Written and maintained by CASRAI Editorial Board
Last updated
General laboratory information for lab and clinic staff. This page is not medical advice and does not replace a laboratory’s own validated procedures or a product’s instructions for use.
Short answer
Phenol red broth is a carbohydrate fermentation medium: a basal broth with one added sugar and the pH indicator phenol red. Yellow means the organism made enough acid from that sugar to drop the pH to 6.8 or below; red or pink means no fermentation; a bubble in the inverted Durham tube means gas. Source: ASM Carbohydrate Fermentation Protocol (checked 2026-10-30).
Definition and scope
The American Society for Microbiology (ASM) protocol states the purpose plainly: carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate, and the pattern of sugars an organism ferments helps differentiate bacterial groups and species. Its examples: all Enterobacteriaceae are glucose fermenters, while maltose fermentation separates Proteus vulgaris (positive) from P. mirabilis (negative).
The broth is a differential medium, meaning a visible change (here a color change of an indicator) distinguishes organisms. Under US device regulation, 21 CFR 866.2320 defines a differential culture medium as a device consisting primarily of liquid biological materials intended to cultivate and identify different types of pathogenic microorganisms, identified by a visible change in a specific biochemical component. It is Class I (general controls) and exempt from premarket notification, subject to the limitations in 866.9 (eCFR, version dated 2026-10-01, checked 2026-10-30). The regulation does not name phenol red broth, and whether a given broth is a regulated device depends on its intended use, so this page makes no product-level claim.
How it works in practice
ASM describes a basal medium to which a single carbohydrate is added, plus an indicator. Because bacteria can also use the peptones in the base and produce alkaline by-products, the pH changes toward acid only when excess acid results from carbohydrate fermentation. Phenol red is used because most end-products of carbohydrate utilization are organic acids.
The ASM reference recipe, per 1,000 ml, uses 10 g proteose peptone no. 3 (or Trypticase), 5 g NaCl, optionally 1 g beef extract, 0.018 g phenol red and 10 g of the test carbohydrate (1.0%), with a final pH of 7.4 ± 0.2 giving a light red broth. A Durham tube (a 6 x 50 mm tube inverted inside a 13 x 100 mm tube) traps gas as a visible bubble. Incubation is generally 35-37 °C for 18-24 hours, and ASM notes that longer incubation may be needed to confirm a negative result.
Phenol red broth results
| Appearance | Meaning (per ASM) |
|---|---|
| Yellow, no bubble | Acid only: enough acid to lower the pH to 6.8 or less; fermenter that does not produce gas (anaerogenic) |
| Yellow, bubble in Durham tube | Acid and gas; even a single bubble counts as gas |
| Red or pink | Nonfermenter of that sugar (negative) |
| Orange | Possibly a delayed reaction; ASM advises reincubating |
ASM’s figure shows E. coli as a glucose fermenter with gas, Shigella sonnei as a glucose fermenter without gas, and Pseudomonas aeruginosa as a nonfermenter.
Types and related concepts: the indicator
ASM lists phenol red alongside bromocresol purple, bromothymol blue and Andrade’s indicator as options. For phenol red it gives these colors:
| Condition | pH (ASM Table 1) | Color |
|---|---|---|
| Uninoculated | 7.4 | Reddish-orange |
| Acid | 6.8 | Yellow |
| Alkaline | 8.4 | Pink-red |
ASM recommends modified Andrade’s indicator instead of phenol red when prolonged incubation is needed (streptococci, enterococci) and for aerobic Gram-negative bacilli such as Enterobacteriaceae. Organisms with special needs change the base: halophilic Vibrio need 2-3% NaCl, and for Neisseria ASM adds 5% sterile rabbit serum or uses cystine-tryptic agar.
Where it matters for labs and supply decisions
Two points from the ASM protocol bear on how a laboratory prepares or qualifies this medium. First, sugars differ in heat tolerance: for arabinose, lactose, maltose, salicin, sucrose, trehalose and xylose, ASM specifies only 3 minutes at 121 °C because they break down, versus 15 minutes at 116-118 °C for the standard broth; the alternative is to autoclave the base and add a separately filter-sterilized (0.45 µm) carbohydrate solution to cooled broth. Second, ASM calls for controls for carbohydrate breakdown and gives a storage range of 4-10 °C with a shelf life of roughly 6-8 weeks for tubes it describes. A purchased or in-house broth should be checked against its own manufacturer’s specification, since the ASM recipe is a reference formulation rather than a product label.
Related CASRAI guides cover other indicator-based differential media: urease test results (pink versus yellow urea medium), triple sugar iron agar results (slant, butt, gas and H2S), MacConkey agar and XLD agar, where phenol red is also the indicator. Contamination control for any broth work is covered in the aseptic technique guide.
Reading results and common misunderstandings
- Red does not always mean negative. ASM warns that a reddish or pink color in a clear tube could indicate a false negative. Turbidity in a negative tube serves as the growth control, showing the organism grew but did not acidify.
- Yellow can revert. Rapid depletion of the carbohydrate lets the reaction revert as the organism turns to peptones. This is why ASM prefers 1.0% carbohydrate over 0.5%.
- Orange is not yellow. It may be a delayed reaction; ASM says to reincubate rather than call it.
- Acid is not gas. Gas is judged only by the bubble in the Durham tube; no bubble means non-gas-producing.
- One sugar per tube. Each broth tests a single carbohydrate, so a result applies only to that sugar.
FAQ
What does a yellow phenol red broth mean?
Per ASM, the organism fermented the added carbohydrate and produced enough acid to lower the pH to 6.8 or below.
What does a bubble in the Durham tube mean?
Gas was produced during fermentation. ASM states that even a single bubble indicates gas.
Why is my phenol red broth orange?
ASM describes an orange tube as a possible delayed reaction and recommends reincubating before reading it.
What does a pink or red result mean?
The organism did not ferment that sugar under the test conditions. Check the tube for turbidity: ASM notes a pink color in a clear tube may be a false negative.
Sources
- American Society for Microbiology, Carbohydrate Fermentation Protocol (K. Reiner), https://asm.org/asm/media/protocol-images/carbohydrate-fermentation-protocol.pdf, date checked 2026-10-30.
- 21 CFR 866.2320, Differential culture medium, https://www.ecfr.gov/current/title-21/section-866.2320, version dated 2026-10-01, date checked 2026-10-30.
