Presence-Absence Broth (PA Broth) (AS-1334) – Medium for Coliform Detection in Water

Presence-Absence (P-A) Broth is the simplest and most widely accepted single-tube method for the presumptive detection of total coliforms and Escherichia coli in 100 mL drinking water samples. Approved under US EPA 40 CFR 141.21 and recognised as an alternative procedure in APHA Standard Methods 9221 B.

The medium is formulated at double strength (71.82 g/L) so that when combined with 100 mL of water sample, it achieves single-strength working concentrations. Lactose fermentation produces acid (yellow colour) and gas (Durham tube). Sodium lauryl sulfate suppresses non-coliforms and Gram-positive organisms.

• Single-tube 100 mL presence/absence test — simple pass/fail result, no enumeration required
• Double-strength (71.82 g/L) — diluted 1:1 with water sample to achieve single-strength
• Bromocresol purple indicator — purple (negative) → yellow (positive acid)
• Durham tube gas detection — ≥10% fill signals coliform fermentation
• US EPA 40 CFR 141.21 approved for drinking-water regulatory compliance
• APHA Standard Methods 9221 B — recognised alternative to MPN multi-tube method
• Field-adaptable — ideal for small labs, remote sites, and routine monitoring

Step 1 — Double-strength stock:

1. Dissolve 71.82 g in 1 litre of purified water with agitation.
2. Place an inverted Durham tube in each 200–250 mL wide-mouth reagent bottle.
3. Dispense 100 mL double-strength broth per bottle. Ensure Durham tube is fully submerged.
4. Autoclave at 121 °C for exactly 12 minutes — do NOT exceed 12 minutes.
5. Cool and inspect: broth should be clear and purple; Durham tube free of air bubbles.
6. Store at 2–8 °C protected from light. Use within 3 months.

Step 2 — Test procedure:

1. Neutralise residual chlorine: add 0.1 mL of 3% sodium thiosulfate per 100 mL before collection.
2. Aseptically add 100 mL of water sample to one prepared bottle.
3. Incubate at 35 ± 0.5 °C. Read at 24 h and 48 h.

📖 Presence-Absence Testing for Drinking Water: A Practical Guide →

• Presumptive test only — all positives must be confirmed per APHA 9221 F
• Presence/absence only — does not enumerate organisms; not an MPN method
• Residual disinfectants inhibit recovery — neutralise chlorine before testing
• Some non-coliforms (e.g. Aeromonas spp.) may produce false positives
• Autoclave strictly 12 minutes only — longer degrades selectivity

Uninoculated: General laboratory waste per local EPA regulations.

Inoculated / used bottles: Autoclave at 121 °C for 30 minutes before disposal. Do not pour untreated coliform-positive media to drain.