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Short answer: Malachite green is the primary dye of the Schaeffer-Fulton endospore stain. A heat-fixed smear is steamed with 0.5% aqueous malachite green for 5 minutes, rinsed with tap water, and counterstained with safranin for 30 seconds. Endospores stay bright green; vegetative cells appear brownish red to pink (ASM Microbe Library protocol).
General laboratory information for staff and students. This page is not medical advice and does not replace your laboratory’s validated procedure.
Definition and scope
The Schaeffer-Fulton method is, in the ASM Microbe Library’s words, “the most common method used to perform endospore staining in undergraduate microbiology courses.” Schaeffer and Fulton modified Dorner’s 1922 method in 1933 to make it faster. This page covers the malachite green step and the stain as a whole; it does not give a bench protocol to follow. For the broader family of differential stains, see differential staining, and for the biology of spore formation, see formation of spores in bacteria.
The stain exists because ordinary stains often miss spores. Gram and simple stains “may or may not reveal” endospores, and only relatively few genera form them, so a positive result is an important identification clue. Introductory courses teach Bacillus and Clostridium (both rods); Sporosarcina is a coccus example. Early dyes such as carmine, methylene blue, carbol fuchsin and safranin could not stain spores, which were seen only as refractile bodies.
How the stain works: steam, tap water, safranin
An endospore has a permeability barrier that blocks dyes unless the barrier is destroyed by heat, chemicals (acid), ultraviolet light or mechanical rupture. The dehydrated state of the spore is the critical feature that makes it hard to stain. In the Schaeffer-Fulton sequence:
- Steaming with malachite green drives the dye into both vegetative cells and endospores.
- Tap water is the decolorizer. It washes the dye out of vegetative cells, but the stain is not released by the endospores and free spores.
- Safranin then colours the now-colourless vegetative cells.
The slide is blotted dry and examined at 1,000X under oil immersion. The ASM protocol covers the smear with blotting paper before saturating it with dye, and warns that a poorly fitted piece of paper can burn or drip, so the paper should be cut to fit only over the specimen area. Consult the safety data sheet (SDS) for malachite green before use.
Formulations compared: ASM versus FDA BAM
Published recipes for the same two reagents differ, and the difference is large enough to matter when you write or audit a procedure. The table sets out the two sources side by side.
| Reagent | ASM Microbe Library (Hussey and Zayaitz, 2016) | FDA BAM R32a (cites BAM 8th ed., Rev A, 1998) |
|---|---|---|
| Malachite green | 0.5% (wt/vol) aqueous: 0.5 g in 100 ml distilled water | Solution A: 10 g in 100 ml distilled water; filter to remove undissolved dye |
| Safranin | Stock 2.5% (wt/vol) in 95% ethanol (2.5 g safranin O per 100 ml); working solution is 10 ml stock plus 90 ml distilled water | Solution B: 0.25 g safranin O in 20 ml distilled water |
| Decolorizer | Tap water | Not listed on the reagent page |
The listed malachite green concentration differs 20-fold between the two sources (0.5 g versus 10 g per 100 ml). These are formulations from different methods and different purposes, not one source correcting the other. The practical lesson is that reagent specifications are method-specific: take them from the laboratory’s validated SOP rather than mixing figures from different sources.
Reading the result
The ASM protocol states the expected result as: “Endospores are bright green and vegetative cells are brownish red to pink.” It also notes the green can be “a little bluish.” A quick key:
- Green bodies (inside or free of cells): endospores that retained malachite green.
- Brownish red to pink cells: vegetative cells that lost the green and took up safranin.
- No green anywhere: not proof that the organism cannot form spores.
The last point has a sourced reason. Young cultures may show no spores, and most methods suggest cultures that are 18 to 36 hours old. The proportion of cells that sporulate also varies among species. Clinical laboratories describe spore shape and position as part of identification. For a related staining question on Gram-stained Bacillus, see Gram stain of Bacillus subtilis.
Where it matters for labs and supply decisions
The ASM protocol itself calls steaming “messy, time-consuming, and variable.” Larger laboratories may use phase-contrast microscopy instead of staining, which trades reagents and hot-plate steaming for different optics. Another staining route is the Dorner method, which uses carbolfuchsin, acid-alcohol and nigrosin and gives red endospores, colourless vegetative cells and a black background. Whichever route a laboratory uses, the reagent list, concentrations and steaming time belong in its written SOP so results are comparable between operators and over time.
Spore staining is also separate from checking that sterilisation works. Biological indicators for steam sterilisers use spores as test organisms; see autoclave validation with biological and chemical indicators. For another acid-based differential stain, see Ziehl-Neelsen staining.
Common misunderstandings
- “Malachite green is a counterstain.” It is the primary dye in this method; safranin is the counterstain.
- “A pink-only slide means no spore-former.” Culture age and species variation can leave spores absent from a given smear.
- “Any published recipe is interchangeable.” Concentrations differ widely between sources, as the table shows.
- “The spore takes up the dye easily.” The reverse is true: the spore barrier resists dye until heat or another treatment breaks it down, which is why the dye then stays put through the water rinse.
FAQ
What color are endospores with malachite green?
Bright green, sometimes slightly bluish, while vegetative cells appear brownish red to pink after the safranin counterstain (ASM Microbe Library).
Why is heat needed?
Heat helps overcome the spore’s permeability barrier so the dye can enter. The ASM protocol steams the malachite green-covered smear for 5 minutes.
Why is safranin used?
Tap water removes malachite green from vegetative cells but not from endospores. Safranin colours the vegetative cells so they contrast with the green spores.
What is the difference between the Schaeffer-Fulton and Dorner methods?
Dorner (1922) uses carbolfuchsin, acid-alcohol and nigrosin and gives red spores on a black background; Schaeffer and Fulton modified it in 1933 to be faster, using malachite green and safranin.
Sources
- ASM Microbe Library, Endospore Stain Protocol, Hussey and Zayaitz, 2016. Checked 2026-11-01.
- US FDA, Bacteriological Analytical Manual R32a: Endospore Stain. Checked 2026-11-01.
