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Short answer
HE agar is Hektoen enteric agar, a selective and differential plate for isolating Gram-negative enteric pathogens such as Salmonella and Shigella. Bile salts suppress Gram-positive growth, three sugars separate fermenters from non-fermenters, and an iron-thiosulfate system turns hydrogen sulfide producers black-centered. Colonies are presumptive only and need confirmation.
Definition and scope
A manufacturer’s instructions for use (IFU) describe Hektoen enteric agar as a “selective and differential medium used in the isolation and differentiation of Gram-negative enteric pathogens.” It belongs to the same family as XLD agar and MacConkey agar, which are both used on enteric specimens, and it is one of several media classes covered in what an agar plate is.
The same IFU states that some strains grow poorly and that “further biochemical or serological testing is required for the identification of organisms grown in this medium.” The plate narrows the field; it does not name the organism.
How the medium works
Selective component
Bile salts inhibit Gram-positive organisms and some Gram-negative strains. The maker’s quality-control (QC) panel shows this directly: Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212, each challenged with more than 1,000 CFU, are expected to be partially or completely inhibited.
Differential components
Three carbohydrates, lactose, sucrose and salicin, give the plate its differential character, and acid fuchsin with bromothymol blue serves as the indicator system. In the maker’s QC panel, E. coli is expected as salmon colonies and E. faecalis as yellow. Ferric ammonium citrate and sodium thiosulfate detect hydrogen sulfide, which produces the black centers associated with Salmonella.
Formulas differ slightly between sources
The base formula is shared but not identical. One maker’s IFU lists agar at 13.5 g and bromothymol blue at 0.064 g per litre, with a total of 75.2 g/L, and notes the formula “may be supplemented and/or adjusted.” The US FDA’s 2026 Salmonella method for soil and sediment lists agar at 14.0 g and bromthymol blue at 0.065 g per litre for its novobiocin variant. Both documents say the medium is not autoclaved. Whichever plate is in use, the label on that lot governs.
Reading colonies: two primary documents side by side
| Organism or class | Expected appearance | Source |
|---|---|---|
| Salmonella Typhimurium ATCC 14028 | Blue to blue-green with black centers | Maker IFU QC (18-24 h, 35 C, aerobic) |
| Shigella flexneri ATCC 12022 | Green to green-blue | Maker IFU QC |
| E. coli ATCC 25922 | Partial or complete inhibition; salmon colonies | Maker IFU QC |
| E. faecalis ATCC 29212 | Partial or complete inhibition; yellow colonies | Maker IFU QC |
| Typical Salmonella on HE+N | Blue-green or green with black centers, or entirely black | FDA 2026 method |
| Atypical Salmonella on HE+N | Blue-green or green without black centers; or yellow, salmon or orange with or without black centers | FDA 2026 method |
Atypical colonies still get picked
A colony that is not green with a black center is not automatically ruled out. The FDA method describes yellow, salmon or orange colonies, with or without black centers, as atypical Salmonella appearances, and it calls for picking five or more typical colonies per plate, or atypical ones when typical ones are absent, for confirmation. It also pairs the plate with XLT4 agar rather than relying on one medium, and notes that screening on TSI or LIA may precede confirmation. For the reaction pattern on the first of those, see triple sugar iron agar results.
HE agar versus HE+N
HE+N is Hektoen enteric agar with novobiocin added, used in the FDA method for Salmonella from agricultural soils and sediment, streaked from tetrathionate and Rappaport-Vassiliadis enrichments and incubated 24 plus or minus 2 hours at 35 plus or minus 2 C. The plain medium in the maker’s IFU has no novobiocin and is a general enteric-pathogen plate. The colony descriptions overlap, but they are different formulations, and the FDA method specifies HE+N by name.
Where it matters for labs
- Method documents: the FDA procedure above names HE+N paired with XLT4; other methods name their own media.
- Quality control: the maker’s IFU lists QC organisms with expected reactions, including inhibition of E. coli and E. faecalis.
- Storage: the dehydrated medium in the maker’s IFU is kept at 2-30 C, dry and sealed, and discarded if not free flowing or discolored. FDA-prepared HE+N plates are stored at 4 plus or minus 2 C for up to 30 days.
- Identification: colonies feed into wider workflows such as those in identifying bacteria; background on the organism is in the Salmonella Gram stain guide.
Common misunderstandings
- A black-centered colony is a confirmed Salmonella. It is a presumptive result; the maker’s IFU requires further biochemical or serological testing.
- Absence of black centers rules Salmonella out. The FDA method lists atypical colonies without black centers.
- HE agar can be autoclaved like other media. Both documents above say not to.
- All HE formulations are identical. Agar and indicator amounts differ between the maker’s IFU and the FDA formula.
FAQ
What does HE stand for in HE agar?
Hektoen enteric. HE agar is Hektoen enteric agar.
What does HE agar select for?
It favors Gram-negative enteric organisms, because bile salts inhibit Gram-positive organisms and some Gram-negative strains.
What color is Salmonella on HE agar?
Typically blue-green or green, often with black centers, and sometimes entirely black, per the FDA method; the maker’s QC strain is blue to blue-green with black centers.
Is HE agar autoclaved?
No. The maker’s IFU and the FDA method both say not to autoclave it.
Sources
- Gold Standard Diagnostics, BACGro Hektoen Enteric Agar IFU, POW4900 Rev 01, effective 13 April 2021: IFU PDF, checked 29 October 2026.
- US FDA, Testing Methodology for Detection and Isolation of Salmonella spp. from Agricultural Soils and Sediment, April 2026, Version 1: FDA method PDF, checked 29 October 2026.
