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Short answer
Bacillus subtilis is a gram-positive, spore-forming rod: in a young, actively growing culture it retains crystal violet and stains purple. Older cultures often give gram-variable smears, with pink cells mixed among purple ones, so a pink B. subtilis cell is not proof of a gram-negative organism. Spores are usually not what the Gram stain is for.
Definition and scope
Bacillus subtilis is a rod-shaped bacterium that has long served as a teaching and research organism for the Gram stain; a 1983 Journal of Bacteriology study by Beveridge and Davies compared B. subtilis and Escherichia coli specifically to follow what the Gram stain does to a gram-positive and a gram-negative cell. In that work, which used a platinum compound in place of iodine as the mordant, the dye complex was retained within the B. subtilis cell wall, whereas ethanol removed the dye from E. coli.
This page is a laboratory and teaching reference on how B. subtilis reads on a Gram stain and why the result can change with culture age. It is not clinical advice and does not repeat the staining method; for reagents and the general procedure, see the CASRAI guide to Gram stain procedure, reagents and how to read the result.
How the Gram stain reads B. subtilis
The American Society for Microbiology (ASM) Gram stain protocol explains the basis of the result: gram-positive cells have a thick peptidoglycan layer (described as about 90% of the cell wall) and hold the purple dye, while gram-negative cells have a thin layer (about 10%) and take up the pink counterstain. For B. subtilis the expected reading on a good smear is therefore purple rods.
Why a correct smear can show pink cells
ASM lists Bacillus among the genera in which a decrease in peptidoglycan thickness during growth coincides with an increasing number of cells that stain gram-negative, and states that in all bacteria the age of the culture may influence the stain. Beveridge’s 1990 study in the Journal of Bacteriology examined the Bacillus–Butyrivibrio–Clostridium group (the Bacillus strain studied was B. brevis) and reported that these cells stained gram-positive in lag and early exponential growth, but as doubling times increased the wall under the surface layer became thinner and the cells more fragile to the Gram stain; by stationary phase the cultures were virtually gram-negative.
ASM therefore recommends young, actively growing cultures for Gram staining, and notes that older cultures may have breaks in the cell wall and often give gram-variable results.
Two other causes of pink gram-positive cells
ASM calls decolorization the most crucial step. Over-decolorizing turns gram-positive cells pink or red, and the right degree depends on smear thickness. A thick smear can under-decolorize, which leaves gram-negative cells purple. ASM recommends a thin smear without clumping.
Gram stain versus endospore stain
Bacillus species form endospores, but ASM states that Gram staining and simple staining may or may not reveal them. The table below compares the two stains as the ASM protocols describe them.
| Question | Gram stain | Endospore stain (Schaeffer-Fulton) |
|---|---|---|
| What it shows | Gram reaction, shape and arrangement of cells | Whether endospores are present, as a clue to identification |
| Colours | Gram-positive purple; gram-negative pink or red | Endospores green (sometimes bluish); vegetative cells brownish-red or pinkish |
| Why it behaves this way | Cell wall peptidoglycan thickness and integrity | The spore’s permeability barrier keeps dyes out unless it is disrupted by heat, chemicals, ultraviolet light or mechanical rupture |
| Culture age | Young, actively growing cultures recommended | Most methods suggest cultures about 18 to 36 hours old; young cultures may show no spores |
ASM adds that the refractility of spores is often evident with simple staining or Gram staining, and that phase-contrast microscopy can show spores without staining. A positive endospore stain is described as an important clue because relatively few species produce endospores.
Where it matters for laboratories
A short checklist drawn from the ASM protocols, useful for a teaching or research laboratory writing the stain into a controlled procedure:
- Record culture age. The same strain can read differently at different ages.
- Run a control on the same slide. ASM recommends including cells of known Gram reaction beside the test culture.
- Keep the smear thin. Smear thickness changes how much decolorizer is enough.
- Record what you saw. Shape, arrangement and Gram reaction; add a spore observation only if a spore stain or phase-contrast was used.
- Know the decolorizer. ASM describes 95% ethanol as the slower option recommended for student use to prevent over-decolorization; acetone is fastest.
- Confirm if in doubt. ASM describes the 3% KOH string test as a confirmatory test for gram reaction.
The reagents, slides and oil-immersion optics behind this work are covered in CASRAI’s guides to microscope slides and microscope objective selection. For containment questions around any bacterial culture, see the entry on biosafety levels, and for background see what bacteriology is.
Common misunderstandings
- “Is Bacillus subtilis gram-negative?” No. It is a gram-positive rod. Pink cells in an older culture reflect a thinning, fragile wall or a staining error, not a change of group.
- “Spores will show up on the Gram stain.” Not reliably. ASM says the Gram stain may or may not reveal them; a spore stain is the dedicated test.
- “No spores seen means it is not a spore-former.” Not necessarily. ASM notes young cultures of spore-formers may show no endospores because there is not enough stress to induce sporulation.
- “Gram-variable means a mixed culture.” It can also mean one culture of one organism that is aging.
FAQ
Is Bacillus subtilis gram-positive or gram-negative?
Gram-positive. A young culture stains purple; older cultures can show pink cells because the cell wall thins.
Why does my Bacillus subtilis Gram stain look pink?
The common causes in the ASM protocol are an old culture, over-decolorization and a smear thicker than the decolorizing time allows. Re-stain from a young culture with a known control on the same slide.
Can a Gram stain show Bacillus subtilis spores?
It may, but not reliably. The Schaeffer-Fulton endospore stain, or phase-contrast microscopy, is the method ASM describes for demonstrating spores.
How old should the culture be?
For the Gram stain, ASM recommends young, actively growing cultures. For an endospore stain, most methods suggest about 18 to 36 hours.
Sources
- American Society for Microbiology, Gram Stain Protocols (Smith and Hussey, 2005/2016). asm.org Gram stain protocols (PDF). Date checked: 2026-10-29.
- American Society for Microbiology, Endospore Stain Protocol (Hussey and Zayaitz, 2007/2016). asm.org endospore stain protocol (PDF). Date checked: 2026-10-29.
- Beveridge TJ, Davies JA. Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain. J Bacteriol 1983;156(2):846-858. PubMed 6195148. Date checked: 2026-10-29.
- Beveridge TJ. Mechanism of gram variability in select bacteria. J Bacteriol 1990;172(3):1609-1620. PubMed 1689718. Date checked: 2026-10-29.
