) materials. Following analysis, all inoculated bottles must be autoclaved at 121 °C for 30 minutes before disposal. Do not pour untreated coliform-positive or suspect media to drain.

Quality Assurance for P-A Testing

Laboratories running P-A testing as part of a quality system should include the following controls with each batch of prepared medium:

Performance failures in controls should prompt rejection of the batch and investigation of preparation conditions, particularly autoclave time and temperature.

When to Use a Local Supplier

For laboratories running routine drinking water monitoring programmes, consistent media supply is operationally critical. A delayed shipment or an out-of-stock situation does not simply delay a test — in regulated water supply monitoring, it can delay a compliance report or a safety sign-off.

Locally manufactured P-A Broth offers a practical advantage: faster replenishment, shorter lead times, and direct technical support for preparation and interpretation questions — without the delays and freight costs associated with international supply chains.

Summary

The Presence-Absence Broth method is a well-established, regulatory-approved approach to drinking water coliform monitoring that balances scientific rigour with operational simplicity. Correct preparation — particularly strict adherence to the 12-minute autoclave time and appropriate residual chlorine neutralisation — is essential for reliable results. All positive results require confirmatory testing before any compliance determination is made.

This article is intended for laboratory professionals and is provided for informational purposes only. Testing of drinking water for regulatory compliance purposes should be performed by accredited laboratories in accordance with applicable national and state standards.